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• IGFBP3 may regulate the growth and development of yak liver. • BgIGFBP3 protein promotes proliferation of yak hepatocytes and growth in mice. • BgIGFBP3 protein exerts its function through PI3K-Akt signaling pathway. IGFBP3 (Insulin-like growth factor binding protein 3) constitutes a crucial constituent of the insulin-like growth factor (IGF), which are intimately associated with the organism's growth and development processes. Despite its significance, the precise function of IGFBP3 in yak liver development remains largely unexplored. In the present study, we systematically examined the expression profile of IGFBP3 in the liver tissues of yaks across various growth stages, elucidated its influence on the activity of yak hepatocytes, and probed its effects on murine liver development. A comparative analysis revealed that the expression of IGFBP3 was significantly higher in the liver tissue of 5-year-old yaks compared to their 15-month-old and 1-day-old counterparts (P < 0.01). To further validate its biological function, pET-28a-BgIGFBP3 prokaryotic expression vector was constructed. Upon exposing yak hepatocytes to varying concentrations of Bos grunniens (Bg) IGFBP3 protein, we observed augmented cellular activities and elevated colony formation rates. Moreover, our investigation revealed the upregulation of key genes within the PI3K-Akt signaling pathway, including ERBB2 , IRS1 , PIK3R1 , AKT1 , RAF1 , MAP2K2 , and MAPK3 , in both yak hepatocyte cultures and murine models. These findings collectively indicate that BgIGFBP3 promotes the proliferation of yak hepatocytes and enhances murine liver development by modulating the PI3K-Akt signaling pathway. The functional relevance of BgIGFBP3 was substantiated through in vivo and in vitro experiments, thereby underscoring its potential as a regulatory factor in liver development processes. [ABSTRACT FROM AUTHOR]

• The IGFBP3 gene knockout cells were successfully established using CRISPR/Cas9 system. • The expression levels of the IGFBP3 gene and protein were remarkably decreased in the KO cells. • IGFBP3 gene knockout cells displayed an increase in the level of SA-ß-Gal staining short telomere length in the KO cells. • The expression levels of senescence-related proteins such as PI3K, AKT1, PDK1, and p53 were higher in the KO cells than in normal cells. The IGF-1 signaling pathway has been deeply involved in the aging mechanism. The insulin-like growth factor binding protein 3 (IGFBP-3) is a protein that binds to IGF-1 that regulates growth, survival, and aging. The purpose of this study was to investigate the impact of the IGFBP3 gene knockout (KO) on the expressions of aging-related proteins and genes using the CRISPR/Cas9 system. The IGFBP3 gene knockout (KO) was performed by the CRISPR/Cas9 system. Sanger DNA sequencing and Indel analyses were used to verify the induction of mutation. First, Sanger DNA sequencing was used to analyze the IGFBP3 gene knockout in murine cells (B16F1). The isolation of three colonies with the mutated DNA sequences in the IGFBP3 gene was validated. In addition, the expression levels of the IGFBP3 gene and protein in the edited B16F1 cells were lower than in those of normal B16F1 cells in western blot analysis as well as RT-PCR and qPCR. Moreover, IGFBP3 gene KO cells enhanced the level of SA-ß-gal staining and short telomere length compared to normal B16F1 cells. In particular, it was found that the expression levels of senescence-related proteins such as PI3K, AKT1, PDK1, and p53 were higher in IGFBP3 gene KO cells than in normal cells in both the absence and presence of IGF-1. Therefore, the above findings could provide a clue that IGFBP3 could play a key role in the aging mechanism. [Display omitted] [ABSTRACT FROM AUTHOR]

Five-year glioblastoma (GBM) survivors (LTS) are the minority of the isocitrate dehydrogenase (IDH)-wild-type GBM patients, and their molecular fingerprint is still largely unexplored. This multicenter retrospective study analyzed a large LTS-GBM cohort from nine Italian institutions and molecularly characterized a subgroup of patients by mutation, DNA methylation (DNAm) and copy number variation (CNV) profiling, comparing it to standard survival GBM. Mutation scan allowed the identification of pathogenic variants in most cases, showing a similar mutational spectrum in both groups, and highlighted TP53 as the most commonly mutated gene in the LTS group. We confirmed DNAm as a valuable tool for GBM classification with a diagnostic refinement by using brain tumor classifier v12.5. LTS were more heterogeneous with more cases classified as diffuse pediatric high-grade glioma subtypes and having peculiar CNVs. We observed a global higher methylation in CpG islands and in gene promoters of LTS with methylation levels of distinct gene promoters correlating with prognosis. • This is a multicenter retrospective study on a large cohort of GBM-LTS patients. • The mutational profile of pathogenic variants and CNV was similar in LTS and STS. • LTS show no episignature but a global higher methylation in CpG islands. • Methylation levels of distinct gene promoters correlated with prognosis. [ABSTRACT FROM AUTHOR]

This study investigates the role of IGFBP3-mediated m6A modification in regulating the miR-23a-3p/SMAD5 axis and its impact on fracture healing, aiming to provide insights into potential therapeutic targets. Utilizing fracture-related datasets, we identified m6A modification-related mRNA and predicted miR-23a-3p as a regulator of SMAD5. We established a mouse fracture healing model and conducted experiments, including Micro–CT, RT-qPCR, Alizarin Red staining, and Alkaline phosphatase (ALP) staining, to assess gene expression and osteogenic differentiation. IGFBP3 emerged as a crucial player in fracture healing, stabilizing miR-23a-3p through m6A modification, leading to SMAD5 downregulation. This, in turn, inhibited osteogenic differentiation and delayed fracture healing. Inhibition of IGFBP3 partially reversed through SMAD5 inhibition, restoring osteogenic differentiation and fracture healing in vivo. The IGFBP3/miR-23a-3p/SMAD5 axis plays a pivotal role in fracture healing, highlighting the relevance of m6A modification. IGFBP3's role in stabilizing miR-23a-3p expression through m6A modification offers a potential therapeutic target for enhancing fracture healing outcomes. [Display omitted] [ABSTRACT FROM AUTHOR]

Steatohepatitis drives fibrogenesis in alcohol-related liver disease. Recent studies have suggested that hepatic stellate cells (HSCs) may regulate the parenchymal cell injury and inflammation that precedes liver fibrosis, although the mechanism remains incompletely defined. Neuropilin-1 (NRP-1) and synectin are membrane proteins implicated in HSC activation. In this study, we disrupted NRP-1 and synectin as models to evaluate the role of HSC activation on the development of steatohepatitis in response to alcohol feeding in mice. Mice with HSC-selective deletion of NRP (ColCre/ Nrp1 loxP) or synectin (ColCre/synectinloxP) vs. paired Nrp1 l oxP or synectinloxP mice were fed a control diet or the chronic/binge alcohol feeding model. Several markers of steatosis and inflammation were evaluated. ColCre/ Nrp1 loxP mice showed less fibrosis, as expected, but also less inflammation and steatosis, with lower hepatic triglyceride content. Similar results were observed in the synectin model. Hepatocytes treated with supernatant of HSCs from ColCre/ Nrp1 loxP mice compared to supernatant from Nrp1 loxP mice were protected against ethanol-induced lipid droplet formation. An adipokine and inflammatory protein array from the supernatant of HSCs with NRP-1 knockdown showed a significant reduction in Igfbp3 (a major insulin-like growth factor-binding protein with multiple metabolic functions) and an increase in SerpinA12 (a serine-protease inhibitor) secretion compared to wild-type HSCs. Recombinant Igfbp3 induced lipid droplets, triglyceride accumulation, and lipogenic genes in hepatocytes in vitro, while SerpinA12 was protective against ethanol-induced steatosis. Finally, Igfbp3 was increased, and SerpinA12 was decreased in serum and liver tissue from patients with alcoholic hepatitis. Selective deletion of NRP-1 from HSCs attenuates alcohol-induced steatohepatitis through regulation of Igfbp3 and SerpinA12 signaling. Hepatic stellate cells are known for their role in fibrosis (scarring of the liver). In this study, we describe their role in the modulation of fat deposition and inflammation in the liver, which occurs secondary to alcohol damage. • HSCs regulate hepatocyte steatosis that precedes liver fibrosis in alcohol-related liver disease. • HSC-derived Igfbp3 and SerpinA12 mediate lipid droplet formation in hepatocytes. • These results are recapitulated in samples from patients with alcoholic hepatitis. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that compared to runners, swimmers carry a higher prevalence of the IL-6 -174C polymorphism and lower single nucleotide polymorphism frequencies of the IGF system. The aim of the present study was to assess the combined frequency of the IL-6 -174G/C and IGFBP3 -202A/C polymorphisms among track and field athletes and swimmers. Track and field athletes were divided into long-distance runners (major event 5000 m-marathon, n = 63) and power athletes (major event 100-200 m sprints and long jump, n = 67). Swimmers were divided into long-distance swimmers (major event: 400-1500 m, n = 50), and short-distance swimmers (major event: 50-100 m, n = 43). All participants had achieved results that ranked them among the top all-time Israeli athletes in their event, and competed at national and/or international level on a regular basis. Carrying both IL-6 C and IGFBP3 C mutations was significantly greater among long-distance swimmers (LDS – 44%) compared to long distance runners (LDR – 21%, p <.01), and among short distance swimmers (SDS – 49%) compared to sprinters and jumpers (S/J – 28%, p <.05). Among runners, the prevalence of those not carrying either of the two mutations was significantly higher among LDR (25%) compared to S/J (10%, p <.03). The prevalence of carrying both IL-6 C and IGFBP3 C mutations was significantly higher among the swimmers compared to runners. It is possible that carrying the IGFBP3 C polymorphism is required to compensate for the potential genetically non-beneficial effects of a higher IL-6C genotype and an attenuated IGF system among the swimmers. • IL-6 CC genotype and C allele frequency were significantly higher among LDS compared to SDS and controls • The prevalence of carrying both IL-6C and IGFBP3C mutations was significantly higher among the swimmers compared to runners. • Carrying the IGFBP3 C might compensate for non-beneficial effects of IL-6 C genotype and an attenuated IGF system among the swimmers [ABSTRACT FROM AUTHOR]

The infection of porcine circovirus type 2 (PCV2) triggers activation of the protein kinase RNA–like endoplasmic reticulum kinase (PERK) pathway and leads to DNA damage. Insulin-like growth factor-binding protein 3 (IGFBP3) may interact with the endoplasmic reticulum (ER). It remains unclear whether IGFBP3 regulates DNA damage via ER stress to mediate PCV2 replication. In this study, we observed an upregulation of porcine IGFBP3 expression during PCV2 infection, and overexpression of IGFBP3 enhanced the expression of PCV2 Cap protein, PCV2 DNA copy number, and viral titers in PK-15 B6 cells and 3D4/21 cells. Additionally, overexpression of IGFBP3 induced an increase in the DNA damage marker γH2AX by activating the PERK/eIF2α pathway without concomitant activation of ATF4, IRE1α, and ATF6α/GRP78 pathways in PK-15 B6 cells and 3D4/21 cells. Knockdown of IGFBP3 had a reverse effect on PCV2 replication in PK-15 B6 cells and 3D4/21 cells. Furthermore, treatment with etoposide enhanced PCV2 replication while KU57788 decreased it. GSK2606414 and salubrinal limited both DNA damage and viral replication. Therefore, our findings suggest that porcine IGFBP3 promotes PCV2 replication through the PERK/eIF2α pathway-mediated induction of DNA damage in PK-15 B6 cells and 3D4/21 cells. Our study provides a basis for exploring novel antiviral strategies via the extensive understanding of the relationships between host cellular proteins and viral replication. • Porcine IGFBP3 expression upregulated in PK-15 and 3D4/21 cells upon PCV2 infection. • Overexpression of IGFBP3 increased DNA damage and PCV2 replication through PERK/eIF2α signaling. • Knockdown of IGFBP3 suppressed DNA damage, PCV2 replication by inhibiting PERK/eIF2α signaling. [ABSTRACT FROM AUTHOR]

The odontogenic differentiation of human dental pulp stem cells (HDPSCs) is associated with reparative dentinogenesis. Transcription factor GATA binding protein 4 (GATA4) is proved to be essential for osteoblast differentiation and bone remodeling. This study clarified the function of GATA4 in HDPSCs odontoblast differentiation. The change in GATA4 expression during reparative dentin formation was detected by immunohistochemistry staining. The expression of GATA4 during HDPSCs odontoblastic differentiation was detected by western blot and quantitative polymerase chain reaction. The effect of GATA4 on odontoblast differentiation was investigated following overexpression lentivirus transfection. RNA sequencing, dual luciferase assay and chromatin immunoprecipitation (CHIP) were conducted to verify downstream targets of GATA4. GATA4 overexpression lentivirus and small interference RNA targeting IGFBP3 were co-transfected to investigate the regulatory mechanism of GATA4. Upregulated GATA4 was observed during reparative dentin formation in vivo and the odontoblastic differentiation of HDPSCs in vitro. GATA4 overexpression suppressed the odontoblastic potential of HDPSCs, demonstrated by decreased alkaline phosphatase activity (p < 0.0001), mineralized nodules formation (p < 0.01), and odonto/osteogenic differentiation markers levels (p < 0.05). RNA sequencing revealed IGFBP3 was a potential target of GATA4. CHIP and dual luciferase assays identified GATA4 could activate IGFBP3 transcription. Additionally, IGFBP3 knockdown recovered the odontoblastic differentiation defect caused by GATA4 overexpression (p < 0.05). GATA4 inhibited odontoblastic differentiation of HDPSCs via activating the transcriptional activity of IGFBP3 , identifying its promising role in regulating HDPSCs odontoblast differentiation and reparative dentinogenesis. • GATA4 expression is upregulated during odontoblastic differentiation both in vivo and in vitro. • Forced expression of GATA4 suppresses the odontoblastic potential of human dental pulp stem cells. • GATA4 overexpression activates the IGFBP3 transcription. [ABSTRACT FROM AUTHOR]

The brain is the most common metastatic site of lung adenocarcinoma; however, the mechanism of this selective metastasis remains unclear. We aimed to verify the hypothesis that exposure of tumor cells to the brain microenvironment leads to changes in their gene expression, which promotes their oriented transfer to the brain. A549 and H1299 lung adenocarcinoma cells were exposed to human astrocyte-conditioned medium to simulate the brain microenvironment. Microarray analysis was used to identify differentially expressed genes, which were confirmed by quantitative real-time PCR and western blotting. Knockdown experiments using microRNAs and the overexpression of genes by cell transfection were performed in addition to migration and invasion assays. In vitro findings were confirmed in clinical specimens using immunohistochemistry. We found and confirmed a significant increase in insulin-like growth factor binding protein-3 (IGFBP3) levels. Our results also showed that the up-regulation of IGFBP3 promoted A549 cell epithelial-mesenchymal transition, migration, and invasion, while the knockdown of IGFBP3 resulted in decreased cell motility. We also found that Transforming growth factor-ß (TGF-ß)/Mothers against decapentaplegic homolog 4 (Smad4)-induced epithelial-mesenchymal transition was likely IGFBP3-dependent in A549 cells. Finally, expression of IGFBP3 was significantly elevated in pulmonary cancer tissues and intracranial metastatic tissues. Our data indicate that up-regulation of IGFBP3 might mediate brain metastasis in lung adenocarcinoma, which makes it a potential therapeutic target. [ABSTRACT FROM AUTHOR]

The molecular pathogenesis of osteonecrosis of the femoral head (ONFH) has been remained obscure so that its prevalence has been increasing in recent decades. Different transcription factors play critical roles in maintaining the balance between osteogenesis and adipogenesis. However, it has been unclear that the genes variants of the transcription factors exert the effects on the imbalance between steogenesis and adipogenesis during the development of ONFH. Here, we selected the 11SNPs from steogenesis, adipogenesis-specific transcription factors RUNX2, Osterix, and PPARγ genes, chondrogenesis or adipogenesis key factors COL2A1, IGFBP3 genes and analysed the genotypes, alleles, haplotypes and their association with the risk and clinical phenotypes of ONFH through Mass ARRAY® platformin in 200 ONFH patients and 177controls. The patients with ONFH (132 males, 68 females; age: 53.46 ± 11.48 yr) were consecutively enrolled at the Department of Orthopedics, the Second Clinical College of Jilin University, from March 2014 to June 2015 and were diagnosed and classified into 10 cases of stage II (5.6%), 54 cases of stage III (30.2%) and 115 cases (64.2%) of stage IV and alcohol-induced (71 cases (39.7%)), idiopathic (64 cases (34.0%)), and steroid-induced osteonecrosis (47 cases (26.3%)) subgroup, respectively. Our results showed that all models of logistical regression analysis, the co-dominants, dominants, and recessives of PPARγrs2920502, significantly associated with the increased risk of ONFH (p = 0.004, p = 0.013, p = 0.016), respectively. Both the minor homozygous CC genotype and the allele C of rs2920502 were evidently correlated with the enhanced risk of ONFH (p = 0.005, p = 0.0005),respectively. The recessives models of IGFBP3rs2132572 (G/A) as well as RUNX2 rs3763190(G/A) were statistically associated with the higher ONFH risk, p = 0.030, p = 0.029, respectively; the minor homozygous(AA) of IGFBP3rs2132572 (G/A) was also related to the increased risk of bilateral hips lesions, p = 0.039. Moreover, the ages on set of major homozygous(GG) and heterozygous(GT) of COL2A1rs2070739(G/A) were significantly younger than that of the minor homozygous(AA) of the SNP(p = 0.008) while the A-T-G-A haplotype of COL2A1 gene revealed significant association with the decreased the risk of bilateral hip lesions, p = 0.01, OR:0.258. More important, the serum HDL-c level and the ratio of LDL-c/HDL-c in the ONFH group were significantly decreased and increased compared with those of the control group (p = 0.02, p = 0.0001), respectively. Particularly, the CC genotype of PPARγ rs2920502 was statistically correlated with the enhanced serum TG level, p = 0.011.These results suggest that the variants of PPARγ, RUNX2, COL2A1, and IGFBP3 genes closely associated with the development of ONFH. [ABSTRACT FROM AUTHOR]

Cardiac fibrosis remains an unresolved problem in heart disease. Its etiology is directly caused by the activation and proliferation of cardiac fibroblasts (CFs). However, there is limited information regarding the biological role of cardiac fibroblasts in cardiac fibrosis. Herein, we screened out a gene, IGFBP3, whose expression significantly increased in TGF-β1-stimulated human primary CFs by mining RNA-Seq data for differential and WGCNA. We verified the IGFBP3's expression in transverse aortic constriction (TAC) surgery, isoproterenol (ISO)-induced cardiac fibrosis models, and TGFβ1-stimulated mouse primary CFs. We also found that the knockdown of IGFBP3 could inhibit the migration and proliferation ability of CFs. Furthermore, we found that aberrant N6-methyladenosine(m6A) mRNA modifications in the animal model and activated CFs may regulate the expression of IGFBP3 in developing cardiac fibrosis. Silencing METTL3 could downregulate the expression of IGFBP3 and inhibit the activation of CFs and the degree of cardiac fibrosis both in vitro and in vivo. Indeed, we also verified the expression of METTL3 and IGFBP3 in the atrial tissues of patients with atrial fibrillation (AF). Thus, METTL3 may regulate IGFBP3's expression and CFs activation via RNA epigenetic modifications, laying the foundation for a specific and novel therapeutic target in cardiac fibrosis. [Display omitted] [ABSTRACT FROM AUTHOR]

Objective Contradictory data between the Insulin-Like Growth Factor System (IGF) system and exercise may be due to alteration in IGF binding proteins. Vitamin D (D) deficiency has been related to muscle weakness and Insulin Like Growth Factor Binding Protein 3 (IGFBP3). A Vit. D and acute exercise merge is proposed to modify the IGF system. Design D insufficient and deficient men (39.0 ± 8.6 yo with serum D (25OH D) 20.0 ± 7.7 ng/mL) did 1 h of stretching (ST), aerobic (AB), and resistance (RT) exercises, before and after 28 d of 4000 IU/d Vit. D3 (D, n = 6) or Placebo (P, n = 7). ST, a time/attention control visit, interchanged unreceptive movements. AB was moderate intensity treadmill walking. RT rotated moderate strength 50% 1-RM repetitions (15, 10) of squat, bench press, leg press, and lat pull down. Serum Total IGF1 (TIGF1), Insulin Like Growth Factor Binding Protein 1 (IGFBP1), and IGFBP3 were measured before (T1, fasting), immediately after (T2), and 2 h post (T3) exercise. Results After ST, IGFBP3 was greater in the D group at T2 (2948, 2130 ng/mL; p < 0.03) and T3 (3087, 2212; p < 0.02). During RT, TIGF1 decreased in the Placebo (P) group from T1 to T3 (151.4, 107.3 ng/mL; p < 0.05), while IGFBP1 increased in the D group from T1 to T3 (26.5, 96.2 ng/mL; p < 0.05). RT IGFBP3 was greater at T1, T2, and T3 in the D group (2932.5, 2110.7; p < 0.03), (3163.9, 2392.5; p < 0.04), and (3355.3, 2353.1; p < 0.01). In AB, IGFBP3 was greater in the D group at T2 (3128.6, 2226.3.0; p < 0.04) and T3 (2949.7, 2135.1; p < 0.05). Conclusion D supplementation amplified IGFBP3 after low or moderate activity which may increase the delivery of IGF1 to tissues. Resistance exercise with D not only increased IGFBP3 and IGFBP1 levels but also conserved TIGF1 levels, possibly shifting the IGF system for enriched muscle well-being. [ABSTRACT FROM AUTHOR]

Background Previous studies have demonstrated the polymorphisms of insulinlike growth factor-1 (IGF-1) and its binding protein-3 (IGFBP3) genes could affect the circulating IGF-1 level. Moreover, the serum IGF-1 level was correlated with muscle size. Objectives This study aimed to explore the effect of polymorphisms of IGF1 , IGFBP3 , and IGFBP5 genes on appendicular skeletal muscle mass in Taiwanese older adults in a metropolitan area. Design A community-based cross-sectional study. Setting and subjects A random sample of 472 elders with complete information of dual energy X-ray absorptiometry examination, genotyping analysis, and serum IGF-1 level from Taichung Community Health Study for Elders (TCHS-E) was included. Measurements Low appendicular skeletal muscle mass index (ASMI) was defined as 2 SDs below the mean of young adults from our TCHS study (n = 471). Seven polymorphisms of IGF1, IGFBP3 , and IGFBP5 were analyzed by using Illumina GoldenGate Genotyping Assay. The χ 2 test, Student t test, and multiple logistic regression were applied for statistical analysis. Results The prevalence of low ASMI was 7.1%, 8.8%, and 23.0% in those aged 70 or younger, 71 to 75, and older than 75 years, respectively. We found that serum IGF-1 level (natural logarithmic transformation) was significantly lower in the low ASMI group compared with the normal ASMI group and the SNP rs2854744 near IGFBP3 gene was significantly associated with low ASMI. Moreover, we discovered the SNP rs6214 on the IGF1 gene would significantly affect the serum IGF-1 level. Therefore, the joint effect of rs6214 and rs2854744 was analyzed. Elders with GG genotype of rs6214 and AC or CC genotypes of rs2854744 had a 3.18-fold (95% CI 1.02–9.89) risk of having low ASMI compared with those with the AA and AA genotype, after adjusting for age, gender, smoking, exercise, hyperlipidemia, and albumin level. Conclusions Our results suggest that rs6214 on the IGF1 gene and rs2854744 near the IGFBP3 gene potentially play an important role with ASMI in Taiwanese older adults in a metropolitan area. [ABSTRACT FROM AUTHOR]

Background IGF1 is responsible for regulation of growth, metabolism and differentiation of human cells. IGFBP3 is the most abundant of the carrier proteins for IGF1 in the blood. IGF1/IGFBP3 molar ratio is an indicator of IGF1 bioavailability. We decided to create a file of reference ranges of IGF1, IGFBP3 and IGF1/IGFBPP3 ratio for the adult Czech population across the age spectrum. Methods We selected a group of 1022 subjects, 467 males and 555 females (ages 20–98 years), from several regions in the Czech Republic. The group consisted of blood donors and patients undergoing regular preventive examinations. Serum levels of IGF1 and IGFBP3 were measured using the following radioimmunoassay kits: IRMA IGF1 (Immunotech, Marseille, France) and IRMA IGFBP3 (Immunotech, Prague, Czech Republic). The IGF1/IGFBP3 ratio was also calculated. The following groups of patients were excluded: patients with diabetes, high blood glucose, high insulin levels, post-surgery patients, polymorbid patients, and subjects with oncological diseases. Subjects were divided into seven age-groups. Changes in the levels of observed analytes in each decade across the age spectrum were evaluated. All statistical analyses were performed by SAS 9.3 (Statistical Analysis Software release 9.3; SAS Institute Inc., Cary, NC, USA). Results All three parameters IGF1, IGFBP3 and IGF1/IGFBP3 decreased in parallel with decrease in age: p < 0.0001, r = − 0.64, − 0.35 and − 0.54, respectively. The dynamics of the decline was different between males and females. Linear regression models with age as independent variable fitted by gender are displayed in Fig. 1. Non-parametric reference interval curves (medians and 2.5th–97.5th percentiles) for IGF1, IGFBP3 and IGF1/IGFBP3 ratio as function of age by gender are displayed in Fig. 2(a,b,c). All medians and 2.5th–97.5th percentiles were plotted by cubic spline. For males, linear regression models were as follows: IGF1 = 291.34619 − 2.41211 × age, IGFBP3 = 2931.62778 − 6.11659 × age, IGF1/IGFBP3 = 0.02897 − 0.00021213 × age. For females, we plotted the following: IGF1 = 241.67406 − 1.98466 × age, IGFBP3 = 3688.60561 − 16.39560 × age, IGF1/IGFBP3 = 0.02029 − 0.00013233 × age. IGF1 was statistically significantly higher in males with p < 0.0001 (Wilcoxon test) but decreased faster ( p = 0.0121). IGFBP3 was statistically significantly higher in females with p = 0.0004 (Wilcoxon test) but decreased faster ( p < 0.0001). IGF1/IGFBP3 was statistically significantly higher in males with p < 0.0001 (Wilcoxon test) but decreased faster ( p < 0.0001). Conclusion Authors recommend using of a linear regression model based reference ranges for IGF1, IGFBP3 and IGF1/IGFBP3 ratio and using different reference ranges for genders. [ABSTRACT FROM AUTHOR]

Background Insulin-like growth factor binding protein 3 (IGFBP3) plays an important role in cellular proliferation, differentiation, apoptosis, and mammary carcinogenesis. Genetic variations in IGFBP3 have been identified to influence the circulating IGFBP3 level. The present study determined the impact of an important promoter polymorphism (A-202C, rs2854744) on susceptibility and progression of breast cancer in a Chinese population. Methods We genotyped the IGFBP3 A-202C polymorphism in a case-control study involving 465 breast cancer patients and 799 age-matched, cancer-free controls using the TaqMan method. Logistic regression was used to assess the genetic association between the occurrence and progression of breast cancer. Results Compared with the wild genotype (-202AA), we found a statistically significant increased risk of breast cancer associated with the variant genotypes (CC vs. AA: OR = 2.00, 95% CI = 1.25–3.21; AC + CC vs. AA: OR = 1.34, 95% CI = 1.06–1.70). In the stratified analysis, the increased risk was more apparent among the subgroups of older subjects (OR = 1.70, 95% CI = 1.20–2.42). Furthermore, we found that patients carrying variant genotypes (AC + CC) had a significantly greater prevalence of large tumor size (OR = 1.72, 95% CI = 1.13–2.64; P = 0.021). Conclusion Our results suggest that the functional IGFBP3 A-202C polymorphism may influence the susceptibility and progression of breast cancer in the Chinese population. [ABSTRACT FROM AUTHOR]

Objective: Studies have reported elevated serum insulin-like growth factor (IGF)-1 levels followed by ω-3 supplementation in various groups. Considering decreased level of IGF1 in patients with cardiovascular disease (CVD) and protective effects of IGF1 against CVD progression and myocar-dial infarctions mortality, this study was performed with the aim of determining effects of ω-3 supplementation on serum levels and gene expression of IGF1 and IGF binding protein-3 (IGFBP3) in men with CVD. Methods: Sixty-two middle-aged (55.9 ± 6.5 y) non-obese men with CVD followed the study protocol in two groups of ω-3 (n = 31 ) or placebo (n = 31 ) supplementation. Participants took ω-3 supplement or placebo (edible paraffin) for 8 wk and were asked not to change their diet or physical activity plan. Anthropometric and lipid profile characteristics, serum IGF1, serum IGFBP3 and also IGF1 and IGFBP3 gene expression in peripheral blood mononuclear cells (PBMCs) were measured in all participants before and after the intervention. Statistical analyses were performed using SPSS software. Results: There were no significant differences between the two study groups in age and body mass index at baseline. The groups also had no difference in baseline serum low-density lipoprotein, high-density lipoprotein, very low-density lipoprotein, triacylglycerols, and IGF1. Compared with placebo, ω-3 supplementation increased serum IGF1 levels (P = 0.01 ), and decreased serum level of IGFBP3 (P = 0.02). There was a trending toward an increase in IGF1 expression and nonsignificant decrease in IGFBP3 expression. Conclusions: ω-3 supplementation in patients with CVD increases serum IGF1 levels and decreases serum IGFBP3. Further research is warranted to investigate the underlying mechanisms. [ABSTRACT FROM AUTHOR]

The signaling axis consisting of GH-IGF1-IGFBP3 is the primary signal taht acts prepubertally to influence height development. Growth plate thinning and even premature closure have been reported in children with tumors treated with retinoid chemotherapy, resulting in long bone dysplasia. Growth failure may occur despite received GH treatment, but the reason is unknown. This study investigate the effect of high-dose all-trans retinoic acid (ATRA) on the development of long bones in growing SD rats. A total of 20 three-week-old male SD rats were randomly divided into a control group and an experimental group (n = 10). Rats were treated by gavage with or without high-dose ATRA for 10 days. The body weights of the rats were recorded daily. At the end of the experiment, we measured the length of nose-tail and tibia, stained the tibia and liver for pathological tissue and RT-PCR reaction, and measured the levels of serum GH, IGF1 and IGFBP3, and so on. Compared with controls, experimental rats exhibited reduced body weight and shortened nasal-tail and radial tibial length. Cyp26b1 enzyme activity in the liver was elevated, and histopathological staining revealed that the cartilaginous epiphyseal plate was narrowed, the medullary cavity of trabecular bone was sparse, the number of trabecular bones was decreased, trabecular separation was increased, bone marrow mineralization was enhanced, osteoclastic activity was increased, and circulating GH-IGF1-IGFBP3 levels were decreased. However, RT-PCR reaction results of localized proximal tibiae showed upregulation of IGF1 and downregulation of IGFBP3. High-dose ATRA intake over a short period of time can reduce GH-IGF1-IGFBP3 levels, affect cartilage and bone homeostasis, and inhibit bone growth in developing animals. • High-dose ATRA affects cartilage and bone homeostasis and inhibits longitudinal bone growth. • High-dose ATRA decreases the level of circulating GH-IGF1-IGFBP3 in rats. • High-dose ATRA may induce overexpression of the hypoxia-related gene IGFBP3 in bone marrow and aggravate bone injury. [ABSTRACT FROM AUTHOR]

To investigate the effects of lead exposure and IGF1R inhibitor AG1024 on the expression of IGF1R and IGFBP3 in PC12 cells. It is clear that the mechanism of the related proteins inducing AD is regulated by them, thus providing theoretical guidance for the prevention and treatment of lead poisoning. This study is mainly used PC12 neuron cell to cultivate and establish a corresponding lead exposure model, deal with cells with different concentrations of lead acetate respectively, divide the experiment into control group, 1 μmoL/L PbAc, 10 μmoL/L PbAc group, IGF1R inhibitor (AG1024) group, IGF1R inhibitor group (AG1024) + 1 μmoL/L PbAc group, IGF1R inhibitor group (AG1024) + 10 μmoL/L PbAc group, respective contamination's three periods of time 24 h, 48 h, and 72 h. Lead exposure dose on cell proliferation was examined by MTT. The protein expression of IGF1R and IGFBP3 in PC12 cells were tested by western blotting and immunohistochemistry, The expression of Aβ40 and Aβ42 in cell supernatant was determined by ELISA. Compared with the control group, the proliferation of the cells in the high-dose lead-exposed group was significantly inhibited (P < 0.05), and the expression of IGF1R and IGFBP3 was significantly decreased (P < 0.05); the contents of Aβ40 and Aβ42 were not statistically significant among the groups (P > 0.05). This study shows that lead can obviously down-regulate the expression of IGF1R and IGFBP3, lead and inhibitor can inhibit the proliferation of cells, promote the tendency of apoptosis, and damage the nervous system. [ABSTRACT FROM AUTHOR]

Abstract: Systemic sclerosis (SSc) or scleroderma is a rare, autoimmune, multi-factorial disease characterized by early microvascular alterations, inflammation, and fibrosis. Chickens from the UCD-200 line develop a hereditary SSc-like disease, showing all the hallmarks of the human disorder, which makes this line a promising model to study genetic factors underlying the disease. A backcross was generated between UCD-200 chickens and its wild ancestor – the red jungle fowl and a genome-scan was performed to identify loci affecting early (21days of age) and late (175days of age) ischemic lesions of the comb. A significant difference in frequency of disease was observed between sexes in the BC population, where the homogametic males were more affected than females, and there was evidence for a protective W chromosome effect. Three suggestive disease predisposing loci were mapped to chromosomes 2, 12 and 14. Three orthologues of genes implicated in human SSc are located in the QTL region on chromosome 2, TGFRB1, EXOC2-IRF4 and COL1A2, as well as CCR8, which is more generally related to immune function. IGFBP3 is also located within the QTL on chromosome 2 and earlier studies have showed increased IGFBP3 serum levels in SSc patients. To our knowledge, this study is the first to reveal a potential genetic association between IGFBP3 and SSc. Another gene with an immunological function, SOCS1, is located in the QTL region on chromosome 14. These results illustrate the usefulness of the UCD-200 chicken as a model of human SSc and motivate further in-depth functional studies of the implicated candidate genes. [Copyright &y& Elsevier]

Abstract: Objective: Approximately 6% of newborns at term are small for gestational age (SGA) and present a birth weight and/or length less than −2SD from the mean. SGA infants are at increased risk for perinatal morbidity, associated psychological and/or mental problems, persistent short stature (about 15% of subjects) and metabolic alterations. Insulin-like growth factors (IGFs), their common receptor (IGF1R) and their binding proteins (IGFBPs) play a critical role in fetal and postnatal growth. In these genes common polymorphisms, such as single nucleotide polymorphisms and variable number of tandem repeats, have been investigated with conflicting results with respect to SGA-related outcomes, and the functional role of these gene variants remains to be elucidated. Design: The study group consisted of 100 pre-pubertal short children born SGA and 94 healthy controls, matched for sex and age, recruited at the Department of Biomedicine of Development Age of the Bari University and at the Paediatric Department of the Messina Hospital. In the present study we analyzed the allelic frequency of the polymorphisms −795 G/A, −667 G/A, −396 C/T in the IGFBP3 in SGA children and their influence on the basal and insulin-stimulated transcriptional activity of the gene. Results: We found that the polymorphisms −667 G/A and −396 C/T in the IGFBP3 promoter region are capable of having an effect on the transcriptional activity of the gene, although with opposing effects. Interestingly, the −667 G/A polymorphism has a negative impact on the IGFBP3 transcription, while the −396 C/T polymorphism determines an increase of the transcriptional activity of the IGFBP3 gene promoter. Interestingly, we found that the −396 C/T polymorphism correlates with lower birth length in SGA children. Most importantly, while the diminished IGFBP3 transcriptional activity induced by the −667A polymorphism was significantly recovered after insulin administration (p-value<0.05), the increased transcriptional activity caused by the −396T polymorphism was not restored to baseline levels by insulin. Conclusions: Altogether our results demonstrated that the −667 G/A and the −396 C/T polymorphisms in IGFBP3 promoter region influence the basal transcriptional activity of the gene. [Copyright &y& Elsevier]

Abstract: Purpose: It has been suggested that hepatocyte growth factor (HGF) and insulin-like growth factor binding protein (IGFBP)-3 are associated with gefitinib resistance in non-small cell lung cancer (NSCLC). We investigated the predictive and prognostic roles of these proteins in NSCLC patients treated with gefitinib. Patients and methods: Of 106 patients enrolled in a randomized phase II study of gefitinib, 97 had plasma samples available for ELISA testing. Of these samples, seven and eight, respectively, had HGF and IGFBP-3 values that could not be measured. Therefore, the correlations between clinical outcomes and plasma levels of HGF and IGFBP-3 were evaluated in 90 and 89 patients, respectively. Results: Plasma HGF levels were significantly higher in older patients, male patients, patients with squamous cell carcinoma, current smokers, and patients with epidermal growth factor receptor (EGFR) wild-type tumors. Low HGF levels were significantly associated with higher response rate, and longer progression-free survival (PFS) and overall survival (OS) irrespective of EGFR mutation status. In a multivariate analysis, the presence of EGFR mutations (P =0.002) and low HGF levels (P =0.031) were independently predictive of longer PFS, and an ECOG PS of 0 (P =0.001) and low HGF levels (P =0.002) were independently predictive of longer OS. No statistically significant differences were found for IGFBP-3. Conclusion: High HGF levels are significantly associated with resistance to gefitinib and can be used as a predictive marker for the differential outcome of gefitinib treatment in NSCLC irrespective of EGFR mutation status. [Copyright &y& Elsevier]

Abstract: Context: IGF-I and IGFBP-3 play a central role in fetal and postnatal growth and levels are low in short SGA children. The -202 A/C and -185 C/T SNPs are located near elements involved in directing IGFBP3 promoter activity and expression. Changes in promoter CpG methylation status affect transcription factor binding and transcriptional activation of IGFBP3 in vitro. Objective: To assess the relationship between IGFBP3 promoter SNPs, IGFBP-3 levels, spontaneous growth and growth response to GH treatment in short prepubertal SGA children. To assess promoter methylation status in a subgroup of short SGA subjects and controls. Patients: 292 Short prepubertal SGA children, 39 short young SGA adults and 85 young adults with normal stature. Intervention: Short prepubertal SGA children received GH 1mg/m2/day. Outcome measures: Fasting levels of IGF-I and IGFBP-3, baseline and delta height SDS. Results: At baseline, IGFBP-3 levels were highest in SGA children with -202 AA genotype and lower in children with 1 or 2 copies of the C-allele (P <0.001). Children with C-202/C-185 haplotype, compared to children with A-202/C-185 haplotype, had lower IGFBP-3 levels (P =0.003) and were shorter (P =0.03). During GH treatment, children with C-202/C-185 haplotype showed a significantly greater increase in IGFBP-3 SDS and in height SDS than children with A-202/C-185 haplotype, resulting in similar IGFBP-3 levels and similar height SDS after 12 months of GH treatment. CpG methylation patterns showed a trend towards more methylation of CpGs involved in transcription factor binding in short young SGA adults compared to controls. Conclusion: Polymorphic variation in the IGFBP3 promoter region is correlated with IGFBP-3 levels, spontaneous growth and response to GH treatment in short SGA children. [Copyright &y& Elsevier]

Insulin-like growth factor binding protein 3 (IGFBP3), which is induced by wild-type p53, regulates IGF and interacts with the TGF-β pathway. IGFBP3 promoter methylation may occur in colorectal cancer with or without the CpG island methylator phenotype (CIMP), which is associated with microsatellite instability (MSI) and TGFBR2 mutation. We examined the relationship between IGFBP3 methylation, p53 expression, CIMP and MSI in 902 population-based colorectal cancers. Utilizing real-time PCR (MethyLight), we quantified promoter methylation in IGFBP3 and eight other CIMP-high--specific promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1). IGFBP3 methylation was far more frequent in non--MSI-high CIMP-high tumors (85% = 35/41) than in MSI-high CIMP-high (49% = 44/90, P < .0001), MSI-high non--CIMP-high (17% = 6/36, P < .0001), and non--MSI-high non--CIMP-high tumors (22% = 152/680, P < .0001). Among CIMP-high tumors, the inverse relationship between MSI and IGFBP3 methylation persisted in p53-negative tumors (P < .0001), but not in p53-positive tumors. IGFBP3 methylation was associated inversely with TGFBR2 mutation in MSI-high non--CIMP-high tumors (P = .02). In conclusion, IGFBP3 methylation is inversely associated with MSI in CIMP-high colorectal cancers, and this relationship is limited to p53-negative tumors. Our data suggest complex relationship between global genomic/epigenomic phenomena (such as MSI/ CIMP), single molecular events (e.g., IGFBP3 methylation, TP53 mutation, and TGFBR2 mutation), and the related pathways. [ABSTRACT FROM AUTHOR]

Abstract: Pulmonary fibrosis is a common response to a variety of lung injuries, characterized by fibroblast/myofibroblast expansion and abnormal accumulation of extracellular matrix. An increased expression of matrix metalloprotease 9 (MMP9) in human and experimental lung fibrosis has been documented, but its role in the fibrotic response is unclear. We studied the effect of MMP9 overexpression in bleomycin-driven lung fibrosis using transgenic mice expressing human MMP9 in alveolar macrophages (hMMP9-TG). At 8 weeks post-bleomycin, the extent of fibrotic lesions and OH-proline content were significantly decreased in the TG mice compared to the WT mice. The decreased fibrosis in hMMP9-TG mice was preceded by a significant reduction of neutrophils and lymphocytes in bronchoalveolar lavage (BAL) at 1 and 4 weeks post-bleomycin, respectively, as well as by significantly less TIMP-1 than the WT mice. From a variety of cytokines/chemokines investigated, we found that BAL levels of insulin-like growth factor binding protein-3 (IGFBP3) as well as the immunoreactive protein in the lungs were significantly lower in hMMP9-TG mice compared with WT mice despite similar levels of gene expression. Using IGFBP-3 substrate zymography we found that BAL from TG mice at 1 week after bleomycin cleaved IGFBP-3. Further, we demonstrated that MMP9 degraded IGFBP-3 into lower molecular mass fragments. These findings suggest that increased activity of MMP9 secreted by alveolar macrophages in the lung microenvironment may have an antifibrotic effect and provide a potential mechanism involving IGFBP3 degradation. [Copyright &y& Elsevier]

Abstract: Background: The insulin-like growth factor1 (IGF1) pathway plays a significant role in both the normal and malignant cell growth. IGF1 binds to the IGF1 receptor and starts a signaling cascade that regulates cell proliferation, differentiation, and apoptosis. The bioavailability of IGF1 is regulated by the binding protein IGFBP3. A CA repeat polymorphism, in the IGF1 gene, which is located 969bp upstream from the transcription start site and the rs2854744 and rs2854746 single nucleotide polymorphisms (SNPs) in the IGFBP3 gene have been associated with the serum levels of the respective proteins and colorectal cancer (CRC), but the results are inconsistent. We wanted to study if these polymorphisms or any haplotypes of the IGF1 and the IGFBP3 genes are associated with CRC. Methods: High linkage disequilibrium was seen in these gene regions. Therefore, the CA repeat along with two SNPs in the IGF1 gene, and rs2854744 (A-202C), rs2854746 (Ala32Gly) and two additional SNPs in the IGFBP3 gene were selected to cover the whole gene regions. A case–control study was carried out using 661 German CRC cases and 607 matched controls. Results: We did not find any association between the CA repeat length or any of the SNPs in the IGF1 and the IGFBP3 genes and the risk of CRC. Nor did the haplotypes of these genes affect the risk of CRC. Conclusion: Our study suggests no major role of the assessed genetic variation within the IGF1 and the IGFBP3 genes in CRC risk. [Copyright &y& Elsevier]

Background/Aims: Insulin-like growth factor I (IGF-I) is an anabolic hormone synthesized in the liver whose levels decrease sharply in liver cirrhosis. Methods: We conducted a randomized double-blind placebo-controlled clinical trial to evaluate the effect of subcutaneous administration of IGF-I (20μg/kg/day with dose escalation to 50–100μg/kg/day) for 4 months in patients with alcoholic or primary biliary cirrhosis (PBC) and subnormal IGF-I levels. Eight alcoholics and one PBC entered the placebo group and seven alcoholics and two PBC the treatment group. Biochemistry, body composition, muscle mass and strength, and resting energy expenditure (REE) were evaluated. Results: Total serum IGF-I and IGF-I/IGFBP-3 ratio (a surrogate marker of IGF-I biovailability) increased in the treatment group but IGF-I values still remained below normal limits in the treated patients. No differences were observed in body composition, muscle strength or muscle mass between groups. However, IGF-I therapy increased significantly serum albumin (P=0.038) and this improvement correlated positively with variation of IGF-I/IGFBP-3 ratio. IGF-I treatment also tended to increase REE (P=0.085); this difference was significant (P=0.049) in the subgroup of alcoholic patients. Conclusions: A short course of IGF-I increased albumin levels and tended to improve energy metabolism in liver cirrhosis. These findings warrant larger clinical trials to assess the clinical benefit of IGF-I in cirrhotic patients. [Copyright &y& Elsevier]

Background/Aims: Hepatocellular carcinoma (HCC) usually develops following chronic liver inflammation caused by hepatitis C or B virus. Through expression profiling in a rare type of HCC, for which the causes are unknown, we sought to find key genes responsible for each step of hepatocarcinogenesis in the absence of viral influence.Methods: We used 68 non-B, non-C liver tissues (20 HCC, 17 non-tumor, 31 normal liver) for expression profiling with PCR-array carrying 3072 genes known to be expressed in liver tissues. To select the differentially expressed genes, we performed random permutation testing. A weighted voting classification algorithm was used to confirm the reliability of gene selection. We then compared these genes with the results of previous expression profiling studies.Results: A total of 220 differentially expressed genes were selected by random permutation tests. The classification accuracies using these genes were 91.8, 92.0 and 100.0% by a leave-one-out cross-validation, an additional PCR-array dataset and a Stanford DNA microarray dataset, respectively. By comparing our results with previous reports on virus-infected HCC, four genes (ALB, A2M, ECHS1 and IGFBP3) were commonly selected in some studies.Conclusions: The 220 differentially expressed genes selected by PCR-array are potentially responsible for hepatocarcinogenesis in the absence of viral influence. [Copyright &y& Elsevier]

• Calcitriol with TGF-βs show higher inhibition of cell growth than each compound. • Combinatory treatment impairs cell growth without affecting apoptosis. • IGFBP3 and CDKNA1 were up regulated by combinatory treatment. • Calcitriol with TGF-β1 reprogram prostate cancer signaling. • RNA signatures confirm the regulation over cell proliferation and cell cycle. Calcitriol and transforming growth factors beta (TGF-β) are involved in several biological pathways such as cell proliferation, differentiation, migration and invasion. Their cellular effects could be similar or opposite depending on the genetic target, cell type and context. Despite the reported association of calcitriol deficiency and disruption of the TGF-β pathway in prostate cancer and the well-known independent effects of calcitriol and TGF-βs on cancer cells, there is limited information regarding the cellular effects of calcitriol and TGF-β in combination. In this study, we in vitro analyze the combinatory effects of calcitriol and TGF-β on cell growth and apoptosis using PC-3 and DU145 human prostate cancer cell lines. Using high-throughput microarray profiling of PC-3 cells upon independent and combinatory treatments, we identified distinct transcriptional landscapes of each intervention, with a higher effect established by the combinatorial treatment, following by TGF-β1 and later by calcitriol. A set of genes and enriched pathways converge among the treatments, mainly between the combinatory scheme and TGF-β1, but the majority were treatment-specific. Of note, CYP24A1, IGFBP3, CDKN1A, NOX4 and UBE2D3 were significantly up-regulated upon the combinatorial treatment whereas CCNA1, members of the CT45A and APOBEC3 family were down-regulated. By public RNA signatures, we were able to confirm the regulation by the co-treatment over cell proliferation and cell cycle. We finally investigated the possible clinical impact of genes modulated by the combinatorial treatment using benchmark prostate cancer data. This comprehensive analysis reveals that the combinatory treatment impairs cell growth without affecting apoptosis and their combinatory actions might synergize and improved their individual effects to reprogram prostate cancer signaling. [ABSTRACT FROM AUTHOR]

Frankincense (Boswellia sacra Fluck.,) is a widely used herbal drug and household medicine for treatment of several diseases. Earlier toxicological studies revealed its proulcerogenic effect and no significant hepatotoxic or nephrotoxic effects in rats. However, some other members of Boswellia species such as Boswellia papyrifera (Caill. ex Delile) Hochst and Boswellia carterii have been reported for testicular toxicity in rats. Testicular toxicity of standardized methanolic extract of B. sacra oleo gum resin was determined through repeated oral dose administration for 28 days. Biochemical, histological and genetic changes in rat testes were evaluated. B. sacra extract was analyzed for its boswellic acid content by high performance liquid chromatography (HPLC) method. The extract was administered at three different doses to rats. Effect on behavior, weight, food and water consumption along with changes in serum testosterone levels and cytoarchitecture of testis, epididymis and adrenal gland were determined. Gene expression of GSTPi , IGFBP3 and HSP70 in testis was also studied. Boswellic acids (α and β) were present in highest concentration whereas acetyl-11-keto beta boswellic acid was present in relatively smaller amounts. The extract did not produce any significant change in the behavior of the animals, food/water consumption or weight gain. Serum testosterone levels were significantly decreased only by highest tested dose of Boswellia extract (1000 mg/kg, p.o). Histological examination did not reveal any variation in the structure of testis, adrenal gland and epididymis after administration of the extract while the expression of all three studied genes was significantly decreased. The results indicate that B. sacra extract does not possess any toxic effect on testis. On the contrary, decrease in gene expression of GSTPi , IGFBP3 and HSP70 revealed its antioxidant potential that may protect testes against effect of toxicants. However, a significant reduction in serum testosterone levels point to mechanisms other than direct testicular toxicity. Image 1 [ABSTRACT FROM AUTHOR]

IGFBPs play a pivotal role in regulating the physiological function of IGFs (insulin-like growth factors). As an important member of IGFBPs, IGFBP3 is involved in the regulation of physiological functions of IGFs. To investigate the functional role of Igfbp3 in a herbivorous fish species, grass carp igfbp3 (GenBank accession no. MN251843) was isolated from the liver by molecular cloning. The ORF of grass carp igfbp3 was 882 bp, which encoded 293 amino acids, and the first 22 amino acids comprised the signal peptide. The predicted molecular weight of grass carp Igfbp3 is 31.95 kDa, and the theoretical isoelectric point is 8.32. Grass carp Igfbp3 displayed a high identity with its counterparts of common carp and zebrafish. And phylogenetic tree analysis showed that the grass carp Igfbp3 was clustered into the Igfbp3 subgroups of common carp and zebrafish. Tissue distribution study showed that igfbp3 was ubiquitously expressed in all tissues examined in the present study. High expression level of igfbp3 was detected in the heart, brain and fat of grass carp. The OGTT demonstrated that igfbp3 mRNA expression was significantly elevated in the liver of grass carp in response to glucose treatment. In vitro study showed that insulin could markedly stimulated igfbp3 mRNA expression in primary grass carp hepatocytes. Moreover, igfbp3 mRNA levels were also regulated by glucagon in grass carp primary hepatocytes. These results may provide the theoretical foundation for investigating the role of Igfbp3 in fish metabolism. Unlabelled Image • IGFBP3 plays a key role in energy homeostasis and glucose metabolism. • igfbp3 was identified and highly expressed in heart, fat and brain of grass carp. • igfbp3 expression was regulated in grass carp liver by OGTT. • igfbp3 mRNA was regulated by insulin or glucagon in primary grass carp hepatocytes. • Igfbp3 was involved in energy/glucose metabolism in grass carp. [ABSTRACT FROM AUTHOR]