Metabolic and mitogenic effects of IGF-I and insulin on muscle cells of rainbow trout

The relative function of IGF-I and insulin on fish muscle metabolism and growth has been investigated by the isolation and culture at different stages (myoblasts at day 1, myocytes at day 4, and myotubes at day 10) of rainbow trout muscle cells. This in vitro model avoids interactions with endogenous peptides, which could interfere with the muscle response. In these cells, the effects of IGF-I and insulin on cell proliferation, 2-deoxyglucose (2-DG), and l-alanine uptake at different development stages, and the use of inhibitors were studied and quantified. Insulin (10-1,000 nM) and IGF-I (10-100 nM) stimulated 2-DG uptake in trout myocytes at day 4 in a similar manner (maximum of 124% for insulin and of 142% for IGF-I), and this stimulation increased when cells differentiated to myotubes (maximum for IGF-I of 193%). When incubating the cells with PD-98059 and especially cytochalasin B, a reduction in 2-DG uptake was observed, suggesting that glucose transport takes place through specific facilitative transporters. IGF-I (1-100 nM) stimulated the l-alanine uptake in myocytes at day 4 (maximum of 239%), reaching higher values of stimulation than insulin (100-1,000 nM) (maximum of 160%). This stimulation decreased when cells developed to myotubes at day 10 (118% for IGF-I and 114% for insulin). IGF-I (0.125-25 nM) had a significant effect on myoblast proliferation, measured by thymidine incorporation (maximum of 170%), and required the presence of 2-5% fetal serum (FBS) to promote thymidine uptake. On the other hand, insulin was totally ineffective in stimulating thymidine uptake. We conclude that IGF-I is more effective than insulin in stimulating glucose and alanine uptake in rainbow trout myosatellite cells and that the degree of stimulation changes when cells differentiate to myotubes. IGF-I stimulates cell proliferation in this model of muscle in vitro and insulin does not. These results indicate the important role of IGF-I on growth and metabolism of fish muscle.

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Regulation of cartilage glycosaminoglycan synthesis in the rainbow trout, Oncorhynchus mykiss, by 3,3′,5-tri-iodo-l-thyronine and IGF-I

Journal of Endocrinology ◽

10.1677/joe.0.1490357 ◽

1996 ◽

Vol 149 (2) ◽

pp. 357-365 ◽

Cited By ~ 13

Author(s):

Y Takagi ◽

B Th Björnsson

Keyword(s):

Rainbow Trout ◽

Oncorhynchus Mykiss ◽

Thymidine Uptake ◽

Post Injection ◽

Sulfate Uptake ◽

Glycosaminoglycan Synthesis ◽

Sulfated Glycosaminoglycan ◽

Igf I ◽

Dose Dependent

Abstract The actions of 3,3′,5-tri-iodo-l-thyronine (T3) and recombinant human IGF-I (rhIGF-I) as well as their interaction on cartilage growth in rainbow trout (Oncorhynchus mykiss) were examined. Uptake of 3H-methyl thymidine and 35S-sulfate by isolated branchial cartilage was measured as a marker for chondrocyte proliferation and sulfated glycosaminoglycan synthesis respectively. When T3 (1·0 μg/g) was injected intraperitoneally, plasma T3 levels reached a transient peak after 1 day and decreased rapidly thereafter. Sulfate and thymidine uptake were not affected by T3 at 1 and 3 days post-injection, but at 6 days post-injection both were significantly higher in T3injected fish than those in controls. The stimulatory effects of a T3 injection on sulfate and thymidine uptake were dose-dependent over the range of 0·01, 0·1 and 1·0 μg/g. In vitro exposure of cartilage to T3 (0·075, 0·75, 7·5, 75 and 750 nm) for 6 days resulted in dose-dependent stimulation of sulfate uptake, with a maximum response at 7·5 nm and higher. T3 exposure (7·5 nm) for 2 or 3 days also increased sulfate uptake, but only slightly. Thymidine uptake was not clearly affected by T3. In vitro addition of rhIGF-I (0·075, 0·75 and 7·5 nm) increased sulfate uptake, but not thymidine uptake, dose-dependently. Compared with T3, rhIGF-I induced a greater maximum level of sulfate uptake: at 7·5 nm rhIGF-I increased the uptake 17-fold whereas T3 increased the uptake fourfold. When T3 (0·075, 0·75 or 7·5 nm) and rhIGF-I (0·1 or 1·0 nm) were added together, stimulative actions of T3 on sulfate uptake were largely additive to those of rhIGF-I. The results indicate that T3 as well as IGF-I are important modulators of sulfated glycosaminoglycan synthesis in rainbow trout cartilage. Journal of Endocrinology (1996) 149, 357–365

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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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Role of GSK3 in Multiple Myeloma Cell Growth and Survival.

Blood ◽

10.1182/blood.v110.11.2509.2509 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2509-2509

Author(s):

Francesco A. Piazza ◽

Carmela Gurrieri ◽

Gino Chioetto ◽

Anna Colpo ◽

Luca Bonanni ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Proliferation ◽

Cell Death ◽

Growth Factors ◽

Cell Growth ◽

Cell Membrane ◽

Critical Role ◽

Igf I

Abstract The serine-threonine GSK3 displays a crucial role in different cancer-pathogenetic pathways, including the PI3K/AKT, Wnt β-catenin and NF-κB signaling cascades, either promoting or counteracting cell survival. The aim of this study was to investigate the role of GSK3 in multiple myeloma (MM) cell growth. GSK3α and β total and phosphorylated protein levels were found differentially expressed in malignant plasma cells as compared to normal resting B-lymphocytes and to normal in vitro generated plasmablasts. Intriguingly, in freshly isolated malignant plasmacells from patients, most of GSK3 was found colocalized with Wnt receptor LRP6 and casein kinase I next to the cell membrane as compared to normal plasmacells or B-cells from other malignancies, wher it was distributed in the cytosol and in the nucleus, thus suggesting a peculiar role of this kinase in these cells. Upon stimulation of MM cells with IL-6 and IGF-I, GSK3 enzymatic activity was hampered, while stimulation with TNFα did not affect GSK3 function nor the early events in NF-κB activation. Basal and IL-6 and IGF-I driven proliferation of MM cells was slightly impaired by GSK3 blockade. Interestingly, GSK3β−/− mouse embryo fibroblasts (MEFs) proliferated slightly slower as compared to GSK3β+/+ cells; however, GSK3α inhibition and IL-6 and IGF-I stimulation, resulted in much higher proliferation of GSK3β −/− cells. Intriguingly, GSK3 inhibition with specific compounds (SB216763 and SB415286) caused a significant rescue from cell death of growth factor-deprived MM cells while resulted in reduced cell proliferation and apoptosis of MM cells grown with serum or growth factors. When GSK3 inhibitors were added to MM cell cultured with bone marrow stromal cells (BMSC), MM cells survival increased and NF-κB and β-catenin-mediated gene transcription (of IAPs and cyclinD1, respectively) was deregulated. GSK3 activity inhibition did not modify the rate of proteasome inhibitor-induced cell death in co-colture experiments with BMSC, suggesting that the sensitivity to bortezomib-induced MM cell apoptosis is independent on GSK3. Altogether, our data indicate thatGSK3 localizes on the cell membrane in primary MM cells;GSK3 is differently regulated downstream from growth factors or TNFα-induced signaling pathways in MM cells;a peculiar role of GSK3 in malignant MM cells as compared to normal MEFs with regard to cell proliferation; anda critical role of this kinase in regulating the MM microenvironment.

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An in vitro model of human bronchial epithelial cells for the investigation of the role of the airway epithelium in asthma exacerbations

Pneumologie ◽

10.1055/s-0037-1619393 ◽

2018 ◽

Vol 72 (S 01) ◽

pp. S101-S102

Author(s):

JC Ehlers ◽

S Bartel ◽

C Vock ◽

H Fehrenbach

Keyword(s):

Epithelial Cells ◽

Airway Epithelium ◽

In Vitro Model ◽

Bronchial Epithelial Cells ◽

Human Bronchial Epithelial Cells ◽

Bronchial Epithelial ◽

Asthma Exacerbations ◽

Vitro Model

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In Vitro Model for Ischemic Stroke: Functional Analysis of Vascular Smooth Muscle Cells

Cellular and Molecular Neurobiology ◽

10.1007/s10571-021-01103-5 ◽

2021 ◽

Author(s):

Melissa Mariana ◽

Claudio Roque ◽

Graça Baltazar ◽

Elisa Cairrao

Keyword(s):

Functional Analysis ◽

Smooth Muscle ◽

Ischemic Stroke ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

In Vitro Model ◽

Muscle Cells ◽

Vitro Model

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KLF7/VPS35 axis contributes to hepatocellular carcinoma progression through CCDC85C-activated β-catenin pathway

Cell & Bioscience ◽

10.1186/s13578-021-00585-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yarong Guo ◽

Bao Chai ◽

Junmei Jia ◽

Mudan Yang ◽

Yanjun Li ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Proliferation ◽

Tumor Growth ◽

Luciferase Reporter ◽

Aberrant Expression ◽

Proliferation And Invasion ◽

Hcc Cells

Abstract Objective Dysregulation of KLF7 participates in the development of various cancers, but it is unclear whether there is a link between HCC and aberrant expression of KLF7. The aim of this study was to investigate the role of KLF7 in proliferation and migration of hepatocellular carcinoma (HCC) cells. Methods CCK8, colony growth, transwell, cell cycle analysis and apoptosis detection were performed to explore the effect of KLF7, VPS35 and Ccdc85c on cell function in vitro. Xenografted tumor growth was used to assess in vivo role of KLF7. Chip-qPCR and luciferase reporter assays were applied to check whether KLF7 regulated VPS35 at transcriptional manner. Co-IP assay was performed to detect the interaction between VPS35 and Ccdc85c. Immunohistochemical staining and qRT-PCR analysis were performed in human HCC sampels to study the clinical significance of KLF7, VPS35 and β-catenin. Results Firstly, KLF7 was highly expressed in human HCC samples and correlated with patients’ differentiation and metastasis status. KLF7 overexpression contributed to cell proliferation and invasion of HCC cells in vitro and in vivo. KLF7 transcriptional activation of VPS35 was necessary for HCC tumor growth and metastasis. Further, co-IP studies revealed that VPS35 could interact with Ccdc85c in HCC cells. Rescue assay confirmed that overexpression of VPS35 and knockdown of Ccdc85c abolished the VPS35-medicated promotion effect on cell proliferation and invasion. Finally, KLF7/VPS35 axis regulated Ccdc85c, which involved in activation of β-catenin signaling pathway, confirmed using β-catenin inhibitor, GK974. Functional studies suggested that downregulation of Ccdc85c partly reversed the capacity of cell proliferation and invasion in HCC cells, which was regulated by VPS35 upregulation. Lastly, there was a positive correlation among KLF7, VPS35 and active-β-catenin in human HCC patients. Conclusion We demonstrated that KLF7/VPS35 axis promoted HCC cell progression by activating Ccdc85c-medicated β-catenin pathway. Targeting this signal axis might be a potential treatment strategy for HCC.

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Antagonism of Adherent Invasive E. coli LF82 With Human α-defensin 5 in the Follicle-associated Epithelium of Patients With Ileal Crohn’s Disease

Inflammatory Bowel Diseases ◽

10.1093/ibd/izaa315 ◽

2020 ◽

Author(s):

Lina Y Alkaissi ◽

Martin E Winberg ◽

Stéphanie DS Heil ◽

Staffan Haapaniemi ◽

Pär Myrelid ◽

...

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Ex Vivo ◽

Ussing Chambers ◽

E Coli ◽

Follicle Associated Epithelium ◽

Vitro Model ◽

Set Up

Abstract Background The first visible signs of Crohn’s disease (CD) are microscopic erosions over the follicle-associated epithelium (FAE). The aim of the study was to investigate the effects of human α-defensin 5 (HD5) on adherent-invasive Escherichia coli LF82 translocation and HD5 secretion after LF82 exposure in an in vitro model of human FAE and in human FAE ex vivo. Methods An in vitro FAE-model was set up by the coculture of Raji B cells and Caco-2-cl1 cells. Ileal FAE from patients with CD and controls were mounted in Ussing chambers. The effect of HD5 on LF82 translocation was studied by LF82 exposure to the cells or tissues with or without incubation with HD5. The HD5 secretion was measured in human FAE exposed to LF82 or Salmonella typhimurium. The HD5 levels were evaluated by immunofluorescence, immunoblotting, and ELISA. Results There was an increased LF82 translocation across the FAE-model compared with Caco-2-cl1 (P < 0.05). Incubation of cell/tissues with HD5 before LF82 exposure reduced bacterial passage in both models. Human FAE showed increased LF82 translocation in CD compared with controls and attenuated passage after incubation with sublethal HD5 in both CD and controls (P < 0.05). LF82 exposure resulted in a lower HD5 secretion in CD FAE compared with controls (P < 0.05), whereas Salmonella exposure caused equal secretion on CD and controls. There were significantly lower HD5 levels in CD tissues compared with controls. Conclusions Sublethal HD5 reduces the ability of LF82 to translocate through FAE. The HD5 is secreted less in CD in response to LF82, despite a normal response to Salmonella. This further implicates the integrated role of antimicrobial factors and barrier function in CD pathogenesis.

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Role of the C5a-C5a receptor axis in the inflammatory responses of the lungs after experimental polytrauma and hemorrhagic shock

Scientific Reports ◽

10.1038/s41598-020-79607-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Shinjini Chakraborty ◽

Veronika Eva Winkelmann ◽

Sonja Braumüller ◽

Annette Palmer ◽

Anke Schultze ◽

...

Keyword(s):

Hemorrhagic Shock ◽

Inflammatory Responses ◽

Experimental Models ◽

Lung Damage ◽

C5a Receptor ◽

Cytokine Levels ◽

Vitro Model

AbstractSingular blockade of C5a in experimental models of sepsis is known to confer protection by rescuing lethality and decreasing pro-inflammatory responses. However, the role of inhibiting C5a has not been evaluated in the context of sterile systemic inflammatory responses, like polytrauma and hemorrhagic shock (PT + HS). In our presented study, a novel and highly specific C5a L-aptamer, NoxD21, was used to block C5a activity in an experimental murine model of PT + HS. The aim of the study was to assess early modulation of inflammatory responses and lung damage 4 h after PT + HS induction. NoxD21-treated PT + HS mice displayed greater polymorphonuclear cell recruitment in the lung, increased pro-inflammatory cytokine levels in the bronchoalveolar lavage fluids (BALF) and reduced myeloperoxidase levels within the lung tissue. An in vitro model of the alveolar-capillary barrier was established to confirm these in vivo observations. Treatment with a polytrauma cocktail induced barrier damage only after 16 h, and NoxD21 treatment in vitro did not rescue this effect. Furthermore, to test the exact role of both the cognate receptors of C5a (C5aR1 and C5aR2), experimental PT + HS was induced in C5aR1 knockout (C5aR1 KO) and C5aR2 KO mice. Following 4 h of PT + HS, C5aR2 KO mice had significantly reduced IL-6 and IL-17 levels in the BALF without significant lung damage, and both, C5aR1 KO and C5aR2 KO PT + HS animals displayed reduced MPO levels within the lungs. In conclusion, the C5aR2 could be a putative driver of early local inflammatory responses in the lung after PT + HS.

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