Insulin-like Growth Factor Igf Research Articles

Molecular Ontogeny of First-Feeding European Eel Larvae.

Digestive system functionality of fish larvae relies on the onset of genetically pre-programmed and extrinsically influenced digestive functions. This study explored how algal supplementation (green-water) until 14 days post hatch (dph) and the ingestion of food [enriched rotifer (Brachionus plicatilis) paste] from 15 dph onward affects molecular maturation and functionality of European eel larval ingestion and digestion mechanisms. For this, we linked larval biometrics to expression of genes relating to appetite [ghrelin (ghrl), cholecystokinin (cck)], food intake [proopiomelanocortin (pomc)], digestion [trypsin (try), triglyceride lipase (tgl), amylase (amyl)], energy metabolism [ATP synthase F0 subunit 6 (atp6), cytochrome-c-oxidase 1 (cox1)], growth [insulin-like growth factor (igf1)] and thyroid metabolism [thyroid hormone receptors (thrαA, thrβB)]. Additionally, we estimated larval nutritional status via nucleic acid analysis during transition from endogenous and throughout the exogenous feeding stage. Results showed increased expression of ghrl and cck on 12 dph, marking the beginning of the first-feeding window, but no benefit of larviculture in green-water was observed. Moreover, expression of genes relating to protein (try) and lipid (tgl) hydrolysis revealed essential digestive processes occurring from 14 to 20 dph. On 16 dph, a molecular response to initiation of exogenous feeding was observed in the expression patterns of pomc, atp6, cox1, igf1, thrαA and thrβB. Additionally, we detected increased DNA contents, which coincided with increased RNA contents and greater body area, reflecting growth in feeding compared to non-feeding larvae. Thus, the here applied nutritional regime facilitated a short-term benefit, where feeding larvae were able to sustain growth and better condition than their non-feeding conspecifics. However, RNA:DNA ratios decreased from 12 dph onward, indicating a generally low larval nutritional condition, probably leading to the point-of-no-return and subsequent irreversible mortality due to unsuccessful utilization of exogenous feeding. In conclusion, this study molecularly identified the first-feeding window in European eel and revealed that exogenous feeding success occurs concurrently with the onset of a broad array of enzymes and hormones, which are known to regulate molecular processes in feeding physiology. This knowledge constitutes essential information to develop efficient larval feeding strategies and will hopefully provide a promising step toward sustainable aquaculture of European eel.

Effects of β2-adrenoceptor agonists on gilthead sea bream (Sparus aurata) cultured muscle cells

β2-adrenoceptors are a subtype of G-protein coupled receptors whose activation leads to increased protein synthesis and decreased degradation in mammalian skeletal muscle, causing hypertrophy. In this study, we compared the effects of the classical β2-agonist noradrenaline (NA) with two representatives of a new generation of agonists (formoterol, FOR and salmeterol, SALM) on growth and metabolism of primary cultured muscle cells of gilthead sea bream. Activation of signaling pathways, cell development and expression of relevant genes were analyzed in day 4 myocytes. The three agonists increased either cAMP levels or PKA phosphorylation, plus TOR phosphorylation, and the proportion of proliferating cell nuclear antigen (PCNA)-positive cells, in parallel with pcna mRNA levels. Thus, demonstrating that these cells are β2-agonists-responsive, and supporting enhanced cell proliferation. The expression of the myogenic factor myf5 was significantly down-regulated, suggesting that the cells were already destined to the muscular linage; while insulin-like growth factors (igf-1 and igf-2) transcript levels were up-regulated, proposing an additional anabolic effect through their local production. Furthermore, SALM treatment up-regulated expression of the lipases (hsl and lipa) and the β-oxidation marker cpt1a, and all three agonists increased mitochondrial dehydrogenase hadh mRNA levels. These data correspond with a situation of enhanced lipolytic and β-oxidation capacity, a fact supported by the higher glycerol released into the media induced by the agonists. Overall, these results suggest a hyperplastic growth condition and a favorable protein/fat ratio profile upon these treatments; consequently, β2-agonists (especially SALM) may be considered good candidates to optimize the growth in this aquaculture species.

Abstract 1852: Combination of the CDK4/6 inhibitor abemaciclib with xentuzumab, a humanized IGF-1 and IGF-2 ligand co-neutralizing monoclonal antibody, results in synergistic antineoplastic effects in human breast cancer cell lines

Abstract Background: The proliferative and pro-survival signals driven by the insulin-like growth factor (IGF) ligands, IGF-1 and IGF-2, are transmitted through their binding to the IGF-1 receptor (IGF-1R). In addition, IGF-2 promotes proliferation through activation of the insulin receptor variant A (IR-A) that is expressed during embryonic development as well as in many cancers. IGF survival signaling has been implicated in driving resistance to cancer therapies with diverse mechanisms of action, due to cross-talk between cellular signaling networks. Recent studies[1][2] suggest that the combination of IGF-1R signaling inhibitors with CDK4/6 inhibitors can result in enhanced anti-tumor activity. The aim of this study was to explore the potential of the IGF-1/-2 ligand blocking antibody, xentuzumab (BI 836845[3]), to enhance the anti-tumor activity of the CDK4/6 inhibitor abemaciclib, alone or in combination with fulvestrant, in human breast cancer (BC) cell lines. Methods: The anti-proliferative activity of the xentuzumab/abemaciclib combination was evaluated using CellTiter-Glo and propidium iodide staining in a panel of 51 and 20 BC cell lines, respectively. Detailed studies of abemaciclib (+/- fulvestrant), xentuzumab and combinations thereof were performed in MCF7 cells. Cell cycle analysis was done by FACS and BrdU ELISA, cellular signaling was assessed by Western blotting, proliferation was evaluated by Incucyte, CellTiter-Glo and alamarBlue assay. Apoptosis was measured by detection of cleaved PARP and caspase 3. Results: Among a panel of BC cell lines, enhanced anti-proliferative activity of xentuzumab+abemaciclib vs. abemaciclib alone was observed specifically in hormone receptor positive (HR+) cell lines. Combined treatment resulted in more pronounced cell cycle arrest in MCF7 cells, associated with synergistic blockade of IGF survival signaling and suppression of cell cycle genes downstream of CDK4/6. The triple combination with fulvestrant more effectively inhibited tumor cell proliferation than the doublet abemaciclib+fulvestrant, and led to induction of apoptosis. Conclusion: The study results show that addition of the IGF-1/-2 neutralizing antibody xentuzumab to abemaciclib, in the absence or presence of fulvestrant, leads to improved anti-proliferative activity and, in the triple combination, results in cellular death in MCF7 HR+ breast cancer cells. A phase Ib trial evaluating the abemaciclib+xentuzumab combination, including triplets with endocrine therapy in HR+BC patients, is currently ongoing.

Characterization of igf1 and igf2 genes during maraena whitefish (Coregonus maraena) ontogeny and the effect of temperature on embryogenesis and igf expression

The insulin-like growth factors IGF-1 and IGF-2 play important roles in the growth, development, and metabolism of teleost fish. We isolated cDNA sequences of igf1, and igf2 genes from maraena whitefish. We quantified the mRNA and protein expressions of IGFs in different tissues of marketable juvenile maraena whitefish. Moreover, we analyzed the gene expression profiles during maraena whitefish development from unfertilized egg to fingerling and examined the effect of incubation temperature on igf1, and igf2 gene expression during embryonic and early larval development.Transcripts encoding IGF-1 or IGF-2 were detected in all tested tissues, with the greatest abundance in the liver. We measured higher igf2 than igf1 copy numbers in all tissues and at all developmental stages examined, even at advanced juvenile stages. Using the Western blot technique, we demonstrated that several isoforms of IGF-1 are expressed in the liver and gills but not in muscle tissue, indicating tissue-specific protein expression of IGF-1. We observed an accelerated embryonic development with increasing temperature, resulting in shortened hatching periods. Out of the three tested temperatures, we observed the highest hatching rate, larval hatching size, and larval growth at 6 °C. At 9 °C, hatching rate, larval hatching size and larval growth were reduced compared to the values we observed at 4 °C and 6 °C, since incubation temperature might have exceeded the optimum. To our knowledge, our data show for the first time that both igf1 and igf2 expression were upregulated due to elevated incubation temperature within embryonic development of fish. Further, we found significantly higher igf expression for the best-developing larvae (6 °C group) at specific life stages of maraena whitefish.

Abstract 20: Xentuzumab, a humanized IGF-1 and IGF-2 ligand neutralizing antibody, improves the antitumor efficacy of enzalutamide in preclinical models of prostate cancer

Abstract Background: The proliferative and pro-survival signals driven by the insulin-like growth factor (IGF) ligands, IGF-1 and IGF-2, are transmitted through their binding to the IGF-1 receptor (IGF-1R). In addition, IGF-2 activates the insulin receptor variant A (IR-A) that is expressed during embryonic development as well as in many cancers. A large body of preclinical evidence suggests that IGF signaling plays a key role in cancer by driving therapy resistance, due to cross-talk with other signaling networks such as androgen receptor signaling. The aim of this study was to explore the potential of the IGF-1/-2 ligand blocking antibody, xentuzumab (BI 836845[1]), to enhance the anti-tumor activity of enzalutamide in prostate cancer cell lines and in a patient-derived prostate cancer xenograft model. Methods: Effects of enzalutamide, xentuzumab and combinations thereof on in vitro proliferation, survival, cell cycle and signaling were evaluated using the prostate cancer cell lines VCaP, DuCaP, MDA PCa 2b, and LNCaP. The in vivo efficacy of enzalutamide, alone and in combination with xentuzumab was investigated using LuCaP 96CR, a patient-derived xenograft model of castration-resistant prostate cancer. Tumors were implanted s.c. into castrate SCID mice. When tumors exceeded 150mm3 animals were randomized into groups: 1) Control; 2) enzalutamide (50 mg/kg QD po), 3) xentuzumab (BI 836845[1], 200 mg/kg QW ip) in combination with enzalutamide. Results: Cell viability was more effectively reduced by the combination of enzalutamide and xentuzumab than either drug alone in three of four cell lines expressing the IGF-1R and the androgen receptor (AR). In VCaP cells, prolonged inhibition of IGF pathway signaling and enhanced blockade of proliferation as well as induction of apoptosis was observed after combination treatment. In vivo, enzalutamide monotherapy did not show significant antitumor efficacy in the LuCaP 96CR model, however, combined treatment with xentuzumab significantly inhibited progression of LuCaP 96CR tumor growth (p<0.001 vs. enzalutamide alone). Reduced serum PSA levels were observed after enzalutamide and combination treatment. Enzalutamide plus xentuzumab inhibited tumor growth at tolerated doses and resulted in significant improvements in survival. Conclusions: These studies demonstrated that addition of the IGF-1/-2 neutralizing antibody xentuzumab to enzalutamide results in improved anti-neoplastic activity in a subset of prostate cancer cell lines in vitro, and to re-sensitization to enzalutamide in a patient-derived xenograft model of CRPC. Reference: [1] Friedbichler K et al. (2014). Mol Cancer Ther 13(2):399-409. Citation Format: Ulrike Weyer-Czernilofsky, Marco H. Hofmann, Paul J. Adam, Flavio Solca, Katrin Friedbichler, Norbert Kraut, Eva Corey, Thomas Bogenrieder. Xentuzumab, a humanized IGF-1 and IGF-2 ligand neutralizing antibody, improves the antitumor efficacy of enzalutamide in preclinical models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 20. doi:10.1158/1538-7445.AM2017-20

Large-scale analysis of variation in the insulin-like growth factor family in humans reveals rare disease links and common polymorphisms

The insulin-like growth factors IGF1 and IGF2 are closely related proteins that are essential for normal growth and development in humans and other species and play critical roles in many physiological and pathophysiological processes. IGF actions are mediated by transmembrane receptors and modulated by IGF-binding proteins. The importance of IGF actions in human physiology is strengthened by the rarity of inactivating mutations in their genes and by the devastating impact caused by such mutations on normal development and somatic growth. Large-scale genome sequencing has the potential to provide new insights into human variation and disease susceptibility. Toward this end, the availability of DNA sequence data from 60,706 people through the Exome Aggregation Consortium has prompted the analyses presented here. Results reveal a broad range of potential missense and other alterations in the coding regions of every IGF family gene, but the vast majority of predicted changes were uncommon. The total number of different alleles detected per gene in the population varied over an ∼15-fold range, from 57 for IGF1 to 872 for IGF2R, although when corrected for protein length the rate ranged from 0.22 to 0.59 changes/codon among the 11 genes evaluated. Previously characterized disease-causing mutations in IGF2, IGF1R, IGF2R, or IGFALS all were found in the general population but with allele frequencies of <1:30,000. A few new highly prevalent amino acid polymorphisms were also identified. Collectively, these data provide a wealth of opportunities to understand the intricacies of IGF signaling and action in both physiological and pathological contexts.

Rebuilding of Carbohydrate and Lipid Metabolism Under Hypoxia: Regularities and Therapeutic Possibilities

Previously, we have found that the effects of chronic (staying in midlands) or moderate periodic hypoxia as well as hypoxic preconditioning is accompanied by beneficial refbuilding in lipid and carbohydrate metabolism in human and animals: a reduction of total cholesterol and its fractions in blood plasma, a hypoglycemic reaction or normalization of elevated blood glucose, and a decrease of impaired glucose tolerance. This metabolic rebuilding has a phase character during the adaptation to hypoxia and following deadaptation. Changes in metabolism during hypoxia occur in a consecutive manner and mediate by induction and repression of target genes following activation of hypoxia responsible signal ways and transcription factors. In particular, we showed the alternate transactivation of HIF-1alpha and -3alpha subunits, and the restructuring of glucose transport by consecutive induction of GLUT-4 and -1 in rat myocardium and lungs. Mitochondrial respiration is consequentially shifted from down - to up- regulation, and from carbohydrate to mainly lipid substrate using. In human, we found attenuation of transcriptional regulator of cholesterol synthesis SREBP-1, leptin and insulin-like growth factor IGF-1 levels in blood plasma under prolonged hypoxic conditions, as well as positive correlation with HIF-dependent protein IGF-1 and HDL-cholesterol level. In middle-aged people with metabolic disorders (metabolic syndrome, diabetes mellitus, dyslipidemia), favorable influence of staying in midlands, moderate acute or periodic hypoxia was found, but metabolic pathology was associated with altered pattern of regulatory protein expression. The data suggest the close relationship between carbohydrate and lipid metabolism under hypoxia, and evidence the possibility of hypoxic correction of metabolic disorders as diabetes, dyslipidemia and metabolic syndrome based on phase processes of metabolic rebuilding.

Regulation of the embryonic erythropoietic niche: a future perspective.

The production of red blood cells, termed erythropoiesis, occurs in two waves in the developing mouse embryo: first primitive erythropoiesis followed by definitive erythropoiesis. In the mouse embryo, both primitive and definitive erythropoiesis originates in the extra-embryonic yolk sac. The definitive wave then migrates to the fetal liver, fetal spleen and fetal bone marrow as these organs form. The fetal liver serves as the major organ for hematopoietic cell expansion and erythroid maturation after mid-gestation. The erythropoietic niche, which expresses critical cytokines such as stem cell factor (SCF), thrombopoietin (TPO) and the insulin-like growth factors IGF1 and IGF2, supports hematopoietic expansion in the fetal liver. Previously, our group demonstrated that DLK1+ hepatoblasts support fetal liver hematopoiesis through erythropoietin and SCF release as well as extracellular matrix deposition. Loss of DLK1+ hepatoblasts in Map2k4−/− mouse embryos resulted in decreased numbers of hematopoietic cells in fetal liver. Genes encoding proteinases and peptidases were found to be highly expressed in DLK1+ hepatoblasts. Capitalizing on this knowledge, and working on the assumption that these proteinases and peptidases are generating small, potentially biologically active peptides, we assessed a range of peptides for their ability to support erythropoiesis in vitro. We identified KS-13 (PCT/JP2010/067011) as an erythropoietic peptide-a peptide which enhances the production of red blood cells from progenitor cells. Here, we discuss the elements regulating embryonic erythropoiesis with special attention to niche cells, and demonstrate how this knowledge can be applied in the identification of niche-derived peptides with potential therapeutic capability.

Appetite, metabolism and growth regulation in Atlantic salmon ( Salmo salar L.) exposed to hypoxia at elevated seawater temperature

High temperature combined with low dissolved oxygen (DO) is one of the most challenging environmental conditions farmed fish experience; thus, understanding their impact on growth regulation is of relevance to cultured and wild populations. This study examines appetite- and growth-regulating mechanisms in Atlantic salmon postsmolt exposed to either high (HO) or low oxygen (LO) at a suboptimally high temperature (17°C). Additionally, the effects of high (HE) and low (LE) dietary energy (DE) were examined. After a month of treatment, analyses of hormones, regulating appetite (ghrelin) and growth (growth hormone receptor ghr1 and insulin-like growth factor IGF-1), and free amino acids (FAA) were measured pre- and postprandially at −4, −2, 0, 2, 4 and 6 h. No preprandial ghrelin peaks were detected despite a significant reduction in feed intake and growth under hypoxia compared to normoxia. LO treatment also had an overall negative effect on survival compared to HO, while nutrient retention efficiency, FCR and plasma FAA concentrations were unaffected (P > 0.05). Feeding HE diet resulted in increased growth (+17%) and improved FCR (−14%) and energy retention efficiency (+26%) independent of DO. Plasma FAA concentrations were unaffected by LO treatment and DE (P > 0.05). Growth regulatory gene expressions possibly reflect an overall lower growth at a high temperature overriding the impacts of DO and DE. This study also indicates that optimal adaptation time to environmental conditions and feeding regime is crucial for establishing a regular hormonal appetite signalling that reflects real feeding anticipation in salmon.

Molecular responses of Walleye (Sander vitreus) embryos to naphthenic acid fraction components extracted from fresh oil sands process-affected water

Naphthenic acid fraction components (NAFCs) are constituents of oil sands process-affected water (OSPW), which is generated as a result of unconventional oil production via surface mining in the Athabasca oil sands region. NAFCs are often considered to be major drivers of OSPW toxicity to various taxa, including fishes. However, the molecular targets of these complex mixtures are not fully elucidated. Here we examined the effects in walleye (Sander vitreus) embryos after exposure to NAFCs extracted from fresh OSPW. Eleutheroembryos (exposed to 0, 4.2 or 8.3mg/L NAFCs from 1day post-fertilization to hatch) were subsampled, measured for growth and deformities, and molecular responses were assessed via real-time polymerase chain reaction (PCR). Fourteen genes were evaluated, with a focus on the aryl-hydrocarbon receptor (AhR) − cytochrome P450 pathway (arnt, cyp1a1), the oxidative stress axis (cat, gst, sod, gpx1b), apoptosis (e.g. casp3, bax and p53), growth factor signaling (e.g. insulin-like growth factors igf1, igf1b, and igf1bp), and tissue differentiation (vim). NAFC exposure was associated with an increase in the expression of cyp1a1, and a decrease in gpx1b and ribosomal protein rps40. These results indicate that NAFC effects on walleye early-life stages may be mediated through oxidative stress via pathways that include AhR.

Molecular performance of Prl and Gh/Igf1 axis in the Mediterranean meager, Argyrosomus regius, acclimated to different rearing salinities.

Aquaculture industry in the Mediterranean region exhibits a growing interest for the Mediterranean meager Argyrosomus regius. Some preliminary works showed a good growth performance of the species in nearly isosmotic salinities. However, the patterns of alteration of prolactin (Prl) as well as growth hormone (Gh)/insulin growth factor-1 (Igf1) axis at the molecular level are not yet described in this species. Therefore, we cloned and sequenced partial cDNAs for pituitary prolactin (prl) and growth hormone (gh), hepatic insulin-like growth factor (igf1), and β-actin (actb). Expression patterns of these transcripts were tested in juveniles of A. regius acclimated to four different environmental salinities: (1) 5‰ (hyposmotic); (2) 12‰ (isosmotic); (3) 38‰ (hyperosmotic; seawater control); and (4) 55‰ (extremely hyperosmotic). All investigated transcripts shared high sequence identities with their counterparts in other perciformes. prl mRNA levels showed inverse pattern with increasing salinities. gh mRNA enhanced significantly in both 12 and 55‰ salinity groups in comparison with the control group, while igf1 showed its maximum expression levels under the nearly isosmotic environment. The results indicated clear sensitivity of prl, gh and igf1 to changes in environmental salinity, which can possibly control the euryhalinity capacity of this species.

Culture conditions have an impact on the maturation of traceable, transplantable mouse embryonic stem cell-derived otic progenitor cells

The generation of replacement inner ear hair cells (HCs) remains a challenge and stem cell therapy holds the potential for developing therapeutic solutions to hearing and balance disorders. Recent developments have made significant strides in producing mouse otic progenitors using cell culture techniques to initiate HC differentiation. However, no consensus has been reached as to efficiency and therefore current methods remain unsatisfactory. In order to address these issues, we compare the generation of otic and HC progenitors from embryonic stem (ES) cells in two cell culture systems: suspension vs. adherent conditions. In the present study, an ES cell line derived from an Atoh1-green fluorescent protein (GFP) transgenic mouse was used to track the generation of otic progenitors, initial HCs and to compare these two differentiation systems. We used a two-step short-term differentiation method involving an induction period of 5days during which ES cells were cultured in the presence of Wnt/transforming growth factor TGF-β inhibitors and insulin-like growth factor IGF-1 to suppress mesoderm and reinforce presumptive ectoderm and otic lineages. The generated embryoid bodies were then differentiated in medium containing basic fibroblast growth factor (bFGF) for an additional 5days using either suspension or adherent culture methods. Upon completion of differentiation, quantitative polymerase chain reaction analysis and immunostaining monitored the expression of otic/HC progenitor lineage markers. The results indicate that cells differentiated in suspension cultures produced cells expressing otic progenitor/HC markers at a higher efficiency compared with the production of these cell types within adherent cultures. Furthermore, we demonstrated that a fraction of these cells can incorporate into ototoxin-injured mouse postnatal cochlea explants and express MYO7A after transplantation. Copyright © 2016 John Wiley & Sons, Ltd.

Експресія генів IGF1R, IGFBP4 та IGFBP5 у клітинах гліоми лінії U87 за умов дефіциту глутаміну

We have studied the expression of insulin-like growth factor receptor (IGF1R) and insulin-like growth factor binding protein (IGFBP4 and IGFBP5) genes in U87 glioma cells upon glutamine deprivation condition in relation to inhibition of ERN1 (endoplasmic reticulum to nuclei signaling 1), a sensor and signaling enzyme of endoplasmic reticulum stress, which control cell proliferation. It was shown that exposure control glioma cells upon glutamine deprivation condition leads to up-regulation of IGFBP4 and down-regulation of IGF1R expression at the mRNA level in control glioma cells, but IGFBP5 gene expression in these cells does not depend upon glutamine deprivation. At the same time, inhibition of IRE1 modifies the effect of glutamine deprivation on the expression of IGFBP5 gene because in glioma cells without functional activity of ERN1 glutamine deprivation leads to suppression of this IGFBP. We have also shown that the expression of all studied genes in glioma cells is regulated by ERN1 signaling enzyme at standard condition because ERN1 inhibition significantly enhances the expression of IGFBP4 and IGFBP5 genes. Proteins encoded by these genes are major inhibitors of pro-proliferative activity of insulin-like growth factors IGF1 and IGF2. We have also shown upregulation of the expression level of IGF1R gene in glioma cells with ERN1 knockdown as compared to control glioma cells. Results of this study shown that glutamine deprivation affects the expression of studied genes and that ERN1 inhibition preferentially changes these genes expression.

Daily rhythms of the expression of genes from the somatotropic axis: The influence on tilapia (Oreochromis niloticus) of feeding and growth hormone administration at different times

The aim of this research was to investigate the presence of daily rhythms in the somatotropic axis of tilapia fed at two times (mid-light, ML or mid-dark, MD) and the influence of the time of day of growth hormone (GH) administration on the response of this axis. Two different GH injection times were tested: ZT 3 (3h after lights on) and ZT 15 (3h after lights off). In both experiments, the mRNA expression levels of hypothalamic pituitary adenylate cyclase-activating polypeptide (pacap), pituitary growth hormone (gh), liver insulin-like growth factors (igf1 and igf2a), and liver and muscle growth hormone receptors (ghr1 and ghr2) and IGF receptors (igf1ra and igf2r) were evaluated by means of qPCR. Daily rhythms were observed in the liver for ghr1, ghr2 and igf2r but only in fish fed at ML, with the acrophases located in the light phase (ZT 3:30, 3:31 and 7:38h, respectively). In the muscle, ghr1 displayed a significant rhythm in both groups and ghr2 in ML fed fish (acrophases at ZT 5:29, 7:14 and 9:23h). The time of both GH administration and feeding influenced the response to GH injection: ML fed fish injected with GH at ZT 15h showed a significant increase in liver igf1, igf2a and ghr2; and muscle ghr2 expression. This is the first report that describes the existence of daily rhythms in the somatotropic axis of tilapia and its time-dependent responses of GH administration. Our results should be considered when investigating the elements of the somatotropic axis in tilapia and GH administration.

Reproductive Behavior of Female University Students in Relation to Endocrine Regulators Concentration in Blood Plasma during Ovarian Cycle Phases

The aim of our work based on answers of Slovak women - university students that are representing an important target group, was to analyse their reproductive behaviour and opinions, because the behaviour and opinions concerning reproduction could differ significantly depending also on education. Our goal was to analyze associations among reproductive behaviour of university student girls, their opinions and concentrations of endocrine regulators (insulin-like growth factors IGF-1, estradiol, testosterone and progesterone) in the blood serum and record their changes during the ovarian cycle. The group under study encompassed 301 female university students aged 19 ± 1.371 years. Analysis of reproductive behaviour characteristics were based on a questionnaire method. Concentrations of the IGF-1, testosterone, estradiol and progesterone in blood plasma according to ovarian cycle phase had been set by biochemical methods and then linked with reproductive behaviour. Average value of sexual debut for girls was 17.21 ± 1.31 years. Most of them get over their first sexual intercourse in the age of 19 and almost half of them had one sexual partner. Differences in hormone concentrations were proved according to ovarian cycle phase for estradiol and progesterone, but no differences were found for testosterone; physiological role of IGF-1 during ovarian cycle did not alternate. IGF-1 influenced regulation of female students´ opinion of optimum age for sexual life beginning, their attitude to gender identity and marriage planning. Satisfaction with sexual identity was associated with higher blood estradiol level (but not with testosterone and progesterone levels). The outputs given in this work can help identifying insufficiently known relations between less known endocrine regulators (IGF-1) and reproductive behaviour of young women. Practical results are assumed e.g. in possible screening tests, diagnostics and subsequent troubleshooting concerning the reproduction behaviour of young women.

Maximal oxygen uptake is associated with allele -202 A of insulin-like growth factor binding protein-3 (IGFBP3) promoter polymorphism and (CA)n tandem repeats of insulin-like growth factor IGF1 in Caucasians from Poland.

Physical fitness is a trait determined by multiple genes, and its genetic basis is modified by numerous environmental factors. The present study examines the effects of the (CA)n tandem repeats polymorphism in IGFI gene and SNP Alw21I restriction site -202 A>C polymorphism in IGF1BP3 on VO2max--a physiological index of aerobic capacity of high heritability. The study sample consisted of 239 (154 male and 85 female) students of the University School of Physical Education in Poznań and athletes practicing various sports, including members of the Polish national team. An association was found between -202 A/C polymorphism of IGFBP3 gene with VO2max in men. Higher VO2max values were attained by men with CC genotype, especially male athletes practicing endurance sports and sports featuring energy metabolism of aerobic/anaerobic character. A statistically significant influence of allele 188 and genotype 188/188 of tandem repeats (CA)n polymorphism of IGF1 gene on VO2max was found in women. Also, lower values of maximal oxygen uptake were noted in individuals with allele 186 or genotype 186/186, and higher VO2max values in athletes with allele 194.

Upregulation of the Na+-Coupled Phosphate Cotransporters NaPi-IIa and NaPi-IIb by B-RAF

B-RAF, a serine/threonine protein kinase, contributes to signaling of insulin-like growth factor IGF1. Effects of IGF1 include stimulation of proximal renal tubular phosphate transport, accomplished in large part by Na⁺-coupled phosphate cotransporter NaPi-IIa. The related Na⁺-coupled phosphate cotransporter NaPi-IIb accomplishes phosphate transport in intestine and tumor cells. The present study explored whether B-RAF influences protein abundance and/or activity of type II Na⁺-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb. cRNA encoding wild-type NaPi-IIa and wild-type NaPi-IIb was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type B-RAF, and electrogenic phosphate transport determined by dual-electrode voltage clamp. NaPi-IIa protein abundance in Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified by chemiluminescence. Moreover, in HEK293 cells, the effect of B-RAF inhibitor PLX-4720 on NaPi-IIa cell surface protein abundance was quantified utilizing biotinylation of cell surface proteins and western blotting. In NaPi-IIa-expressing Xenopus oocytes, but not in oocytes injected with water, addition of phosphate to extracellular bath generated a current (I P), which was significantly increased following coexpression of B-RAF. According to kinetic analysis, coexpression of B-RAF enhanced the maximal IP. Coexpression of B-RAF further enhanced NaPi-IIa protein abundance in the Xenopus oocyte cell membrane. Treatment of HEK293 cells for 24 h with PLX-4720 significantly decreased NaPi-IIa cell membrane protein abundance. Coexpression of B-RAF, further significantly increased IP in NaPi-IIb-expressing Xenopus oocytes. Again, B-RAF coexpression enhanced the maximal IP. In conclusion, B-RAF is a powerful stimulator of the renal and intestinal type II Na⁺-coupled phosphate cotransporters NaPi-IIa and NaPi-IIb, respectively.

Metabolic endocrine factors involved in spawning recovery and rematuration of iteroparous female rainbow trout (Oncorhynchus mykiss)

To determine how energy balance affects metabolic hormones hypothesized to play a role in the onset of a new reproductive cycle in iteroparous salmonids, food availability after spawning was restricted in female rainbow trout. These fish were compared with a control group that was fed a standard brood stock ration. Bodyweight, length, and muscle lipid content were determined, and blood was collected from fish at regular intervals; a subset of fish from each group was sacrificed at each sampling time for the collection of liver and ovary tissue, and to calculate hepatosomatic index (HSI) and gonadosomatic index (GSI). Plasma hormone levels were quantified by radioimmunoassay, and tissue gene expression levels were analyzed using q-RT-PCR. The experiment was conducted twice, using two-year-old and three-year-old post-spawned fish. Food-restriction arrested ovarian growth and development within 15–20weeks, as evidenced by lower GSI in restricted-ration fish. Food restriction also reduced Fulton’s condition factor, muscle lipid content, and specific growth rate from one month onward, and reduced HSI after 3months. In the liver, insulin-like growth factor (igf1 and igf2) gene expression was reduced in three-year-old food-restricted fish within 2months; however, no effect of ration on igf1 or igf2 expression was detected in two-year-old fish. In both years, IGF binding protein-1 (igfbp1) gene expression decreased over time in both treatment groups. Liver leptin (slepA1) gene expression was lower in two-year-old food-restricted fish at 4months. These results show that this feed restriction regime arrested reproductive development and affected factors associated with energy balance purported to play a role in initiating reproductive development within 2–4months after spawning.

Expression levels of mRNA for insulin-like growth factors 1 and 2, IGF receptors and IGF binding proteins in in vivo and in vitro grown bovine follicles

This study investigated mRNA levels for insulin-like growth factors (IGFs) IGF1 (IGF-I) and IGF2 (IGF-II), IGF receptors (IGF1R and IGF2R), and binding proteins (IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6) in bovine follicles of 0.2, 0.5 or 1.0 mm in diameter. mRNA expression levels in in vitro cultured follicles that reached approximately 0.5 mm were compared with that of in vivo grown follicles. IGF1R and IGF2R expression levels in 0.5 mm in vivo follicles were higher than in 1.0 or 0.2 mm follicles, respectively. IGFBP-1, IGFBP-2. IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6 showed variable expression in the follicular size classes analyzed. In vitro grown follicles had significantly reduced expression levels for IGF1, IGF1R, IGFBP-3, IGFBP-5 and IGFBP-6 mRNA when compared with 0.2 mm follicles, but, when compared with in vivo grown follicles (0.5 mm), only IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-6 showed a reduction in their expression. In conclusion, IGFs, their receptors and IGFBPs showed variable expression of mRNA levels in the follicular size classes analyzed.

Development of a multiplex gene expression assay for components of the endocrine growth axis in coho salmon

This study explores the efficacy of the Quantigene plex (QGP) technology for measuring a panel of endocrine growth-related transcripts in coho salmon, Oncorhynchus kisutch. The QGP technology permits the simultaneous quantification of multiple targeted mRNAs within a single tissue homogenate using sequence-specific probes and requires no reverse transcription (RT) or amplification as is required for RT-quantitative PCR (RT-qPCR). Using liver homogenates from coho salmon under fed and fasted conditions, we compared the detectable fold differences of steady-state mRNA levels between the QGP and probe-based RT-qPCR assays for insulin-like growth factors (igf1 and igf2), insulin-like growth factor binding proteins (igfbp1b, igfbp2a, and igfbp2b), somatolactin receptor (slr), and growth hormone receptors (ghr1 and ghr2). Significant, positive correlations for all genes between the two assays were found. In addition, the relatively low variance of results from the QGP assay suggests that this is a suitable method for a comprehensive analysis of endocrine growth-related transcripts and could potentially be used to develop assays for other gene networks in teleosts.