Bovine IGF-1 Research Articles

Correlation of Bovine Dairy Igf-1 And the Neoplasia Development

Cow's milk and its derivatives are among the most consumed foods in the world being part of the daily diet of several people. However, its consumption is linked to factors that were associated with neoplastic development, such as IGF-1. IGF-1 acts by stimulating growth effects on most cells of the body via the IGF1R receptor. In this context, this work aimed to investigate studies that correlate these variables: IGF-1, cow's milk, and neoplasias. The results showed that IGF-1 in cow's milk may increase serum IGF-1 levels in humans from milk consumption. Also, we have shown it that some supplements such as choline and casein do not alter the amount of IGF-1, but when the cow undergoes recombinant bovine somatotropin treatment, there is a significant increase in circulating IGF-1. However, further studies are needed to explain the cause-and-effect relationship noted in this study.

The Effect of In Vitro-digestion on Mammalian Milks’ Insulin and IGF-1

Abstract Objectives This study investigated the impact of in vitro-digestion and spray-dry processing on insulin concentration and insulin growth factor-1 (IGF-1) in milk from several species. Diabetes is a growing health problem in the U.S. and worldwide, predicted to affect 642 million people by 2040. To date, insulin therapy is considered to be the best treatment due to its low side effects. However, insulin treatment in type-2 diabetes can lead to insulin resistance. Natural alternatives are used to treat diabetes in many regions of the world, and mechanisms of action need investigating. Literature shows that daily camel milk consumption led to a decrease in insulin dose in patients with type 1 diabetes and can improve glucose control in type-2 diabetes. The presence of bioactive insulin has been hypothesized as a mechanism for this effect. Methods Human Insulin and Bovine IGF-1 immunoactivity were measured in milk from Human, Bovine, Goat, and Camel and spray dried milk from goat and camel by ELISA (RayBioTech) with microplate reader. Raw and pasteurized samples subjected to in vitro digestion were also measured. Results Raw goat and camel milk showed insulin immunoreactivity with approximately 31, and 20 µIU/mL, respectively. Spray-dried goat and camel milk from different animals showed insulin immunoreactivity of approximately 26, and 20 µIU/mL, respectively. Insulin immunoactivity in bovine and human milk was 16 and 20 µIU/mL. In vitro digestion did not decrease insulin levels in either goat or camel milk, but significantly reduced human and bovine milk insulin immunoactivity. Bovine IGF1 did not cross react with this anti-human insulin antibody. IGF-1 immunoactivity was present in goat milk at very low concentration but not detected in other samples. Conclusions Goat and camel milk (fresh and spray dried) had high insulin concentrations. There was no impact of in vitro-digestion and spray dried processing on insulin levels. IGF-1 was only present in raw goat milk. IGF-1 was not detected in camel milk samples suggesting that the antidiabetic effects of camel milk is due to its insulin concentration and the ability of the insulin to survive gastric and intestinal digestion, as well as pasteurization. Funding Sources NIFA; NCSU.

Knock-in Vector for Expression of Insulin-like Growth Factor 1 on the Bovine β-casein Gene Locus

The production of therapeutic protein and improve of productivity of the domestic animal from transgenic animal is the major technology of biotechnology. Lactoferrin has its highest content in the colostrum, and is known as the antiviral substance. Insulin like growth factor1 is known to play an important role in the growth of the animal. The objective of this study is construction of knock-in vector that insulin like growth factor1 gene is inserted into the β-casein gene locus for expression of bovine insulin like growth factor1 (bIGF1) on the bovine β-casein gene. The knock-in vector consists of 5’ arm region (1.02 kb), insulin like growth factor 1 cDNA, CMV-EGFP, and 3’ arm region (1.83 kb). To express bIGF1 gene as transgene, the F2A sequence was fused to the 5’ terminal of bIGF1 gene and inserted into exon 7 of the β-casein gene. As a result, the knock-in vector is confirmed that the amino acids are synthesized without termination from the β-casein exon 7 region to the bIGF1 gene. These knock-in vectors may help to create transgenic dairy cattle expressing bovine IGF1 protein in the mammary gland via the expression system of the bovine β-casein gene.

Assessment on dietary protein requirement of sandfish (Holothuria scabra) through growth response and plasma insulin-like growth factor-1 profile

A growth trial was undertaken to investigate dietary protein requirement of the sub adult sandfish, Holothuria scabra, using correlation of growth rate and plasma IGF-1 concentrations. The sand fish (n = 6 per treatment, average wet weight 21.5g ± 2.5 S.D.) were fed one of the seven diets containing various levels of proteins at the ration of 3% of the body weight, once a day for 6 weeks. The analysis of IGF-1 was performed from pooled data per treatment using Bovine IGF-1 kit. Results revealed that protein doses affected the specific growth rate (SGR), and the food conversion ratio (FCR) with the 10% protein dose gave the best result. Plasma concentration of IGF-1 was positively correlated with protein dose and specific growth rate in the sandfish. The 10% protein dose produced highest SGR, but the IGF-1 concentration was not significantly different from other treatments. It could be concluded that both SGR and plasma IGF-1 concentration were affected by protein dose in the diet. The sandfish was expected to require 10-35% protein in the diet as suitable rearing dose.

Association between <i>IGF1/Tas</i>I polymorphism and milk traits of Polish Holstein Friesian cows

Abstract. The study was carried out on 658 Polish Holstein Friesian cows. A transversion of A→C in the P1 promoter region of bovine IGF1 gene at position 977 bp upstream from the start codon in exon 1 was identified using ACRS-PCR method. Reverse primer introduced an artificial TasI restriction site. Three genotypes (AA, AC, CC) were found in the analysed herd of dairy cows occurred with a frequency of 0.766, 0.213 and 0.021, respectively. In the presented study, the statistically significant differences between individuals of different IGF1/TasI genotypes were found in milk, fat and protein yield. In the 2nd and 3rd lactations, the cows carrying the CC genotype produced more milk than the AA individuals (P≤0.05). In the case of protein and fat yield, similar tendencies were observed.

Preparation of Insulin-Like Growth Factor-1 from Bovine Colostrums

This experiment achieved a purpose that bovine colostrums IGF-1 were extracted from bovine colostrums rapidly and efficiently. Fresh bovine colostrums were heated at 40 °C for 1h and centrifuged at 2500 rpm for 20min, then they were discarded the upper fat and adjusted the pH value of skim milk at 4.2 to remove casein, the salting saturation was 35% to remove high molecular proteins. We extracted IGF-1 concentrated liquid by the ultra filtration which were 50ku and 3ku molecular weight, and the IGF-1 concentrated liquid were purified further by chromatography which was filled up weak cation exchanger (CM-SephadexC-25), the equilibrium buffer was NaCl solution （0.02 mol / L buffer, pH 7.25, the flow rate was 80cm / h）. Finally we used the HPLC and the ELISA to determine the purity and the yield of the IGF-1 concentrated liquid. The results were that the yield of the bovine colostrums IGF-1 was 71.98%, and the purity was 65.51%.

Association of IGF-1 gene polymorphism with milk production traits and paternal genetic trends in Iranian Holstein bulls.

The effects of Insulin-Like Growth Factor-1 polymorphism were examined on milk production traits and genetic trends in the Iranian Holstein bulls. A total of 282 bulls were genotyped for SnaBI restriction site in the 5' flanking region of IGF-1 by applying PCR-RFLP method. The allele frequencies for C and T alleles were 0.562 and 0.438, respectively. The results indicated that genetic variants at the 5'-noncoding region of the bovine IGF-1 gene had a marked effect on estimated breeding values of milk (EBVM) and fat yields (EBVF). The heterozygous bulls (CT genotype) had higher EBVM and EBVF than homozygous bulls (P 0.1). The studied polymorphism of IGF-1 showed no significant association with genetic trends of milk related traits (P > 0.1). These results suggest that allelic interaction in IGF-1 polymorphism as over dominance and might be effectiveness in animal improving by crossbreeding.

The Scaffolding Adapter Gab1 Mediates Vascular Endothelial Growth Factor Signaling and Is Required for Endothelial Cell Migration and Capillary Formation

Vascular endothelial growth factor (VEGF) is involved in the promotion of endothelial cell proliferation, migration, and capillary formation. These activities are mainly mediated by the VEGFR2 receptor tyrosine kinase that upon stimulation, promotes the activation of numerous proteins including phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase (PI3K), Akt, Src, and ERK1/2. However, the VEGFR2-proximal signaling events leading to the activation of these targets remain ill defined. We have identified the Gab1 adapter as a novel tyrosine-phosphorylated protein in VEGF-stimulated cells. In bovine aortic endothelial cells, Gab1 associates with VEGFR2, Grb2, PI3K, SHP2, Shc, and PLCgamma, and its overexpression enhances VEGF-dependent cell migration. Importantly, silencing of Gab1 using small interfering RNAs leads to the impaired activation of PLCgamma, ERK1/2, Src, and Akt; blocks VEGF-induced endothelial cell migration; and perturbs actin reorganization and capillary formation. In addition, co-expression of VEGFR2 with Gab1 mutants unable to bind SHP2 or PI3K in human embryonic kidney 293 cells and bovine aortic endothelial cells mimics the defects observed in Gab1-depleted cells. Our work thus identifies Gab1 as a novel critical regulatory component of endothelial cell migration and capillary formation and reveals its key role in the activation of VEGF-evoked signaling pathways required for angiogenesis.

Stem Cells Combined With Gene Transfer for Therapeutic Vasculogenesis

Discoveries in one scientific field sometimes have the unexpected result of igniting a major step forward or new direction in another field. We are in the early phases of one of these sea changes. Advances in stem cell therapies are progressing at a rapid clip and are providing the field of gene therapy with an expanded platform of technologies for the introduction of recombinant genes into human tissues. Likewise, a decade worth of experience in gene therapy provides stem cell researchers with a jump-start. Indeed, it is likely and probable that these 2 fields will eventually merge due to significant scientific and technical overlap. See p 732 The major contribution of the study by Iwaguro et al1 in this issue of Circulation is the demonstration that gene transfer of vectors encoding VEGF into endothelial progenitor cells (EPCs) ex vivo and injection of gene modified EPCs into ischemic muscle in vivo increases neovascularization in an animal model of hindlimb ischemia. Peripheral blood mononuclear cells from human volunteers were cultured in a special cocktail that facilitates growth of EPCs—plating on fibronectin and grown in the presence of multiple growth factors, such as endothelial cell basal medium, fetal bovine serum, human VEGF-A, human FGF-2, human EGF, IGF-1, and ascorbic acid—as previously characterized by this group.2 After 7 days in culture, EPCs were transduced with adenoviral vectors encoding a murine VEGF164 gene or a control reporter gene. Proliferative indices and adhesion properties were measured ex vivo in order to characterize the phenotype of the cells. The authors then conducted pilot studies in vivo to determine the dose of gene-modified cells required to produce a similar magnitude of neovascularization achieved with nontransduced EPCs. Interestingly, they found that 30 times fewer VEGF-transduced EPCs (1.5×104 cells) produced equivalent neovascularization compared with 4.5×105 …

Effect of interferon-γ (IFN-γ) on transforming growth factor-β (TGF-β) regulation of sialomucin complex/Muc4

Sialomucin complex (SMC, rat Muc4) is a heterodimeric glycoprotein complex consisting of a mucin subunit ASGP-1 (Ascites sialoglycoprotein-1) and a transmembrane subunit ASGP-2, which is highly overexpressed on the surface of ascites 13762 rat mammary adenocarcinoma cells. The complex is produced from a single gene and polypeptide precursor. SMC is developmentally regulated in normal rat mammary gland by multiple and complex mechanisms, with levels in the lactating gland being 100-fold those in the virgin gland. SMC transcript levels are enhanced in normal rat mammary epithelial cells by fetal bovine serum, insulin, and IGF-1 by an ERK-1/2-dependent pathway. SMC is post-transcriptionally regulated by Matrigel (extracellular matrix) by inhibition of SMC precursor synthesis. SMC is also post-transcriptionally regulated by TGF-β by disruption of SMC precursor processing into mature ASGP-1 and ASGP-2. The inhibition of SMC levels by TGF-β occurs by an ERK1/2-independent pathway, suggesting that the SMAD or another pathway may be involved in this effect. Interestingly, the inhibition of SMC levels by TGF-β can be blocked by treatment with IFN-γ, which has been shown to block TGF-β effects via a Jak/Stat-dependent pathway. This effect is dose-responsive and is dependent on the order in which the cytokines are added, suggesting that the balance of signaling inputs is important in determining the expression level of SMC. Thus, SMC is regulated by multiple mechanisms, and the delicate interplay of the pathways involved serves to maintain normal levels of the complex and repress potential deleterious effects of overexpression.

Determination of Transforming Growth Factor-β1 (TGF-β1) and Insulin-Like Growth Factor 1 (IGF-1) in Bovine Colostrum Samples

The major growth factors in bovine colostrum are transforming growth factor-βs (TGF-β1 and TGF-β2) and insulin-like growth factors (IGF-1 and IGF-2). Recently, TGF-β2 content of bovine colostrum was measured using a TGF-β2 specific ELISA (1) and now we have validated ELISAs for for bovine TGF-β1 and IGF-1. The concentrations of IGF-1 and TGF-β1 in the first milking after calving were 248–1 850 ng/ml and 12.4–42.6 ng/ml, respectively, and they declined in correlation with total protein concentration to 27.0–101 ng/ml (IGF-1) and 0.80–3.49 ng/ml (TGF-β1) by the fifth milkings. The amount of TGF-β1 was on average 5.3±1.4% of that of TGF-β2 and there is a high correlation (r=0.966) between the concentrations of these growth factors in the same samples. No free TGF-β1 form of could be detected.

All- trans-Retinoic Acid Blocks Cell Cycle Progression of Human Ovarian Adenocarcinoma Cells at Late G 1

We prepared single cell clones from two ovarian carcinoma cell lines, CA-OV3 and SK-OV3, and analyzed the effect of all- trans-RA treatment on cell division, DNA synthesis, and cell cycle stage distribution of these single cell clones. Our results show that despite the well-known heterogeneous nature of these cell lines, all single cell clones of SK-OV3 cells are resistant to the growth inhibitory effects of all- trans-RA. In contrast, all single cell clones of CA-OV3 cells were growth inhibited by all- trans-RA. However, the extent of growth inhibition did vary somewhat from clone to clone. Additional studies employing flow cytometry showed that all- trans-RA blocked CA-OV3 cell cycle progression in the G 1stage. Finally, all- trans-RA was able to inhibit G 1progression in growth-arrested CA-OV3 cells following stimulation with fetal bovine serum, insulin, IGF-1, or estrogen. Since each of these growth factors is known to act via distinct signal transduction pathways, our results suggest that all- trans-RA blocks G 1progression by targeting a downstream process or event which occurs at a point after the insulin/IGF-1, estrogen, and serum signal transduction pathways converge.

Characterizing mutagenesis in the hprt gene of rat alveolar epithelial cells.

A clonal selection assay was developed for mutation in the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene of rat alveolar epithelial cells. Studies were conducted to establish methods for isolation and long-term culture of rat alveolar epithelial cells. When isolated by pronase digestion purified on a Nycodenz gradient and cultured in media containing 7.5% fetal bovine serum (FBS), pituitary extract, EGF, insulin, and IGF-1, rat alveolar epithelial cells could be maintained in culture for several weeks with cell doubling times of 2-4 days. The rat alveolar epithelial cell cultures were exposed in vitro to the mutagens ethylnitrosourea (ENU) and H2O2, and mutation in the hprt gene was selected for by culture in the presence of the toxic purine analog, 6-thioguanine (6TG). In vitro exposure to ENU or H2O2 produced a dose-dependent increase in hprt mutation frequency in the alveolar epithelial cells. To determine if the assay system could be used to evaluate mutagenesis in alveolar type II cells after in vivo mutagen or carcinogen exposure, cells were isolated from rats treated previously with ENU or alpha-quartz. A significant increase in hprt mutation frequency was detected in alveolar epithelial cells obtained from rats exposed to ENU or alpha-quartz; the latter observation is the first demonstration that crystalline silica exposure is mutagenic in vivo. In summary, these studies show that rat alveolar epithelial cells isolated by pronase digestion and Nycodenz separation techniques and cultured in a defined media can be used in a clonal selection assay for mutation in the hprt gene. This assay demonstrates that ENU and H2O2 in vitro and ENU and alpha-quartz in vivo are mutagenic for rat alveolar epithelial cells. This model should be useful for investigating the genotoxic effects of chemical and physical agents on an important lung cell target for neoplastic transformation.

Insulin-like growth factors 1 and 2 in bovine colostrum. Sequences and biological activities compared with those of a potent truncated form.

1. Insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) together with a truncated form of IGF-1 were purified to homogeneity from bovine colostrum. 2. Two forms of IGF-1 were totally resolved from IGF-2 in the purification by h.p.l.c. involving cation-exchange and reverse-phase columns. 3. The complete amino acid sequences for all three forms of IGF were determined. The sequence of bovine IGF-1 was found to be identical with that of human IGF-1, and that of the variant lacked the N-terminal tripeptide Gly-Pro-Glu (-3N:IGF-1). Bovine IGF-2 was found to differ in three residues of the C-domain compared with human IGF-2, with serine, isoleucine and asparagine substituted for alanine, valine and serine respectively at positions 32, 35 and 36. 4. Protein synthesis in L6 rat myoblasts was stimulated and protein degradation inhibited in a co-ordinate response with all three IGFs. The relative potency in both processes was -3N:IGF-1 greater than IGF-1 greater than IGF-2. A similar order of potency was obtained for the stimulation of DNA synthesis by -3N:IGF-1 and IGF-1. The approximately 10-fold effect on biological activity of removing the N-terminal tripeptide is unexpected in view of current information on IGF-1 structure and function.

The bovine insulin-like growth factor (IGF) binding protein purified from conditioned medium requires the N-terminal tripeptide in IGF-1 for binding

The insulin-like growth factor binding protein (BP) secreted by bovine kidney (MDBK) cells has been purified by affinity chromatography on a rat IGF-2 Sepharose column. Purified BP migrated as a single band of M r 40,000 upon SDS polyacrylamide gel electrophoresis. An N-terminal sequence of 53 residues was obtained which was very similar up to residue 21 to the corresponding rat BRL-3A BP sequence. In competitive binding experiments with bovine IGF-1 and IGF-2, and recombinant human IGF-1, BP had a similar affinity for these ligands when IGF-1 tracer was used, but a higher affinity for IGF-2 with IGF-2 as radioligand. The N-terminal destripeptide truncated form of bovine IGF-1, which has enhanced biological activity, was found to have a markedly reduced affinity for BP compared to intact IGF-1. The increased bioactivity of destripeptide IGF-1 can be explained by this reduced affinity for BP.

Comparative binding of bovine, human and rat insulin-like growth factors to membrane receptors and to antibodies against human insulin-like growth factor-1.

The immunological properties of human, bovine and rat insulin-like growth factors (IGF) and insulin were compared in competitive binding studies with Tr10 and NPA polyclonal antisera raised in rabbits against human IGF-1. Bovine IGF-1 was 11-19% as effective as human IGF-1 in competing for binding with 125I-labelled human IGF-1, whereas IGF-2 reacted poorly and insulin did not compete. Similar competitive binding curves were obtained with the mouse monoclonal anti-(human IGF-1) antibody 3D1, except that bovine IGF-1 showed a severalfold greater affinity for the monoclonal antibody than for either polyclonal antiserum. Membranes isolated from human placenta, sheep placenta and foetal-human liver were used as sources of cellular receptors. In human placental membranes, most of the binding of IGF-1 tracers could be attributed to a type-1 receptor, because insulin inhibited up to 65% of tracer binding. The other two tissues apparently contain only type-2 receptors, as evidenced by the very low potency of bovine or human IGF-1 in competing for binding with IGF-2 tracers and the absence of any competition by insulin. In competition for binding with labelled bovine or human IGF-1 to human placental membranes, bovine IGF-1 had a similar potency to human IGF-1, whereas bovine IGF-1 was more potent in binding studies with tissues rich in type-2 receptors. Rat IGF-2 was considerably less effective than human IGF-2 in competition for receptors on any of the membrane preparations.

Binding properties and biological potencies of insulin-like growth factors in L6 myoblasts.

Protein synthesis in rat L6 myoblasts is stimulated and protein breakdown inhibited in a co-ordinate manner by insulin-like growth factors (IGF) or insulin. For both processes, bovine IGF-1 was somewhat more potent than human IGF-1, which was effective at a tenth the concentration of insulin, rat IGF-2 or human IGF-2. A similar order of potency is noted when DNA synthesis or protein accumulation is monitored over a 24 h period, but between 20- and 50-fold higher concentrations of each growth factor are required than those needed to produce effects in the 4 h protein-synthesis or -breakdown measurements. Binding experiments with labelled human or bovine IGF-1 as ligand demonstrated competition at concentrations of IGF-2, especially human IGF-2, lower than that of either IGF-1 preparation. This pattern was much more pronounced when the radioligand was either human IGF-2 or rat IGF-2. Insulin competed 10-15% for the binding of labelled IGF-1, but not at all with labelled IGF-2. Ligand-receptor cross-linking experiments showed that labelled bovine IGF-1 bound approximately equally to the type 1 IGF receptor (Mr 130000 after reduction) and to the type 2 IGF receptor (Mr 270000 after reduction), and that unlabelled IGF-1 competed equally with radioligand binding to both receptors. On the other hand, rat IGF-2 competed more effectively for binding to the type-2 receptor, and insulin competed only for binding to the type-1 receptor. Further cross-linking experiments with rat IGF-2 as radioligand demonstrated binding only to the type-2 receptor and to proteins with Mr values after reduction of 230000 and 200000. This binding was prevented by high rat IGF-2 concentrations, less effectively by bovine IGF-1 and not at all by insulin. The apparently conflicting biological potencies and receptor binding of the different growth factors can be explained if all the biological actions are mediated via the type-1 IGF receptor, rather than through the abundant type-2 receptor.