Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.

Insights into the xylan degradation system of Cellulomonas sp. B6: biochemical characterization of rCsXyn10A and rCsAbf62A.

Valorization of the hemicellulose fraction of plant biomass is crucial for the sustainability of lignocellulosic biorefineries. The Cellulomonas genus comprises Gram-positive Actinobacteria that degrade cellulose and other polysaccharides by secreting a complex array of enzymes. In this work, we studied the specificity and synergy of two enzymes, CsXyn10A and CsAbf62A, which were identified as highly abundant in the extracellular proteome of Cellulomonas sp. B6 when grown on wheat bran. To explore their potential for bioprocessing, the recombinant enzymes were expressed and their activities were thoroughly characterized. rCsXyn10A is a GH10 endo-xylanase (EC 3.2.1.8), active across a broad pH range (5 to 9), at temperatures up to 55 °C. rCsAbf62A is an α-L-arabinofuranosidase (ABF) (EC 3.2.1.55) that specifically removes α-1,2 and α-1,3-L-arabinosyl substituents from arabino-xylo-oligosaccharides (AXOS), xylan, and arabinan backbones, but it cannot act on double-substituted residues. It also has activity on pNPA. No differences were observed regarding activity when CsAbf62A was expressed with its appended CBM13 module or only the catalytic domain. The amount of xylobiose released from either wheat arabinoxylan or arabino-xylo-oligosaccharides increased significantly when rCsXyn10A was supplemented with rCsAbf62A, indicating that the removal of arabinosyl residues by rCsAbf62A improved rCsXyn10A accessibility to ß-1,4-xylose linkages, but no synergism was observed in the deconstruction of wheat bran. These results contribute to designing tailor-made, substrate-specific, enzymatic cocktails for xylan valorization. KEY POINTS: • rCsAbf62A removes α-1,2 and α-1,3-L-arabinosyl substituents from arabino-xylo-oligosaccharides, xylan, and arabinan backbones. • The appended CBM13 of rCsAbf62A did not affect the specific activity of the enzyme. • Supplementation of rCsXyn10A with rCsAbf62A improves the degradation of AXOS and xylan.

Optimisation of xylanases production by two Cellulomonas strains and their use for biomass deconstruction.

One of the main distinguishing features of bacteria belonging to the Cellulomonas genus is their ability to secrete multiple polysaccharide degrading enzymes. However, their application in biomass deconstruction still constitutes a challenge. We addressed the optimisation of the xylanolytic activities in extracellular enzymatic extracts of Cellulomonas sp. B6 and Cellulomonas fimi B-402 for their subsequent application in lignocellulosic biomass hydrolysis by culture in several substrates. As demonstrated by secretomic profiling, wheat bran and waste paper resulted to be suitable inducers for the secretion of xylanases of Cellulomonas sp. B6 and C. fimi B-402, respectively. Both strains showed high xylanolytic activity in culture supernatant although Cellulomonas sp. B6 was the most efficient xylanolytic strain. Upscaling from flasks to fermentation in a bench scale bioreactor resulted in equivalent production of extracellular xylanolytic enzymatic extracts and freeze drying was a successful method for concentration and conservation of the extracellular enzymes, retaining 80% activity. Moreover, enzymatic cocktails composed of combined extra and intracellular extracts effectively hydrolysed the hemicellulose fraction of extruded barley straw into xylose and xylooligosaccharides. KEY POINTS: • Secreted xylanase activity of Cellulomonas sp. B6 and C. fimi was maximised. • Biomass-induced extracellular enzymes were identified by proteomic profiling. • Combinations of extra and intracellular extracts were used for barley straw hydrolysis.

PsAA9A, a C1-specific AA9 lytic polysaccharide monooxygenase from the white-rot basidiomycete Pycnoporus sanguineus.

Woody biomass represents an important source of carbon on earth, and its global recycling is highly dependent on Agaricomycetes fungi. White-rot Basidiomycetes are a very important group in this regard, as they possess a large and diverse enzymatic repertoire for biomass decomposition. Among these enzymes, the recently discovered lytic polysaccharide monooxygenases (LPMOs) have revolutionized biomass processing with their novel oxidative mechanism of action. The strikingly high representation of LPMOs in fungal genomes raises the question of their functional versatility. In this work, we studied an AA9 LPMO from the white-rot basidiomycete Pycnoporus sanguineus, PsAA9A. Successfully produced as a recombinant secreted protein in Pichia pastoris, PsAA9A was found to be a C1-specific LPMO active on cellulosic substrates, generating native and oxidized cello-oligosaccharides in the presence of an external electron donor. PsAA9A boosted cellulolytic activity of glysoside hydrolases from families GH1, GH5, and GH6.This study serves as a starting point towards understanding the functional versatility and biotechnological potential of this enzymatic family, highly represented in wood decay fungi, in Pycnoporus genus. KEY POINTS: • PsAA9A is the first AA9 from P. sanguineus to be characterized. • PsAA9A has activity on cellulose, producing C1-oxidized cello-oligosaccharides. • Boosting activity with GH1, GH5, and GH6 was proven.

SOCS2 deletion protects against hepatic steatosis but worsens insulin resistance in high-fat-diet-fed mice.

Hepatic steatosis is a prominent feature in patients with growth hormone (GH) deficiency. The ubiquitin ligase SOCS2 attenuates hepatic GH signaling by inhibiting the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5b (STAT5b) axis. Here, we investigated the role of SOCS2 in the development of diet-induced hepatic steatosis and insulin resistance. SOCS2-knockout (SOCS2(-/-)) mice and wild-type littermates were fed for 4 mo with control or high-fat diet, followed by assessment of insulin sensitivity, hepatic lipid content, and expression of inflammatory cytokines. SOCS2(-/-) mice exhibited increased hepatic TG secretion by 77.6% (P<0.001) as compared with wild-type control mice and were protected from high-fat-diet (HFD)-induced hepatic steatosis, showing 49.3% (P<0.01) reduction in liver TG levels compared to HFD-fed wild-type littermates. In contrast, we found that HFD-triggered attenuation of systemic insulin sensitivity was more marked in SOCS2(-/-) mice. Livers from the HFD-fed SOCS2(-/-) mice showed increased NF-κB activity as well as elevated expression of genes for the inflammatory cytokines IFN-γ and IL-6. An inhibitory role of SOCS2 on Toll-like receptor 4 signaling was demonstrated in macrophages obtained from the SOCS2(-/-) and wild-type mice. This study identified SOCS2 as an important regulator of hepatic homeostasis under conditions of high-fat dietary stress.

SOCS2 protects against chemical-induced hepatocellular carcinoma progression by modulating inflammation and cell proliferation in the liver.

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers worldwide, but the precise intracellular mechanisms underlying the progression of this inflammation associated cancer are not well established. SOCS2 protein plays an important role in the carcinogenesis of different tumors by regulating cytokine signalling through the JAK/STAT axis. However, its role in HCC is unclear. Here, we investigate the role of SOCS2 in HCC progression and its potential as HCC biomarker. The effects of SOCS2 in HCC progression were evaluated in an experimental model of diethylnitrosamine (DEN)-induced HCC in C57BL/6 and SOCS2 deficient mice, in cultured hepatic cells, and in liver samples from HCC patients. Mice lacking SOCS2 showed higher liver tumor burden with increased malignancy grade, inflammation, fibrosis, and proliferation than their controls. Protein and gene expression analysis reported higher pSTAT5 and pSTAT3 activation, upregulation of different proteins involved in survival and proliferation, and increased levels of proinflammatory and pro-tumoral mediators in the absence of SOCS2. Clinically relevant, downregulated expression of SOCS2 was found in neoplasia from HCC patients compared to healthy liver tissue, correlating with the malignancy grade. In summary, our data show that lack of SOCS2 increases susceptibility to chemical-induced HCC and suggest the tumor suppressor role of this protein by regulating the oncogenic and inflammatory responses mediated by STAT5 and STAT3 in the liver. Hence, SOCS2 emerges as an attractive target molecule and potential biomarker to deepen in the study of HCC treatment.

Synergic activity of Cel8Pa ß-1,4 endoglucanase and Bg1Pa ß-glucosidase from Paenibacillus xylanivorans A59 in beta-glucan conversion.

In the efficient bioconversion of polysaccharides from lignocellulosic biomass, endoglucanases and ß-glucosidases are key enzymes for the deconstruction of ß-glucans. In this work, we focused on a GH8 endoglucanase (Cel8Pa) and a GH1 ß-glucosidase (Bg1Pa) from Paenibacillus xylanivorans A59. Cel8Pa was active on a broad range of substrates, such as ß-glucan from barley (24.5 IU/mg), lichenan (17.9 IU/mg), phosphoric acid swollen cellulose (PASC) (9.7 IU/mg), carboxi-methylcellulose (CMC) (7.3 IU/mg), chitosan (1.4 IU/mg) and xylan (0.4 IU/mg). Bg1Pa was active on cellobiose (C2) and cello-oligosaccharides up to C6, releasing glucose as the main product. When both enzymes were used jointly, there was a synergic effect in the conversion rate of polysaccharides to glucose. Cel8Pa and Bg1Pa presented important properties for simultaneous saccharification and fermentation (SSF) processes in second generation bioethanol production, such as tolerance to high concentration of glucose and ethanol.

A thermostable GH8 endoglucanase of Enterobacter sp. R1 is suitable for ß-glucan deconstruction.

Glycoside hydrolase family 8 (GH8) includes endoglucanases, lichenases, chitosanases and xylanases, which are essential for polysaccharides breakdown. In this work, we studied a thermally stable GH8 from the cellulose synthase complex of Enterobacter sp. R1, for deconstruction of ß-glucans. The biochemical characterization of the recombinant GH8ErCel showed high specificity towards barley ß-glucan and lichenan and lower activity on carboxymethylcellulose and swollen cellulose, yielding different length oligosaccharides. By molecular modeling, six conserved subsites for glucose binding and some possible determinants for its lack of xylanase and chitosanase activity were identified. GH8ErCel was active at a broad range of pH and temperature and presented remarkable stability at 60 °C. Additionally, it hydrolyzed ß-glucan from oat and wheat brans mainly to tri- and tetraoligosaccharides. Therefore, GH8ErCel may be a good candidate for enzymatic deconstruction of ß-glucans at high temperature in food and feed industries, including the production of prebiotics and functional foods.

LXR Signaling Regulates Macrophage Survival and Inflammation in Response to Ionizing Radiation.

PURPOSE: To evaluate the role of liver X receptor (LXR) nuclear receptors on irradiation-induced cell death and polarization of macrophages and the potential implications in the context of radiation therapy treatment of cancer. METHODS AND MATERIALS: Primary and immortalized murine bone marrow-derived macrophages (BMDMs) from wild type or LXR double knock-out mice were exposed to gamma irradiation. Subsequently, analysis of LXR signaling on cell proliferation and cytotoxicity induced by ionizing radiation was determined by time-lapse photomicroscopy. Genotoxic cell damage was evaluated by Western blot of γ-H2AX and p53. Pyroptosis was analyzed through cell viability assay, lactate dehydrogenase release assay, and Western blot of caspase-1 active protein. Expression of inflammatory markers was measured by real-time quantitative polymerase chain reaction. RESULTS: Genetic and pharmacologic inactivation of LXR induced radiosensitivity of macrophages. LXR deficiency decreased cell proliferation and enhanced cytotoxicity induced by ionizing radiation in both immortalized and primary BMDMs. Protein levels of γ-H2AX and p53, both involved in response to cell damage, were exacerbated in LXR-deficient macrophages exposed to irradiation. Cell membrane damage was augmented and cell viability was decreased in LXR-deficient macrophages compared with LXR wild type macrophages in response to irradiation. In addition, LXR deficiency enhanced both caspase-1 activation and lactate dehydrogenase release in BMDM exposed inflammasome activators. LXR inactivation or deficiency markedly increased the expression of proinflammatory markers IL-1ß, IL-6, and inducible nitric oxide synthase in irradiated macrophages. CONCLUSIONS: The present work identifies LXR transcription factors as potential therapeutic targets to enhance the suppressive effects of radiation therapy on tumor growth through induction of macrophage cell death and activation of the inflammatory cascade.

Signal transducer and activator of transcription (STAT)-5: an opportunity for drug development in oncohematology.

The signal transducer and activator of transcription (STAT) are transcription factors that work via JAK/STAT pathway regulating the expression of genes involved in cell survival, proliferation, differentiation, development, immune response, and, among other essential biological functions, hematopoiesis. JAK/STAT signaling is strictly regulated under normal physiological conditions. However, a large group of diverse diseases has been associated to an aberrant regulation of STAT factors. Erroneous modulation of the pathway leads to constitutive STAT activation, thereby driving proliferation, inflammation, and an uncontrolled immune response. Deregulated STAT5 activation has been found in the development of many hematopoietic tumors, including chronic and acute leukemias, polycythemia vera, and lymphoma. Mutations in the kinases that phosphorylate STAT5, and/or overexpression of the upstream receptor-associated tyrosine kinases have been suggested as the main drivers of constitutive STAT5 activation. Hyper-activated STAT5 leads to the aberrant expression of its target genes including antiapoptotic, proliferative, and pro-inflammatory genes, favouring tumorigenesis. In this review, we intent to discuss the biology of JAK/STAT pathway, with particular focus on STAT5 and its crucial role in the development and progression of hematologic malignancies. Furthermore, we provide a synopsis of potential therapeutic strategies based on STAT5 activity inhibition that may represent an excellent opportunity for drug development in oncohematology.

Migración de dispositivo intrauterino a vejiga / Intrauterine device migration to bladder

Introducción: La litiasis vesical secundaria se forma en el reservorio vesical y requiere la existencia de condiciones patológicas previas como lo son los cuerpos extraños. Objetivo: Describir dos casos clínicos de litiasis vesical secundaria a migración de un dispositivo intrauterino. Caso clínico: Se presentan dos casos de migración intravesical de dispositivo intrauterino con litiasis vesical secundaria. Se diagnosticaron años después de su inserción, ante la aparición de dolor pélvico, cistitis a repetición y hematuria. La laparoscopia no fue útil para su diagnóstico. En consulta de Urología la ultrasonografía y la radiografía de pelvis fueron herramientas diagnósticas útiles ante la sospecha inicial de esta patología. Presentaron buena evolución y regresión total de los síntomas tras cistolitotomía suprapúbica. Conclusión: Se debe pensar en la posibilidad de migración de un dispositivo intrauterino a vejiga ante la cronicidad de síntomas urinarios irritativos bajos en toda mujer que emplee este método anticonceptivo y desconozca su paradero(AU)

Introduction: Secondary bladder lithiasis is formed in the bladder reservoir and requires the existence of previous pathological conditions such as foreign bodies. Objective: To describe two clinical cases of bladder lithiasis secondary to intrauterine device migration. Clinical case report: Two cases of intravesical migration of an intrauterine device with secondary bladder stones are reported. They were diagnosed years after insertion, due to the appearance of pelvic pain, recurrent cystitis and hematuria. Laparoscopy was not helpful for its diagnosis. In Urology consultation, ultrasound and pelvic radiography were useful diagnostic tools in the event of the initial suspicion of this pathology. They showed good evolution and total regression of symptoms after suprapubic cystolithotomy. Conclusion: The possibility of an intrauterine device migration to the bladder should be considered when chronicity of irritative low urinary symptoms in every woman who uses this contraceptive method and which locations are unknown(AU)

Feocromocitoma Benigno vs Tumor Renal. Presentación de un caso / Benign Pheochromocytoma vs Kidney Tumor. A case presentation

RESUMEN Introducción: El feocromocitoma es una neoplasia neuroendocrina localizada en la médula adrenal. Objetivo: Destacar la importancia que todavía posee en la práctica clínica el interrogatorio y el examen físico minucioso, a pesar de los novedosos medios diagnósticos con que se cuenta en la actualidad. Presentación del caso : Se presenta caso clínico de paciente masculino de 17 años de edad, con incidentaloma suprarrenal izquierdo, catalogado inicialmente como tumor renal abscedado, cuya biopsia de pieza operatoria demostró finalmente que correspondía a un feocromocitoma benigno. La determinación de catecolaminas fue normal, en la tomografía computarizada se evidenció una masa retroperitoneal que involucraba riñón y suprarrenal izquierdos. Se realizó la resección de la neoplasia y fue dado de alta médica en buenas condiciones. Conclusiones: El feocromocitoma es una patología infrecuente y compleja, cuya resolución quirúrgica es mandatoria.

ABSTRACT Background: Pheochromocytoma is a neuroendocrine neoplasm located in the adrenal medulla. Objective: To highlight the importance of physical examination and the detailed clinical practice, despite the new diagnostic means that are currently available. Case presentation: A 17-years-old male patient with left adrenal incidentaloma, initially classified as an abscessed renal tumor, whose surgical specimen biopsy finally showed that it corresponded to a benign pheochromocytoma it is presented. The catecholamine determination was normal, the computed tomography revealed a retroperitoneal mass that involved the left kidney and adrenal. The neoplasm was resected and he was discharged in good condition. Conclusions: Pheochromocytoma is an infrequent and complex pathology, whose surgical resolution is necessary.

Sex steroids and growth hormone interactions.

GH and sex hormones are critical regulators of body growth and composition, somatic development, intermediate metabolism, and sexual dimorphism. Deficiencies in GH- or sex hormone-dependent signaling and the influence of sex hormones on GH biology may have a dramatic impact on liver physiology during somatic development and in adulthood. Effects of sex hormones on the liver may be direct, through hepatic receptors, or indirect by modulating endocrine, metabolic, and gender-differentiated functions of GH. Sex hormones can modulate GH actions by acting centrally, regulating pituitary GH secretion, and peripherally, by modulating GH signaling pathways. The endocrine and/or metabolic consequences of long-term exposure to sex hormone-related compounds and their influence on the GH-liver axis are largely unknown. A better understanding of these interactions in physiological and pathological states will contribute to preserve health and to improve clinical management of patients with growth, developmental, and metabolic disorders.

Suppressor of cytokine signaling 2 (SOCS2) deletion protects against multiple low dose streptozotocin-induced type 1 diabetes in adult male mice.

BACKGROUND: Diabetes type 1 is characterized by the failure of beta cells to produce insulin. Suppressor of cytokine signaling (SOCS) proteins are important regulators of the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway. Previous studies have shown that GH can prevent the development of type I diabetes in mice and that SOCS2 deficiency mimics a state of increased GH sensitivity. METHODOLOGY: The elevated sensitivity of SOCS2-/- mice to GH and possibly to PRL was the rationale to analyze the effects of multiple low dose streptozotocin (MLDSTZ)-induced diabetes in SOCS2-/- mice. RESULTS: We show that 6-month-old SOCS2-/- mice, but not 2-month-old mice, were less sensitive to MLDSTZ-induced diabetes, compared to controls. MLDSTZ treatment induced glucose intolerance in both SOCS2+/+ and SOCS2-/- mice, as shown by glucose tolerance tests, with SOCS2+/+ mice showing a more marked intolerance, compared to SOCS2-/- mice. Furthermore, insulin tolerance tests showed that the SOCS2-/- mice have an improved hypoglycemic response to exogenous insulin, compared to SOCS2+/+ mice. Moreover, in isolated islets, lipotoxic effects on insulin release could partly be overcome by ligands, which bind to GH or PRL receptors. CONCLUSION: Knockdown of SOCS2 makes mice less sensitive to MLDSTZ. These results are consistent with the proposal that elimination of SOCS2 in pancreatic islets creates a state of ß-cell hypersensitivity to GH/PRL that mimics events in pregnancy, and which is protective against MLDSTZ-induced type I diabetes in mice. SOCS2-dependent control of ß-cell survival may be of relevance to islet regeneration and survival in transplantation.

[Foodborne outbreak by Clostridium perfringens in a school dining room]. / Brote de toxiinfección alimentaria por Clostridium perfringens en un comedor escolar.

BACKGROUND AND OBJECTIVE: The purpose was to investigate the etiology and epidemiological characteristics of a food-borne infection outbreak in a school dining room. SUBJECTS AND METHOD: Retrospective cohort study. Information about gastrointestinal symptoms and the use of the service of the school dining room was obtained. Coprocultures and detection of Clostridium perfringens enterotoxin in stool samples by means of reverse passive latex agglutination were carried out in 7 ill persons. Relative risks (RR) at 95% confidence intervals (95% CI) were calculated. RESULTS: The attack rate was 17.5% (48/275) and the probability of becoming sick was higher in students who ate at the second shift than in those eating at the first one (RR=13.8; 95% CI: 4.4-43.1). C perfringens enterotoxin was detected in 6 stool samples from patients. A high recount of C perfringens was not observed in those food samples kept frozen after their elaboration. CONCLUSIONS: The determination of C perfringens enterotoxin in feces allowed to confirm the etiologic agent of the outbreak.

[Factors related to perceived poor health condition or poor quality of life among those over 65 years of age]. / Factores asociados a mal estado de salud percibido o a mala calidad de vida en personas mayores de 65 años.

BACKGROUND: Nowadays, in the developed countries, a long lifespan is no longer the exception to the rule, however there are still many people who even today do not manage to age with a good quality of life. The objectives of this study are, first of all, to contribute to a better knowledge of the main factors which have an impact on the quality of life and the perceived health condition of those over age 65 and, secondly, to determine what advantages and disadvantages involved in each one of the tools for gauging health and quality of life as compared to the other two tools employed. METHODS: Based on 911 home surveys of non-institutionalized individuals over age 65, a multivariate analysis was made using Logistic regression, relating the results obtained in the Nottingham Health Profile (NHP), the EuroQol and the Self Perceived Health Status to the socio-demographic characteristics, the level of economic resources, the degree of social-family support, the physical and mental health condition and the functional capacity. RESULTS: The main factors related to the perception of a poor health condition and a poor quality of life in the EuroQol and the NHP are anxiety disorders (Odds Ratio ranging from 1.8(IC:1.2-2.8) for mobility and 7.9(IC:4.5-13.9) for Profile*11111), depressive disorders (OR:1.8(IC:1.3-2.6) for pain/discomfort-3.3(IC:2.1-5.1) for social isolation), lack of exercise (OR:1.4 (IC:1-2.1) for anxiety/depression -3.9(IC:2.5-6.2) for everyday activities), dependence for basic everyday living activities (OR:0.5(IC:0.3-0.9) for emotional reaction -4.8(IC:3-7.6) for everyday activities) and dependence for the instrumental daily living activities (OR:1.5(IC:1.1-2.1) for Analog Visual Scale c < 70-7.1(IC:2.9-17.2) for personal care). CONCLUSIONS: Mental health and functioning capacity are the factors which have the greatest bearing on the perception of health condition and quality of life of individuals over age 65. Given that the three tools used have led to similar results, the EuroQol has advantages to offer due to its short length, including an overall evaluation by dimensions.

Lipid profiling and transcriptomic analysis reveals a functional interplay between estradiol and growth hormone in liver.

17ß-estradiol (E2) may interfere with endocrine, metabolic, and gender-differentiated functions in liver in both females and males. Indirect mechanisms play a crucial role because of the E2 influence on the pituitary GH secretion and the GHR-JAK2-STAT5 signaling pathway in the target tissues. E2, through its interaction with the estrogen receptor, exerts direct effects on liver. Hypothyroidism also affects endocrine and metabolic functions of the liver, rendering a metabolic phenotype with features that mimic deficiencies in E2 or GH. In this work, we combined the lipid and transcriptomic analysis to obtain comprehensive information on the molecular mechanisms of E2 effects, alone and in combination with GH, to regulate liver functions in males. We used the adult hypothyroid-orchidectomized rat model to minimize the influence of internal hormones on E2 treatment and to explore its role in male-differentiated functions. E2 influenced genes involved in metabolism of lipids and endo-xenobiotics, and the GH-regulated endocrine, metabolic, immune, and male-specific responses. E2 induced a female-pattern of gene expression and inhibited GH-regulated STAT5b targeted genes. E2 did not prevent the inhibitory effects of GH on urea and amino acid metabolism-related genes. The combination of E2 and GH decreased transcriptional immune responses. E2 decreased the hepatic content of saturated fatty acids and induced a transcriptional program that seems to be mediated by the activation of PPARα. In contrast, GH inhibited fatty acid oxidation. Both E2 and GH replacements reduced hepatic CHO levels and increased the formation of cholesterol esters and triacylglycerols. Notably, the hepatic lipid profiles were endowed with singular fingerprints that may be used to segregate the effects of different hormonal replacements. In summary, we provide in vivo evidence that E2 has a significant impact on lipid content and transcriptome in male liver and that E2 exerts a marked influence on GH physiology, with implications in human therapy.

The influence of estrogens on the biological and therapeutic actions of growth hormone in the liver.

GH is main regulator of body growth and composition, somatic development, intermediate metabolism and gender-dependent dimorphism in mammals. The liver is a direct target of estrogens because it expresses estrogen receptors which are connected with development, lipid metabolism and insulin sensitivity, hepatic carcinogenesis, protection from drug-induced toxicity and fertility. In addition, estrogens can modulate GH actions in liver by acting centrally, regulating pituitary GH secretion, and, peripherally, by modulating GHR-JAK2-STAT5 signalling pathway. Therefore, the interactions of estrogens with GH actions in liver are biologically and clinically relevant because disruption of GH signaling may cause alterations of its endocrine, metabolic, and gender differentiated functions and it could be linked to dramatic impact in liver physiology during development as well as in adulthood. Finally, the interplay of estrogens with GH is relevant because physiological roles these hormones have in human, and the widespread exposition of estrogen or estrogen-related compounds in human. This review highlights the importance of these hormones in liver physiology as well as how estrogens modulate GH actions in liver which will help to improve the clinical use of these hormones.

Influence of neonatal hypothyroidism on hepatic gene expression and lipid metabolism in adulthood.

Thyroid hormones are required for normal growth and development in mammals. Congenital-neonatal hypothyroidism (CH) has a profound impact on physiology, but its specific influence in liver is less understood. Here, we studied how CH influences the liver gene expression program in adulthood. Pregnant rats were given the antithyroid drug methimazole (MMI) from GD12 until PND30 to induce CH in male offspring. Growth defects due to CH were evident as reductions in body weight and tail length from the second week of life. Once the MMI treatment was discontinued, the feed efficiency increased in CH, and this was accompanied by significant catch-up growth. On PND80, significant reductions in body mass, tail length, and circulating IGF-I levels remained in CH rats. Conversely, the mRNA levels of known GH target genes were significantly upregulated. The serum levels of thyroid hormones, cholesterol, and triglycerides showed no significant differences. In contrast, CH rats showed significant changes in the expression of hepatic genes involved in lipid metabolism, including an increased transcription of PPARα and a reduced expression of genes involved in fatty acid and cholesterol uptake, cellular sterol efflux, triglyceride assembly, bile acid synthesis, and lipogenesis. These changes were associated with a decrease of intrahepatic lipids. Finally, CH rats responded to the onset of hypothyroidism in adulthood with a reduction of serum fatty acids and hepatic cholesteryl esters and to T3 replacement with an enhanced activation of malic enzyme. In summary, we provide in vivo evidence that neonatal hypothyroidism influences the hepatic transcriptional program and tissue sensitivity to hormone treatment in adulthood. This highlights the critical role that a euthyroid state during development plays on normal liver physiology in adulthood.

Liver X receptor agonist downregulates growth hormone signaling in the liver.

Liver X receptor (LXR) agonists have been shown to influence the development of hyperlipidemia and atherosclerosis in mouse models. It has also been demonstrated that some LXR agonists can cause hepatic steatosis in experimental animals. Growth hormone (GH) is known to regulate hepatic metabolism and the absence of hepatic GH receptors (GHR) leads to hepatic steatosis. In this study, we analyzed whether the actions of LXR agonists could involve interference with GH signaling. We showed that LXR agonists impair GH signaling in hepatocytes. LXR agonist treatment attenuated GH induction of suppressor of cytokine signaling 2 (SOCS2), SOCS3, and CIS mRNA levels in BRL-4 cells. Likewise, the activity of a luciferase reporter vector driven by the GH response element (GHRE) of the SOCS2 gene was inhibited by simultaneous treatment with an LXR agonist. The inhibitory effect of LXR agonists on GH signals can be mimicked by overexpression of the LXR regulated factors, sterol regulatory element binding protein 1 (SREBP1) and SREBP2, in hepatic cells. In both cases total and phosphorylated signal transducers and activators of transcription 5b (STAT5b) protein levels were significantly reduced. DNA binding assays demonstrated that SREBP1 binds to an E-box within a previously defined GHRE in the SOCS2 gene promoter, but does not compete with STAT5b binding to a nearby site in the same promoter construct. Taken together, our findings indicate that the inhibitory effects of LXR agonists on GH signaling are mediated by SREBP1, through the downregulation of STAT5b gene transcription and stimulation of STAT5b protein degradation. The findings provide a new insight into the understanding of the molecular actions of LXR agonists, which may be of relevance to their pharmacological actions.