Biochemical and functional analysis of a conserved IGF-binding protein isolated from rainbow trout (Oncorhynchus mykiss) hepatoma cells

Rainbow trout (Oncorhynchus mykiss) serum contains several IGF-binding proteins (IGFBPs) that specifically bind to IGFs. The structures of these fish IGFBPs have not been determined and their physiological functions are poorly defined. In this study, we identified a 30 kDa IGFBP present in rainbow trout serum and secreted by cultured trout hepatoma cells. This IGFBP binds to IGFs but not to insulin. This IGFBP was purified to homogeneity using a three-step procedure involving Phenyl-Sepharose chromatography, IGF-I affinity chromatography and reverse-phase HPLC. Affinity cross-linking studies indicated that this IGFBP binds to IGF-I with a higher affinity than to IGF-II. N-terminal sequence analysis of the trout IGFBP suggests that it shares high sequence identity with that of human IGFBP-1 in the N-terminal region. When added to cultured fish and human cells, the trout IGFBP inhibited IGF-I-stimulated DNA synthesis and cell proliferation in a concentration-dependent manner. The inhibitory effect of the fish IGFBP was comparable to those of human IGFBP-1 and -4. These results indicate that the IGFBP molecule is structurally and functionally conserved in evolutionarily ancient vertebrate species such as bony fish.

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Effects of IGF-I bioavailability on bovine preantral follicular development in vitro

Reproduction ◽

10.1530/rep-06-0382 ◽

2007 ◽

Vol 133 (6) ◽

pp. 1121-1128 ◽

Cited By ~ 55

Author(s):

Fiona H Thomas ◽

Bruce K Campbell ◽

David G Armstrong ◽

Evelyn E Telfer

Keyword(s):

Follicular Development ◽

Follicle Development ◽

Dependent Manner ◽

Igf Binding Proteins ◽

Preantral Follicles ◽

Follicle Diameter ◽

Igf I ◽

Igf Binding ◽

Development In Vitro

The aim of this study was to determine the effect of regulation of IGF-I bioavailability on preantral follicle development in vitro. Bovine preantral follicles were cultured for 6 days in serum-free medium with increasing doses of Long R3 (LR3) IGF-I (an analog with low affinity for IGF-binding proteins (IGFBPs)), or human recombinant IGF-I (hrIGF-I). Follicle diameter and estradiol production were measured every second day. On day 6, ratios of oocyte/follicle diameter and oocyte morphology were assessed by histological examination, and IGFBP-2 and -3 were detected by immunocytochemistry and in situ hybridization respectively. Both types of IGF-I increased follicle diameter in a dose-dependent manner (P < 0.05) and increased estradiol production over control levels (P < 0.05). However, follicles treated with LR3 IGF-I and the highest concentration of hrIGF-I (1000 ng/ml) had smaller oocyte/follicle ratios, and increased oocyte degeneration, compared with controls or follicles treated with physiological concentrations of hrIGF-I (P < 0.05). IGFBPs were detected in cultured preantral follicles, indicating a requirement for regulation of IGF bioavailability during the early stages of follicular development. Specifically, IGFBP-3 mRNA was found to be expressed in oocytes, and IGFBP-2 immunoreactivity was detected in oocytes and granulosa cells of cultured follicles. In summary, the regulation of IGF-I bioavailability by IGFBPs is necessary for the co-ordination of oocyte and follicle development in vitro.

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Insulin and insulin-like growth factor I signaling pathways in rainbow trout (Oncorhynchus mykiss) during adipogenesis and their implication in glucose uptake

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00457.2009 ◽

2010 ◽

Vol 299 (1) ◽

pp. R33-R41 ◽

Cited By ~ 30

Author(s):

L. Bouraoui ◽

E. Capilla ◽

J. Gutiérrez ◽

I. Navarro

Keyword(s):

Rainbow Trout ◽

Growth Factor ◽

Oncorhynchus Mykiss ◽

Glucose Uptake ◽

Signaling Pathways ◽

Mapk Phosphorylation ◽

Factor I ◽

Rainbow Trout Oncorhynchus Mykiss ◽

Igf I ◽

Stimulation Of

Primary cultures of rainbow trout ( Oncorhynchus mykiss ) adipocytes were used to examine the main signaling pathways of insulin and insulin-like growth factor I (IGF-I) during adipogenesis. We first determined the presence of IGF-I receptors (IGF-IR) and insulin receptors (IR) in trout preadipocytes ( day 5) and adipocytes ( day 14). IGF-IRs were more abundant and appeared to be in higher levels in differentiated cells than in preadipocytes, whereas IRs were detected in lower but constant levels throughout the culture. The cells were immunoreactive against ERK1/2 MAPK, and AKT/PI3K, components of the two main signal transduction pathways for insulin and IGF-I receptors. Stimulation of MAPK phosphorylation by IGF-I was higher in preadipocytes than in adipocytes, while no effects were observed in MAPK phosphorylation after incubation of cells with insulin. AKT phosphorylation increased in the presence of both insulin and IGF-I, with higher levels of stimulation in adipocytes than in preadipocytes. Activation of both pathways was blocked by the use of specific inhibitors of MAPK (PD98059) and AKT (wortmannin). We describe here, for the first time, the effects of IGF-I and insulin on 2-deoxyglucose uptake in primary culture of trout adipocytes. IGF-I was more potent in stimulating glucose uptake than insulin, and PD98059 and wortmannin inhibited the stimulation of glucose uptake by this growth factor, suggesting that IGF-I plays an important metabolic role in trout adipocytes. Our results suggest that differential activation of the MAPK and AKT pathways are involved in the IGF-I- and insulin-induced effects of trout adipocytes during the various stages of adipogenesis.

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Insulin-like growth factor (IGF)-II binding to IGF-binding proteins and IGF receptors is modified by deletion of the N-terminal hexapeptide or substitution of arginine for glutamate-6 in IGF-II

Biochemical Journal ◽

10.1042/bj2930713 ◽

1993 ◽

Vol 293 (3) ◽

pp. 713-719 ◽

Cited By ~ 48

Author(s):

G L Francis ◽

S E Aplin ◽

S J Milner ◽

K A McNeil ◽

F J Ballard ◽

...

Keyword(s):

Growth Factor ◽

Binding Proteins ◽

Biological Activities ◽

Biological Properties ◽

Hepatoma Cells ◽

Insulin Like Growth Factor ◽

Igf Binding Proteins ◽

Anabolic Response ◽

Igf I ◽

Igf Binding

Recombinant insulin-like growth factor-II (IGF-II) and two structural analogues, des(1-6)IGF-II and [Arg6]-IGF-II, were produced to investigate the role of N-terminal residues in binding to IGF-binding proteins (IGFBPs) and hence the biological properties of the modified peptides. The growth factors were modelled on two previously characterized variants of IGF-I, des(1-3)IGF-I and [Arg3]-IGF-I, which both show substantially decreased binding to IGFBPs and were expressed as fusion proteins in Escherichia coli. The biological activities of the corresponding analogues of IGF-I and IGF-II were compared in rat L6 myoblasts and H35B hepatoma cells. In the L6-myoblast protein-synthesis assay, the IGF-II analogues, des(1-6)IGF-II and [Arg6]-IGF-II, were slightly more potent than IGF-II but about 10-fold less potent than IGF-I and 100-fold less potent than the respective IGF-I analogues, des(1-3)IGF-I and [Arg3]IGF-I. In H35 hepatoma cells the anabolic response measured was the inhibition of protein breakdown, and the potency order was insulin >>> [Arg3]-IGF-I > des(1-3)IGF-I > [Arg6]-IGF-II > des(1-6)IGF-II > IGF-I > IGF-II. Binding of the IGFs and their analogues to the type 1 IGF receptor in L6 myoblasts and to the insulin receptor in H35 hepatoma cells did not fully explain the observed anabolic potency differences. Moreover, binding of all four analogues to the IGFBPs secreted by L6 myoblasts and H35B hepatoma cells was greatly decreased compared with the parent IGF. We conclude that the observed anabolic response to each IGF was determined by their relative binding to the competing cell receptor and IGFBP binding sites present.

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Partial Purification and Characterization of Rhodanese from Rainbow Trout (Oncorhynchus mykiss) Liver

The Scientific World JOURNAL ◽

10.1100/2012/648085 ◽

2012 ◽

Vol 2012 ◽

pp. 1-5 ◽

Cited By ~ 3

Author(s):

Hossein Tayefi-Nasrabadi ◽

Reza Rahmani

Keyword(s):

Rainbow Trout ◽

Relative Activity ◽

Tissue Homogenate ◽

Partial Purification ◽

Toxic Substances ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Rainbow Trout Oncorhynchus Mykiss ◽

Liver Tissue Homogenate

Cyanide is one of the most toxic substances present in a wide variety of food materials that are consumed by animals. Rhodanese, a ubiquitous enzyme, can catalyse the detoxification of cyanide by sulphuration reaction. In this study, rhodanese was partially purified and characterized from the liver tissue homogenate of the rainbow trout. The enzyme was active in a broad range of pH, from 5 to 12. The optimal activity was found at a high pH (pH 10.5), and the temperature optimum was25∘C. The enzyme was heat labile, losing > 50% of relative activity after only 5 min of incubation at40∘C. TheKmvalues for KCN and Na2S2O3as substrates were 36.81 mM and 19.84 mM, respectively. Studies on the enzyme with a number of cations showed that the activity of the enzyme was not affected by Sn2+, but Hg2+, Ba2+, Pb2+, and Ca2+inhibited and Cu2+activated the enzyme with a concentration-dependent manner.

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Endocrine control of oleic acid and glucose metabolism in rainbow trout (Oncorhynchus mykiss) muscle cells in culture

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00696.2009 ◽

2010 ◽

Vol 299 (2) ◽

pp. R562-R572 ◽

Cited By ~ 24

Author(s):

Joan Sánchez-Gurmaches ◽

Lourdes Cruz-Garcia ◽

Joaquím Gutiérrez ◽

Isabel Navarro

Keyword(s):

Oleic Acid ◽

Rainbow Trout ◽

Glucose Metabolism ◽

Oncorhynchus Mykiss ◽

Acid Metabolism ◽

Peroxisome Proliferator ◽

Endocrine Control ◽

Peroxisome Proliferator Activated Receptor ◽

Rainbow Trout Oncorhynchus Mykiss ◽

Igf I

The effects of insulin and IGF-I on fatty acid (FA) and glucose metabolism were examined using oleic acid or glucose as tracers in differentiated rainbow trout ( Oncorhynchus mykiss ) myotubes. Insulin and IGF-I significantly reduced the production of CO2 from oleic acid with respect to the control values. IGF-I also significantly reduced the production of acid-soluble products (ASP) and the concentration of FA in the medium, while cellular triacylglycerols (TAG) tended to increase. Only insulin produced a significant accumulation of glycogen inside the cells in glucose distribution experiments. Incubation with catecholamines did not affect oleic acid metabolism. Cells treated with rapamycin [a target of rapamycin (TOR) inhibitor] significantly increased the oxidation of oleic acid to CO2 and ASP, while the accumulation of TAG diminished. Rosiglitazone (a peroxisome proliferator-activated receptor γ agonist) and etomoxir (a CPT-1 inhibitor) produced a severe and significant reduction in the production of CO2 and ASP. Rosiglitazone and etomoxir also produced a significant accumulation of FA outside and inside the cells, respectively. No significant effects of these drugs on glucose distribution were observed. These data indicate that insulin and IGF-I act as anabolic hormones in trout myotubes in both oleic acid and glucose metabolism, although glucose oxidation appears to be less sensitive than FA oxidation to insulin and IGF-I. The use of rapamycin, etomoxir, and rosiglitazone may help us to understand the mechanisms of regulation of lipid metabolism in fish.

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Differential effects of IGF-binding proteins, IGFBP-3 and IGFBP-5, on IGF-I action and binding to cell membranes of immortalized human chondrocytes

Journal of Endocrinology ◽

10.1677/joe.0.1660029 ◽

2000 ◽

Vol 166 (1) ◽

pp. 29-37 ◽

Cited By ~ 23

Author(s):

T Matsumoto ◽

T Tsurumoto ◽

MB Goldring ◽

H Shindo

Keyword(s):

Binding Proteins ◽

Type Ii Collagen ◽

Cartilage Tissue ◽

Dependent Manner ◽

Igf Binding Proteins ◽

Culture Model ◽

Igf I ◽

Igf Binding ◽

Model Treatment ◽

Igfbp 3

Insulin-like growth factor-I (IGF-I) is an important anabolic factor for cartilage tissue and its action is, in part, regulated by IGF-binding proteins (IGFBPs). The object of this study was to investigate the effects of IGFBPs on IGF-I action and on binding of IGF-I to cells using a reproducible immortalized human chondrocyte culture model. Treatment of the C-28/I2 cells with IGF-I or des(1-3)IGF-I in serum-free medium stimulated cell proliferation in a dose-dependent manner. However, the effect of des(1-3)IGF-I was more potent, thereby suggesting that endogenously produced IGFBPs inhibited IGF action. The stimulatory effect of IGF-I was inhibited significantly by addition of IGFBP-3 but enhanced slightly by IGFBP-5. However, neither IGFBP-3 nor IGFBP-5 had an effect on basal cell growth. Binding of (125)I-labeled IGF-I to the cells was displaced by both IGFBP-3 and IGFBP-5, although higher concentrations of unlabeled IGFBP-5 were required to displace IGF-I to the same extent as IGFBP-3. Treatment of the cells with IGF-I increased the levels of IGFBP-5 protein measured by Western ligand blotting, and stimulated a corresponding increase in IGFBP-5 mRNA while increasing type II collagen mRNA. Our findings indicate that the balance between IGFBP-3 and IGFBP-5 influences IGF receptor binding and its action on chondrocyte proliferation, and may thereby modulate cartilage metabolism.

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Endoplasmic reticulum stress as a key mechanism in stunted growth of seawater rainbow trout (Oncorhynchus mykiss)

BMC Genomics ◽

10.1186/s12864-021-08153-5 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Bernat Morro ◽

Richard Broughton ◽

Pablo Balseiro ◽

Sigurd O. Handeland ◽

Simon Mackenzie ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Rainbow Trout ◽

Endoplasmic Reticulum Stress ◽

Atpase Activity ◽

Oncorhynchus Mykiss ◽

Plasma Cortisol ◽

Stunted Growth ◽

Rainbow Trout Oncorhynchus Mykiss ◽

Igf I ◽

Liver Proteome

Abstract Background Rainbow trout (Oncorhynchus mykiss) is a salmonid species with a complex life-history. Wild populations are naturally divided into freshwater residents and sea-run migrants. Migrants undergo an energy-demanding adaptation for life in seawater, known as smoltification, while freshwater residents display these changes in an attenuated magnitude and rate. Despite this, in seawater rainbow trout farming all fish are transferred to seawater. Under these circumstances, weeks after seawater transfer, a significant portion of the fish die (around 10%) or experience growth stunting (GS; around 10%), which represents an important profitability and welfare issue. The underlying causes leading to GS in seawater-transferred rainbow trout remain unknown. In this study, we aimed at characterising the GS phenotype in seawater-transferred rainbow trout using untargeted and targeted approaches. To this end, the liver proteome (LC-MS/MS) and lipidome (LC-MS) of GS and fast-growing phenotypes were profiled to identify molecules and processes that are characteristic of the GS phenotype. Moreover, the transcription, abundance or activity of key proteins and hormones related to osmoregulation (Gill Na+, K + –ATPase activity), growth (plasma IGF-I, and liver igf1, igfbp1b, ghr1 and ctsl) and stress (plasma cortisol) were measured using targeted approaches. Results No differences in Gill Na+, K + –ATPase activity and plasma cortisol were detected between the two groups. However, a significant downregulation in plasma IGF-I and liver igf1 transcription pointed at this growth factor as an important pathomechanism for GS. Changes in the liver proteome revealed reactive-oxygen-species-mediated endoplasmic reticulum stress as a key mechanism underlying the GS phenotype. From the lipidomic analysis, key observations include a reduction in triacylglycerols and elevated amounts of cardiolipins, a characteristic lipid class associated with oxidative stress, in GS phenotype. Conclusion While the triggers to the activation of endoplasmic reticulum stress are still unknown, data from this study point towards a nutritional deficiency as an underlying driver of this phenotype.

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IGF-I binding in primary culture of muscle cells of rainbow trout: changes during in vitro development

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00121.2002 ◽

2002 ◽

Vol 283 (3) ◽

pp. R647-R652 ◽

Cited By ~ 38

Author(s):

Juan Castillo ◽

Pierre-Yves Le Bail ◽

Gilles Paboeuf ◽

Isabel Navarro ◽

Claudine Weil ◽

...

Keyword(s):

Rainbow Trout ◽

Mononuclear Cells ◽

Muscle Cells ◽

Binding Assays ◽

Igf Binding Proteins ◽

Igf I ◽

Igf Binding ◽

High Concentrations ◽

Trout Muscle

To characterize and study the variations of IGF-I binding during the development of trout muscle cells, in vitro experiments were conducted using myocyte cultures, and IGF-I binding assays were performed in three stages of cell development: mononuclear cells ( day 1), small myotubes ( day 4), and large myotubes ( day 10). Binding experiments were done by incubating cells with IGF-I for 12 h at 4°C. Specific IGF-I binding increased with the concentration of labeled IGF-I and reached a plateau at 32 pM. The displacement of cold human and trout IGF-I showed a very similar curve (EC50 = 1.19 ± 0.05 and 0.95 ± 0.05 nM, respectively). IGF binding proteins did not interfere significantly because displacement of labeled IGF-I by either cold trout recombinant IGF-I or Des (1–3) IGF-I resulted in similar curves. Insulin did not displace labeled IGF-I even at very high concentrations (>1 μM), which indicates the specificity of IGF-I binding. The amount of receptor (R0) increased from 253 ± 51 fmol/mg DNA on day 1 to 766 ± 107 fmol/mg DNA on day 10. However, the affinity ( K d) of IGF-I receptors did not change significantly during this development (from 1.29 ± 0.19 to 0.79 ± 0.13 nM). On the basis of our results, we conclude that rainbow trout muscle cells in culture express specific IGF-I receptors, which increase their number with development from mononuclear cells to large myotubes.

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Effects of endotoxin lipopolysaccharide administration on the somatotropic axis

Journal of Endocrinology ◽

10.1677/joe.0.1590239 ◽

1998 ◽

Vol 159 (2) ◽

pp. 239-246 ◽

Cited By ~ 46

Author(s):

L Soto ◽

AI Martin ◽

S Millan ◽

E Vara ◽

A Lopez-Calderon

Keyword(s):

Body Weight ◽

Body Weight Loss ◽

Serum Levels ◽

Dependent Manner ◽

Igf Binding Proteins ◽

Somatotropic Axis ◽

Gh Secretion ◽

Igf I ◽

Male Wistar Rats ◽

Igf Binding

The aim of this work was to study the effect of chronic activation of the immune system on the somatotropic axis. Accordingly, the changes in growth hormone (GH) secretion, circulating insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) in response to endotoxin lipopolysaccharide (LPS) administration were examined in adult male Wistar rats. Acute LPS injection (2.5, 25 or 250 microg/kg) increased serum corticosterone in a dose-dependent manner and decreased serum levels of insulin and IGF-I, serum GH concentration declined linearly as the LPS dose increased. Western ligand blot showed an increase in the 33 kDa band (corresponding to IGFBP-1 and IGFBP-2) in the rats that received the highest dose of LPS (250 microg/kg). Chronic LPS administration (250 microg/kg daily for 8 days) significantly decreased body weight, serum levels of IGF-I and pituitary GH content, whereas it increased circulating IGFBP-3 (47 kDa band), IGFBP-1 and IGFBP-2 (33 kDa band) and the 24 kDa band (which possibly corresponds to IGFBP-4). Serum concentration of corticosterone and hypothalamic somatostatin content were also increased by chronic LPS treatment. These data suggest that the decrease in GH and IGF-I secretion and the increase in circulating IGFBPs are important mechanisms in body weight loss during chronic inflammation.

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