IGF-I Receptor mRNA Research Articles

P41 Exploring the biological role of insulin and insulin-like growth factor-1 hybrid receptors in the endothelium

Endothelial cells co-express insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R) homodimers. Furthermore, association of insulin αβ half receptors and IGF-1 αβ half receptors also occur resulting in insulin/IGF-1 hybrid receptors. An abundance of hybrid receptors has been associated with reduced insulin sensitivity in metabolic tissue. The aim of this project is to study the differential expression of vascular derived insulin receptors, IGF-1 receptors and insulin/IGF-1 hybrids during insulin resistance in a diet-induced model of obesity (DIO). C57BL/6 mice were fed on high-fat or low-fat diets ad libitum for 16-weeks. Obese mice showed significant metabolic abnormalities indicative of insulin resistance when compared with lean littermates. A significant increase in hybrid receptor levels were observed in aorta from obese mice (immunoprecipitation and Western blotting). Total IR and IGF-1R protein levels were reduced in obese mice (Western blotting) however, no change was observed in IR and IGF-1R mRNA levels (real time-PCR). Insulin-stimulated (4.5 nmol/kg injected) in vivo serine phosphorylation of Akt was significantly blunted in aortae of obese mice, whereas IGF-1-stimulated (90 nmol/kg injected) remained unaffected. We have shown that IR and IGF1R homodimers are preferentially titrated into hybrid receptors in a model of DIO and this is accompanied with a reduction in insulin sensitivity but not IGF-1 sensitivity in the aorta. Hybrid receptors may provide a novel target for the prevention of CVD progression in vulnerable patients.

MicroRNA-214 Reduces Insulin-like Growth Factor-1 (IGF-1) Receptor Expression and Downstream mTORC1 Signaling in Renal Carcinoma Cells

Elevated IGF-1/insulin-like growth factor-1 receptor (IGF-1R) autocrine/paracrine signaling in patients with renal cell carcinoma is associated with poor prognosis of the disease independent of their von Hippel-Lindau (VHL) status. Increased expression of IGF-1R in renal cancer cells correlates with their potency of tumor development and progression. The mechanism by which expression of IGF-1R is increased in renal carcinoma is not known. We report that VHL-deficient and VHL-positive renal cancer cells possess significantly decreased levels of mature, pre-, and pri-miR-214 than normal proximal tubular epithelial cells. We identified an miR-214 recognition element in the 3'UTR of IGF-1R mRNA and confirmed its responsiveness to miR-214. Overexpression of miR-214 decreased the IGF-1R protein levels, resulting in the inhibition of Akt kinase activity in both types of renal cancer cells. IGF-1 provoked phosphorylation and inactivation of PRAS40 in an Akt-dependent manner, leading to the activation of mTORC1 signal transduction to increase phosphorylation of S6 kinase and 4EBP-1. Phosphorylation-deficient mutants of PRAS40 and 4EBP-1 significantly inhibited IGF-1R-driven proliferation of renal cancer cells. Expression of miR-214 suppressed IGF-1R-induced phosphorylation of PRAS40, S6 kinase, and 4EBP-1, indicating inhibition of mTORC1 activity. Finally, miR-214 significantly blocked IGF-1R-forced renal cancer cell proliferation, which was reversed by expression of 3'UTR-less IGF-1R and constitutively active mTORC1. Together, our results identify a reciprocal regulation of IGF-1R levels and miR-214 expression in renal cancer cells independent of VHL status. Our data provide evidence for a novel mechanism for IGF-1R-driven renal cancer cell proliferation involving miR-214 and mTORC1.

MiR-139 is up-regulated in osteoarthritis and inhibits chondrocyte proliferation and migration possibly via suppressing EIF4G2 and IGF1R

Osteoarthritis (OA) is one of the most progressive articular cartilage erosions. microRNAs (miRNAs) play pivotal roles in OA modulation, but the role of miR-139 in OA remains elusive. This study aims to reveal the effects and possible mechanism of miR-139 in OA and chondrocytes. The levels of miR-139 and its possible targets eukaryotic translation initiation factor 4 gamma 2 (EIF4G2) and insulin-like growth factor 1 receptor (IGF1R) were detected by qRT-PCR in the articular cartilages of 20 OA patients and 20 non-OA patients. Human chondrocyte CHON-001 cells were transfected with miR-139 mimic or inhibitor, as well as the siRNAs of EIF4G2 and IGF1R. Cell viability by MTT assay, proliferation by colony formation assay and migration by Transwell assay were performed. Results showed that miR-139 was up-regulated, while EIF4G2 and IGF1R mRNAs down-regulated in OA cartilages (P < 0.001), and negative correlations existed between the level of miR-139 and EIF4G2 or IGF1R. Overexpression of miR-139 in CHON-001 cells suppressed both mRNA and protein levels of EIF4G2 and IGF1R, and inhibited cell viability, colony formation number and cell migration, while miR-139 inhibitor induced the opposite effects. Knockdown of EIF4G2 or IGF1R in CHON-001 cells reversed the effects of miR-139 inhibitor on cell viability, colony formation and cell migration. These results indicate that miR-139 is capable of inhibiting chondrocyte proliferation and migration, thus being a possible therapeutic target for OA. The mechanism of miR-139 in chondrocytes may be related to its regulation on EIF4G2 and IGF1R.

Differential Expression of IR-A, IR-B and IGF-1R in Endometrial Physiology and Distinct Signature in Adenocarcinoma.

Type 2 diabetes and obesity are risk factors for endometrial hyperplasia and cancer, suggesting that hyperinsulinemia contributes to pathogenesis. Insulin action through insulin receptor (IR) splice variants IR-A and IR-B regulates cellular mitogenesis and metabolism, respectively. We hypothesized that IR-A and IR-B are differentially regulated in normal endometrium, according to mitogenic and metabolic requirements through the menstrual cycle, as well as in endometrial hyperplasia and cancer. IR-A, IR-B, and IGF-1 receptor (IGF-1R) mRNA was quantified in endometrium, endometrial epithelial and stromal cells, and in vitro after hormone stimulation. Academic center. Endometrium was collected from women with regular cycles (n = 71), complex hyperplasia (n = 5), or endometrioid adenocarcinoma (n = 11). In vitro sex-steroid treatment. IR-A and IR-B expression Results: IR-A increased dramatically during the early proliferative phase, 20-fold more than IR-B. In early secretory phase, IR-B and IGF-1R expression increased, reaching maximal expression, whereas IR-A decreased. In adenocarcinoma, IR-B and IGF-1R expression was 5- to 6-fold higher than normal endometrium, whereas IR-A expression was similar to IR-B. Receptor expression was unrelated to body mass index. IR-A was elevated during the normal proliferative phase, and in endometrial hyperplasia and adenocarcinoma. The dramatic early rise of IR-A in normal endometrium indicates IR-A is the predominant isoform responsible for initial estrogen-independent endometrial proliferation as well as that of cancer. IR-B is elevated during the normal secretory phase when glucose uptake and glycogen synthesis support embryo development. Differing from other cancers, IR-B expression equals mitogenic IR-A in endometrial adenocarcinoma. Differential IR isoform expression suggests a distinct role for each in endometrial physiology and cancer.

Prepubertal tamoxifen treatment affects development of heifer reproductive tissues and related signaling pathways

Prepubertal exposure of the developing ovaries and reproductive tract (RT) to estrogen or xenoestrogens can have acute and long-term consequences that compromise the reproductive performance of cattle. This research examined effects of the selective estrogen receptor modulator tamoxifen (TAM) on gene and protein abundance in prepubertal ovaries and RT, with a particular focus on signaling pathways that affect morphology. Tamoxifen was administered to Holstein heifer calves (n=8) daily (0.3mg/kg subcutaneously) from 28 to 120 d of age, when tissues were collected. Control calves (n=7) received an equal volume of excipient. Weight, gross measurements, and samples of reproductive tissues were collected, and protein and mRNA were extracted from snap-frozen samples of vagina, cervix, uterus, oviduct, ovary, and liver. Neither estradiol nor insulin-like growth factor I (IGFI) concentrations in the serum were affected by TAM treatment. Tamoxifen treatment reduced ovarian weight independently from effects on antral follicle populations, as there was no difference in visible antral follicle numbers on the day of collection. Estrogen receptor α (ESR1) and β (ESR2) mRNA, ESR1 protein, IGFI, progesterone receptor, total growth hormone receptor, WNT4, WNT5A, and WNT7A mRNA, in addition to mitogen-activated protein kinase (MAPK) and phosphorylated MAPK proteins were affected differently depending on the tissue examined. However, neither IGFI receptor mRNA nor protein abundance were affected by TAM treatment. Results indicate that reproductive development in prepubertal Holstein heifer calves is TAM-sensitive, and that bovine RT and ovarian development are supported, in part, by estrogen receptor-dependent mechanisms during the period studied here. Potential long-term consequences of such developmental disruption remain to be defined.

MiR-223 reverses the resistance of EGFR-TKIs through IGF1R/PI3K/Akt signaling pathway.

Acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, is a critical issue for the treatment of EGFR mutant-positive non-small cell lung cancer (NSCLC). Recent evidence supports the role of microRNA-223 (miR-223) in modulating chemotherapeutic drug sensitivity, but its role in the resistance to EGFR-TKIs in NSCLC remains unclear. To this end, we investigated the involvement of miR-223 in erlotinib resistance, using two pairs of TKI-sensitive or resistant cell lines, PC9 vs PC9/ER, and HCC827 vs HCC827/ER, as well as PC9/CD133+, which are lung cancer stem-like cells derived from PC9 cells. Downregulation of miR-223 expression in PC9/ER and PC9/CD133+ cells was detected, and the reverse correlation of miR-233 and insulin-like growth factor 1 receptor (IGF1R) in these cells was also revealed. Next, levels of IGF1R mRNA and p-Akt were significantly reduced in miR-223 stably transfected PC9/ER and PC9/CD133+ cells. However, the sensitivity of PC9/ER and PC9/CD133+ cells to erlotinib was partially restored, after overexpression of miR-223 in those cells. Similar results were also observed in vivo. Furthermore, miR-223-mediated inhibition of the IGF1R/PI3K/Akt signaling pathway may have been reversed by the agonist of IGF1R in miR-223 transfected cells. Our findings indicated that downregulation of miR-223, which can induce activation of the IGF1R/phosphatidylinositol 3-kinase (PI3K)/Akt pathway in PC9/ER and PC9/CD133+ cells, may be responsible for the resistance of PC9/ER and PC9/CD133+ cells to erlotinib, suggesting that miR-223 is a potential therapeutic target for overcoming EGFR-TKIs resistance.

Development of a Quantitative PCR Assay for Detection of Human Insulin-Like Growth Factor Receptor and Insulin Receptor Isoforms.

The biological activity of insulin and the insulin-like growth factor (IGF) ligands, IGF-I and IGF-II, is based in part on the relative abundance and distribution of their target receptors: the insulin receptor (IR) splice variants A (IR-A) and B (IR-B) and IGF 1 receptor (IGF-1R). However, the relative quantity of all three receptors in human tissues has never been measured together on the same scale. Due to the high homology between insulin receptor (IR)-A and IR-B proteins and lack of antibodies that discern the two IR splice variants, their mRNA sequence is the most reliable means of distinguishing between the receptors. Hence, highly specific primers for IR-A, IR-B, and IGF-1R mRNA were designed to accurately detect all three receptors by quantitative RT-PCR and enable direct quantification of relative receptor expression levels. A standard concentration curve of cDNA from each receptor was performed. Assay specificity was tested using competition assays and postamplification analysis by gel electrophoresis and cloning. Forward and reverse primer concentrations were optimized to ensure equal efficiencies across primer pairs. This assay enables a specific molecular signature of IGF/insulin signaling receptors to be assayed in different tissues, cell types, or cancers.

Equine insulin receptor and insulin‐like growth factor‐1 receptor expression in digital lamellar tissue and insulin target tissues

Hyperinsulinaemia is implicated in the pathogenesis of endocrinopathic laminitis. Insulin can bind to different receptors: two insulin receptor isoforms (InsR-A and InsR-B), insulin-like growth factor-1 receptor (IGF-1R) and InsR/IGF-1R hybrid receptor (Hybrid). Currently, mRNA expression of these receptors in equine tissues and the influence of body type and dietary carbohydrate intake on expression of these receptors is not known. The study objectives were to characterise InsR-A, InsR-B, IGF-1R and Hybrid expression in lamellar tissue (LT) and insulin responsive tissues from horses and examine the effect of dietary nonstructural carbohydrate (NSC) on mRNA expression of these receptors in LT, skeletal muscle, liver and two adipose tissue (AT) depots of lean and obese ponies. In vivo experiment. Lamellar tissue samples were evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) for receptor mRNA expression (n=8) and immunoblotting for protein expression (n=3). Archived LT, skeletal muscle, liver and AT from lean and obese mixed-breed ponies fed either a low (~7% NSC as dry matter; 5 lean, 5 obese) or high NSC diet (~42% NSC as dry matter; 6 lean, 6 obese) for 7days were evaluated by RT-qPCR to determine the effect of body condition and diet on expression of the receptors in different tissues. Significance was set at P≤0.05. Lamellar tissue expresses both InsR isoforms, IGF-1R and Hybrid. LT IGF-1R gene expression was greater than either InsR isoform and InsR-A expression was greater than InsR-B (P≤0.05). Obesity significantly lowered IGF-1R, InsR-A and InsR-B mRNA expression in LT and InsR-A in tailhead AT. High NSC diet lowered expression of all three receptor types in liver; IGF-1R and InsR-A in LT and InsR-A in tailhead AT. Lamellar tissue expresses IGF-1R, InsR isoforms and Hybrids. The functional characteristics of these receptors and their role in endocrinopathic laminitis warrants further investigation.

Inhibition of lung adenocarcinoma cells by insulin-like growth factor-I receptor and Kirsten rat sarcoma mutations: A mutation analysis with antisense oligodeoxynucleotide.

BackgroundKirsten rat sarcoma (KRAS) mutations are widespread in lung adenocarcinoma patients. The combined utilization of KRAS antisense oligodeoxynucleotide (ASODN) and insulin-like growth factor-I receptor (IGF-IR) may inhibit the proliferation of A549 cell lines of lung adenocarcinoma.MethodsPoint mutations of the KRAS gene in A549 cells were detected by polymerase chain reaction with special sequence primers (PCR-SSP) and gene sequence analysis; ASODN was designed and synthesized according to the mutation specialty of KRAS; and the correlation of gene mutations and clinicopathological features were analyzed. Inhibition on the proliferation and morphostructure change were measured by methyl thiazolyl tetrazolium and colony-forming unit assays. Flow cytometry was used to evaluate the expression of KRAS and IGF-IR proteins and cell apoptosis and reverse transcriptase-polymerase chain reaction were used to detect the expression of KRAS and IGF-IR messenger ribonucleic acid (mRNA). Male nude mice were used to form the mice-human lung cancer model and show the inhibition of KRAS ASODN on A549 cells.ResultsPCR-SSP and gene sequence analysis results showed that the codon 12 of KRAS had changed from GGT to GTT. KRAS ASODN or IGF-IR ASODN could inhibit cell proliferation and promote apoptosis of A549 cells. However, the combined utilization of KRAS ASODN and IGF-IR ASODN could inhibit cell proliferation and promote apoptosis more powerfully than exclusive use of KRAS ASODN or IGF-IR ASODN.ConclusionThe two ASODNs can inhibit the proliferation of lung adenocarcinoma cells through decreasing the expression of KRAS and IGF-IR mRNA and protein.

Metformin inhibits the proliferation of human prostate cancer PC-3 cells via the downregulation of insulin-like growth factor 1 receptor

Metformin is a biguanide drug that is widely used for the treatment of type 2 diabetes. Recent studies have shown that metformin inhibits cancer cell proliferation and tumor growth both in vitro and in vivo. The anti-tumor mechanisms of metformin include activation of the AMP-activated protein kinase/mTOR pathway and direct inhibition of insulin/insulin-like growth factor (IGF)-mediated cellular proliferation. However, the anti-tumor mechanism in prostate cancer remains unclear. Because activation of the IGF-1 receptor (IGF-1R) is required for prostate cell proliferation, IGF-1R inhibitors may be of therapeutic value. Accordingly, we examined the effects of metformin on IGF-1R signaling in prostate cancer cells. Metformin significantly inhibited PC-3 cell proliferation, migration, and invasion. IGF-1R mRNA expression decreased significantly after 48 h of treatment, and IGF-1R protein expression decreased in a similar manner. IGF-1R knockdown by siRNA transfection led to inhibited proliferation, migration and invasion of PC-3 cells. IGF-1 activated both ERK1/2 and Akt, but these effects were attenuated by metformin treatment. In addition, intraperitoneal treatment with metformin significantly reduced tumor growth and IGF-1R mRNA expression in PC-3 xenografts. Our results suggest that metformin is a potent inhibitor of the IGF-1/IGF-1R system and may be beneficial in prostate cancer treatment.

IGF-2R-Gαq signaling and cardiac hypertrophy in the low-birth-weight lamb.

The cardiac insulin-like growth factor 2 receptor (IGF-2R) can induce cardiomyocyte hypertrophy in a heterotrimeric G protein receptor-coupled manner involving αq (Gαq) or αs (Gαs). We have previously shown increased left ventricular weight and cardiac IGF-2 and IGF-2R gene expression in low-birth-weight (LBW) compared with average-birth-weight (ABW) lambs. Here, we have investigated the cardiac expression of IGF-2 gene variants, the degree of histone acetylation, and the abundance of proteins in the IGF-2R downstream signaling pathway in ABW and LBW lambs. Samples from the left ventricle of ABW and LBW lambs were collected at 21 days of age. There was increased phospho-CaMKII protein with decreased HDAC 4 abundance in the LBW compared with ABW lambs. There was increased GATA 4 and decreased phospho-troponin I abundance in LBW compared with ABW lambs, which are markers of pathological cardiac hypertrophy and impaired or reduced contractility, respectively. There was increased histone acetylation of H3K9 at IGF-2R promoter and IGF-2R intron 2 differentially methylated region in the LBW lamb. In conclusion, histone acetylation of IGF-2R may lead to increased IGF-2R mRNA expression and subsequently mediate Gαq signaling early in life via CaMKII, resulting in an increased risk of left ventricular hypertrophy and cardiovascular disease in adult life.

Insulin-like growth factor-1 mRNA isoforms and insulin-like growth factor-1 receptor mRNA expression in chronic hepatitis C.

To evaluate the expression of different insulin-like growth factor (IGF)-1 mRNA isoforms and IGF-1 receptor (IGF-1R) mRNA in hepatitis C virus (HCV)-infected livers. Thirty-four liver biopsy specimens from chronic hepatitis C (CH-C) patients were obtained before anti-viral therapy. Inflammatory activity (grading) and advancement of fibrosis (staging) were evaluated using a modified point scale of METAVIR. The samples were analyzed using quantitative real-time PCR technique. From fragments of liver biopsies and control liver that were divided and ground in liquid nitrogen, RNA was isolated using RNeasy Fibrous Tissue Mini Kit according to the manufacturer's instruction. Expression levels of IGF-1 mRNA isoforms (IGF-1A, IGF-1B, IGF-1C, P1, and P2) and IGF-1R mRNA were determined through normalization of copy numbers in samples as related to reference genes: glyceraldehyde-3-phosphate dehydrogenase and hydroxymethylbilane synthase. Results on liver expression of the IGF-1 mRNA isoforms and IGF-1R transcript were compared to histological alterations in liver biopsies and with selected clinical data in the patients. Statistical analysis was performed using Statistica PL v. 9 software. The study showed differences in quantitative expression of IGF-1 mRNA variants in HCV-infected livers, as compared to the control. Higher relative expression of total IGF-1 mRNA and of IGF-1 mRNAs isoforms (P1, A, and C) in HCV-infected livers as compared to the control were detected. Within both groups, expression of the IGF-1A mRNA isoform significantly prevailed over expressions of B and C isoforms. Expression of P1 mRNA was higher than that of P2 only in CH-C. Very high positive correlations were detected between reciprocal expressions of IGF-1 mRNA isoforms P1 and P2 (r = 0.876). Expression of P1 and P2 mRNA correlated with IGF-1A mRNA (r = 0.891; r = 0.821, respectively), with IGF-1B mRNA (r = 0.854; r = 0.813, respectively), and with IGF-1C mRNA (r = 0.839; r = 0.741, respectively). Expression of IGF-1A mRNA significantly correlated with isoform B and C mRNA (r = 0.956; r = 0.869, respectively), and B with C isoforms (r = 0.868) (P < 0.05 in all cases). Lower expression of IGF-1A and B transcripts was noted in the more advanced liver grading (G2) as compared to G1. Multiple negative correlations were detected between expression of various IGF-1 transcripts and clinical data (e.g., alpha fetoprotein, HCV RNA, steatosis, grading, and staging). Expression of IGF-1R mRNA manifested positive correlation with grading and HCV-RNA. Differences in quantitative expression of IGF-1 mRNA isoforms in HCV-infected livers, as compared to the control, suggest that HCV may induce alteration of IGF-1 splicing profile.

Potassium diformate influences gene expression of GH/IGF-I axis and glucose homeostasis in weaning piglets

The objective of this study was to determine the effects of potassium diformate (KDF) on growth performance of weaning piglets and genes expression related with growth axis and hepatic gluconeogenesis. A total of 180 piglets weaned at 28d of age were allocated randomly into two groups, with 6 pens in each group and 15 piglets in each pen. Piglets in the control group were fed basal diet, whereas the KDF-treated group was fed basal diet supplemented with 10g/kg KDF. After 5 weeks feeding, KDF-treated piglets showed higher average daily feed intake (ADFI) and average daily bodyweight gain (ADG) than those of the control (P<0.05), whereas the feed conversion ratio (FCR) was lower than that of the control (P=0.02). The apparent digestibility of dry matter and crude protein of the KDF-treated group were higher than those of the control group (P<0.01). KDF treatment did not change the blood indices level of insulin-like growth factor-I (IGF-I), high density lipoprotein (HDL), low density lipoprotein (LDL), total cholesterol (TC), triglyceride (TG) and blood glucose (BG) in plasma. KDF treatment did not influence the mRNA expression of growth hormone releasing hormone (GHRH) and somatostatin (SS) in hypothalamus, and the levels of growth hormone receptor (GHR) and IGF-I receptor (IGF-IR) mRNA in pituitary. Whereas, it increased the abundance of GH mRNA in pituitary, and the mRNA of GHR, IGF-I, IGF-IR, glucose-6-phosphatase (G6PC), fructose-1,6-bisphosphatase (FBP) and phosphoenolpyruvate carboxykinase (PCK) in liver (P<0.05). Furthermore, KDF treatment increased the concentration of IGF-I peptide in liver (P=0.000). These results suggest that, the beneficial effect of KDF on the growth performance may relate to the regulation of the GH axis and glucose homeostasis in piglets.

200 STAGE-SPECIFIC EXPRESSION PATTERNS OF THE INSULIN-LIKE GROWTH FACTOR 1 RECEPTOR DURING BOVINE PRE-IMPLANTATION EMBRYONIC DEVELOPMENT

Insulin-like growth factor 1 (IGF1) is a key regulator in early embryonic development, influencing physiological processes and stimulating growth and development (Fowden et al. 2003). Supplementing IGF1 during in vitro culture of bovine embryos improved cleavage and developmental rates while it reduced apoptosis (Byrne et al. 2002). The signal transduction of IGF1 is performed by its binding to the insulin-like growth factor 1 receptor (IGF1R). At the mRNA level, IGF1R is expressed throughout pre-implantation embryonic development and was identified as a potential marker of good quality embryos (Yaseen et al. 2001). However, information on protein level is rare. Therefore, protein expression of the IGF1R during early embryonic development in vitro was analysed in the present study by immunofluorescence staining. Furthermore, the mRNA expression of the IGF1R was investigated by RT-qPCR. In vitro derived embryos of different stages (2-cell, 4-cell, 8-cell, 16-cell stage, morula, blastocyst, and expanded blastocyst) were either directly subjected to immunofluorescence staining or frozen at –80°C for use in RT-qPCR. Staining was performed with a peptide antibody against two peptide sequences of the bovine IGF1R α unit, which was specifically produced. Pixel intensity of immunofluorescence was measured and a mean grey value was calculated using the cellsens® software (Olympus, Hamburg, Germany). Data were analysed by one-way ANOVA followed by a Tukey's test using SigmaStat 3.5 Software (Systat Software GmbH, Erkrath, Germany). The detection of the IGF1R mRNA and protein was possible in all stages of embryonic development beginning at the 2-cell stage up to the expanded blastocyst. The maximal mRNA expression could be observed in 2- and 4-cell embryos. It significantly decreased to the 8-cell stage, followed by an increase up to the expanded blastocyst. The IGF1R protein was mainly localised in the plasma membrane of single blastomeres and also weakly in the cytoplasm. Mean grey values are highest in the 2-cell stage, showing a significant decline up to the 16-cell stage and an increase again until the expanded blastocyst. The mRNA and protein expression showed similar patterns during early embryonic development. IGF1R expression started to increase at the 8-cell stage (mRNA) and 16-cell stage (protein) indicating a link to the maternal-embryonic transition. For the first time, these results show that in bovine embryos, the IGF1R expression is related to the activation of the embryonic genome. We gratefully acknowledge the financial support of the H. Wilhelm Schaumann Foundation (Hamburg, Germany).

Reduced insulin-like growth factor I receptor and altered insulin receptor isoform mRNAs in normal mucosa predict colorectal adenoma risk.

Hyperinsulinemia resulting from obesity and insulin resistance is associated with increased risk of many cancers, but the biology underlying this risk is unclear. We hypothesized that increased mRNA levels of the insulin-like growth factor I receptor (IGFIR) versus the insulin receptor (IR) or elevated ratio of IR-A:IR-B isoforms in normal rectal mucosa would predict adenoma risk, particularly in individuals with high body mass index (BMI) or plasma insulin. Biopsies from normal rectal mucosa were obtained from consenting patients undergoing routine colonoscopy at University of North Carolina Hospitals (Chapel Hill, NC). Subjects with colorectal adenomas were classified as cases (n = 100) and were matched to adenoma-free controls (n = 98) based on age, sex, and BMI. IGFIR and IR mRNA levels were assessed by qRT-PCR, and IR-A:IR-B mRNA ratios by standard PCR. Plasma insulin and crypt apoptosis were measured by ELISA and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), respectively. Logistic regression models examined relationships between receptor mRNAs, BMI, plasma insulin, and adenoma risk. Unexpectedly, cases were significantly more likely to have lower IGFIR mRNA levels than controls. No overall differences in total IR mRNA or IR-A:IR-B ratios were observed between cases and controls. Interestingly, in patients with high plasma insulin, increased IR-A:IR-B ratio was associated with increased likelihood of having adenomas. Our work shows novel findings that reduced IGFIR mRNA and, during high plasma insulin, increased IR-A:IR-B ratios in normal rectal mucosa are associated with colorectal adenoma risk. Our work provides evidence supporting a link between IGFIR and IR isoform expression levels and colorectal adenoma risk.

Increased mRNA levels of apolipoprotein M and apolipoprotein AI in the placental tissues with fetal macrosomia.

The present study examined mRNA levels of apolipoprotein M (apoM) and apolipoprotein AI (apoAI) in the term placental tissues obtained from 37 women with normal birth weight neonates and from 37 women with macrosomic neonates (birth body weight ≥4,000 g), and further discussed possible clinical significance of these observations. The mRNA levels of apoM and apoAI in the placental tissues were determined by the real time RT-PCR, which demonstrated that both apoM and apoAI mRNA levels were significantly higher in the placentas from macrosomia than those from normal birth. Moreover, we analyzed the overexpressions of apoM and apoAI with the clinical data. Meanwhile we examined several known risk factors of macrosomia including the mRNA levels of insulin-like growth factor I (IGF-I), IGF-II, insulin-like growth factor I receptor (IGF-IR) and IGF-IIR. It demonstrated that apoM expression was significantly positively correlated to the placental weight, fetal birth weight, pregestational body mass index (BMI), weight gain during pregnancy, maternal weight, maternal BMI and the mRNA levels of IGF-IR as well as IGF-IIR. The apoAI mRNA level was statistically significantly correlated to the placental weight, fetal birth weight, IGF-I and IGF-IR mRNA levels. Binary logistic regression analysis suggested that both apoM and apoAI mRNA may considered as independent risk factors for macrosomia. The clinical significance needs further investigation.

Insulin-like growth factor 1 receptor and response to anti-IGF1R antibody therapy in osteosarcoma.

BackgroundSurvival outcomes for patients with osteosarcoma (OS) have remained stagnant over the past three decades. Insulin-like growth factor 1 receptor (IGF1R) is over-expressed in a number of malignancies, and anti-IGF1R antibodies have and are currently being studied in clinical trials. Understanding the molecular aberrations which result in increased tumor response to anti-IGF1R therapy could allow for the selection of patients most likely to benefit from IGF1R targeted therapy.Methods IGF1R mRNA expression was assessed by RT PCR in OS patient primary tumors, cell lines, and xenograft tumors. IGF1R copy number was assessed by 3 approaches: PCR, FISH, and dot blot analysis. Exons 1–20 of IGF1R were sequenced in xenograft tumors and 87 primary OS tumors, and surface expression of IGF1R was assessed by flow cytometry. Levels of mRNA and protein expression, copy number, and mutation status were compared with tumor response to anti-IGF1R antibody therapy in 4 OS xenograft models.Results IGF1R mRNA is expressed in OS. Primary patient samples and xenograft samples had higher mRNA expression and copy number compared with corresponding cell lines. IGF1R mRNA expression, cell surface expression, copy number, and mutation status were not associated with tumor responsiveness to anti-IGF1R antibody therapy.Conclusions IGF1R is expressed in OS, however, no clear molecular markers predict response to IGF1R antibody-mediated therapy. Additional pre-clinical studies assessing potential predictive biomarkers and investigating targetable molecular pathways critical to the proliferation of OS cells are needed.

Urocortin 3 activates AMPK and AKT pathways and enhances glucose disposal in rat skeletal muscle.

Insulin resistance (IR) in skeletal muscle is an important component of both type 2 diabetes and the syndrome of sarcopaenic obesity, for which there are no effective therapies. Urocortins (UCNs) are not only well established as neuropeptides but also have their roles in metabolism in peripheral tissues. We have shown recently that global overexpression of UCN3 resulted in muscular hypertrophy and resistance to the adverse metabolic effects of a high-fat diet. Herein, we aimed to establish whether short-term local UCN3 expression could enhance glucose disposal and insulin signalling in skeletal muscle. UCN3 was found to be expressed in right tibialis cranialis and extensor digitorum longus muscles of rats by in vivo electrotransfer and the effects studied vs the contralateral muscles after 1 week. No increase in muscle mass was detected, but test muscles showed 19% larger muscle fibre diameter (P=0.030), associated with increased IGF1 and IGF1 receptor mRNA and increased SER256 phosphorylation of forkhead transcription factor. Glucose clearance into the test muscles after an intraperitoneal glucose load was increased by 23% (P=0.018) per unit mass, associated with increased GLUT1 (34% increase; P=0.026) and GLUT4 (48% increase; P=0.0009) proteins, and significantly increased phosphorylation of insulin receptor substrate-1, AKT, AKT substrate of 160 kDa, glycogen synthase kinase-3β, AMP-activated protein kinase and its substrate acetyl coA carboxylase. Thus, UCN3 expression enhances glucose disposal and signalling in muscle by an autocrine/paracrine mechanism that is separate from its pro-hypertrophic effects, implying that such a manipulation may have promised for the treatment of IR syndromes including sarcopaenic obesity.

MRNA Expression of IGF-1 and IGF-1R in Patients with Colorectal Adenocarcinoma and Type 2 Diabetes

Increasing studies show that messenger RNA (mRNA) levels of local IGF-system are overexpressed in cancer tissue of patients with colorectal cancer (CRC). However, the influence of type 2 diabetes (T2DM) on the expression of insulin-like growth factor-1 (IGF-1) and IGF-1 receptor (IGF-1R) mRNA in colorectal cancer tissue and adjacent non-cancerous tissue (ANCT) is unknown. The aim of this study was to assess mRNA expression of IGF-1 and IGF-1R in paired samples of cancer tissue and ANCT between colorectal adenocarcinoma (CA) patients with and without T2DM. To quantify the levels of IGF-1 and IGF-1R mRNA in CA, we analyzed the expression of IGF-1 and IGF-1R mRNA levels in paired samples of cancer tissue and ANCT in CA patients with and without T2DM using real-time reverse transcription-polymerase chain reaction (RT-PCR). mRNA levels of IGF-1 and IGF-1R were significantly higher in cancer tissue compared with its ANCT in CA patients with and without T2DM. Compared with the CA group, significantly higher levels of IGF-1 and IGF-1R mRNA were observed in cancer tissue in CA with T2DM group. No significant differences were observed in the role of cancer locations, Dukes stages and diabetes duration on mRNA expression of IGF-1. After adjusting for age, gender and Dukes stages, multivariate analysis indicated IGF-1 mRNA level was a risk factor for prognosis (p <0.05). Our results support the hypothesis that IGF system plays an important role in CRC. Further larger studies are needed.

Insulin-like growth factor-1 (IGF-1) enhances developmental competence of cat embryos cultured singly by modulating the expression of its receptor (IGF-1R) and reducing developmental block

ObjectiveThe aim of this study is to determine the effects of insulin-like growth factor-1 (IGF-1) and the mRNA expression of IGF-1 receptor (IGF-1R) during the in vitro development of cat embryos cultured in groups versus singly. MethodsCumulus-oocyte complexes (COCs) were matured and fertilized in vitro with frozen-thawed semen. Cleaved embryos (48h post-fertilization) were randomly assigned to one of the following treatments: 1) group embryo culture without IGF-1 (10 embryos per 50μl droplet), 2) single-embryo culture without IGF-1, and 3) to 6) single-embryo culture (50μl droplet per embryo) supplemented with different concentrations of IGF-1 (5, 25, 50 and 100ng/ml, respectively). During in vitro culture, the embryos were analyzed for development to the morula, blastocyst and hatching blastocyst stage. Relative mRNA expression of IGF-1R was also examined by qPCR at the morula and blastocyst stages. In addition, the mRNA expression of IGF-1R in morula-stage embryos treated with IGF-1 was determined. The influence of IGF-1 to preimplantation embryo development was then explored by co-incubation with 0.5μM IGF-1R inhibitor (Picropodophyllin; PPP). ResultsGroup embryo culture led to a significantly higher blastocyst development rate compared with single-embryo culture (P<0.05). The poor development of singly cultured embryos coincided with the significantly lower IGF-1R expression in morulae than in group-cultured morulae. IGF-1 (25 or 50ng/ml) supplementation significantly improved the blastocyst formation rate of single embryos to a level similar to group culture by promoting the morula-to-blastocyst transition. IGF-1 supplementation (25 or 50ng/ml) of singly cultured embryos upregulated the expression of IGF-1R mRNA in morula-stage embryos to the same level as that observed in group-cultured embryos (without IGF-1). The beneficial effects of IGF-1 on singly cultured embryo were (P<0.05) suppressed by PPP even in the group culture embryo without growth factor supplementation. ConclusionIGF-1 supplementation improves the developmental competence of feline embryos cultured individually and also increases IGF-1R gene expression to levels similar to group-cultured embryos.