IGF-I Receptor mRNA Research Articles

Effects of Dietary Supplementation with Cysteamine on Growth Hormone Receptor and Insulin-Like Growth Factor System in Finishing Pigs

The present study was conducted to test the hypothesis that chronic cysteamine (CS) supplementation may affect serum insulin-like growth factor (IGF)-I concentrations and growth hormone (GH) receptor (GHR), IGF-I, IGF-I receptor (IGF-IR), IGF binding protein (IGFBP)-3, and insulin receptor (IR) mRNA levels in different tissues of finishing pigs. A total of 24 finishing pigs (60.05 +/- 1.24 kg; 12 gilts and 12 barrows) were assigned randomly to one of the three dietary groups, with four pens/group (per pen: one gilt, one barrow). The pigs were fed a basal diet containing 0 (control), 70, or 140 mg/kg cysteamine feed additive (containing 28% cysteamine hydrochloride) for 47 days. The results indicated that CS supplementation (70 mg/kg) increased the average daily gain (ADG) and serum IGF-I level, upregulated mRNA levels of GHR and IGF-I (liver, stomach, muscle), IGF-IR (stomach, duodenum, muscle), and IGFBP-3 (liver) but downregulated IGFBP-3 (stomach, duodenum, muscle). CS supplementation (70 mg/kg) did not affect mRNA levels of GHR and IGF-I (duodenum), IGF-IR (liver), and IR (liver, stomach, duodenum, muscle). CS supplementation (140 mg/kg) downregulated GHR (duodenum), IGF-I, and IGF-IR mRNA (liver, stomach, duodenum, muscle) but upregulated IGFBP-3 and IR mRNA (liver, stomach, duodenum, muscle) and did not affect ADG and serum IGF-I concentration. Collectively, the results suggest that dietary CS supplementation modulates the growth rate, serum IGF-I concentrations, and the gene expression of GHR, IGF-I, IGF-IR, IGFBP-3, and IR in a dose-dependent manner. CS supplementation has tissue-specific regulation of GHR, IGF-I, IGF-IR, and IGFBP-3 mRNA levels. Moreover, the results also imply the possible physiologic role of the GH-IGF axis in mediating the dietary CS supplementation-supported growth of finishing pigs.

Inhibitory effects of nordihydroguaiaretic acid (NDGA) on the IGF‐1 receptor and androgen dependent growth of LAPC‐4 prostate cancer cells

Nordihydroguaiaretic acid (NDGA) is an inhibitor of the IGF-1 receptor (IGF-1R) in breast and other cancers, and concomitantly inhibits tumor growth both in cultured cells and animals. The current study evaluates the effect of NDGA on the androgen-stimulated growth of human prostate cancer cells. LAPC-4 prostate cancer cells in tissue culture were androgen starved for 3 days, 1 nM dihydrotestosterone (DHT) and other androgens were then added for up to 7 days, and cell proliferation measured. IGF-1R protein expression was measured by Western blot, and IGF-1R mRNA expression by quantitative PCR. IGF-1R receptor kinase activation was measured by ELISA. After 7 days, LAPC-4 growth was doubled by 1 nM DHT. NDGA had a rapid effect to inhibit IGF-1R autophosphorylation induced by IGF-1. DHT increased the expression of IGF-1R protein and mRNA levels. Maximal IGF-1R protein levels were observed 3 days after the addition of androgen. In addition, NDGA, at 10 microM or less, inhibited DHT-induced proliferation in both cells grown in plates and cells grown in soft agar. Androgen receptor (AR) studies by FRET revealed that NDGA had no conformational effects on the AR in response to ligand. NDGA blocks the DHT-induced growth of LAPC-4 prostate cancer cells by several mechanisms including rapid inhibition of the IGF-1R kinase, and a dose-dependent inhibition of androgen stimulation of IGF-1R expression. Clinical studies of this agent will determine its efficacy in the setting of androgen-dependent prostate cancer.

Effect of Dietary Fiber and Growth Hormone on Colonic Adaptation in Short Bowel Syndrome Treated by Enteral Nutrition

Colon adaptation can partially compensate for the reduced capacity of nutrient absorption in patients with short bowel syndrome (SBS). The aim of this study was to assess the effect of combined treatment with enteral nutrition (EN), dietary fiber, and recombinant human growth hormone (rhGH) on promoting colonic adaptation. A group of 40 male Sprague-Dawley rats undergoing up to 80% to 85% small intestine resection were randomly assigned to four groups of 10 rats each: enteral nutrition (EN, the control); enteral nutrition/dietary fiber (EF); enteral nutrition/rhGH (EG); and enteral nutrition/dietary fiber/rhGH (EFG). All groups received isonitrogenous, isocaloric enteral feeding for 21 days. Body weight, daily nitrogen balance, colonic morphology, DNA, insulin-like growth factor-1/IGF-1 receptor (IGF-1)/IGF-1R) expression were determined. Morphologic adaptation of the colon (including increased mucosal thickness and plica height, enlarged surface area, increased hyditloid cells) was observed on postoperative day 21. GH is superior to fiber in several aspects: increasing colon diameters (0.46 +/- 0.03 vs. 0.38 +/- 0.02 cm, P < 0.05), villous height (356 +/- 23 vs. 307 +/- 21 microm, P < 0.05) and total surface area (15,222 +/- 1344 vs. 13,178 +/- 1727 microm(2), P < 0.05). Increased DNA content-1.66 +/- 0.13 (EG) and 1.71 +/- 0.13 (EGF) vs. 1.28 +/- 0.11(EF), P < 0.05-in the colon was also found in the EG and EGF groups. GH administration led to a significant increase in plasma IGF-1 (439.6 +/- 88.3 ng/ml in the EG group, 455.4 +/- 107.4 ng/ml in the EGF group) and growth hormone (9.29 +/- 6.49 ng/ml in the EG group, 9.68 +/- 3.26 ng/ml in the EGF group) as compared to the EN group (IGF-1, 328.7 +/- 68.1 ng/ml; GH, 5.81 +/- 2.41 ng/ml) and the EF group (IGF-1, 356.4 +/- 52.1 ng/ml; GH, 6.51 +/- 4.66 ng/ml). Analysis of IGF-1 and IGF-1 receptor mRNA also demonstrated a significantly higher IGF-1 mRNA in the EG and EFG groups than in the EN and EF groups. Colon functional adaptation was also associated with accelerated absorptive function of water in the EF, EG, and EGF groups. Improved nutritional status (body weight, nitrogen retention, plasma protein) were seen in the EG and EGF groups. Dietary fiber, in combination with growth factor, synergistically promoted colon adaptation in the SBS animal model and facilitated maintenance of daily nutritional needs in rodents.

Suppression of insulin-like growth factor-1 receptor by RNA interference inhibits cell growth in vitro and induces chemosensitization of HO8910PM cell to cisplatin

To assess the effect of suppression of insulin-like growth factor-1 receptor (IGF1R) in HO8910PM cell line by small interference RNA (siRNA). Transfection of siRNA using lipofectamine 2000 was conducted to silence IGF1R gene expression, the expression levels of IGF1R mRNA and protein were evaluated, and the effects on the cell cycles at 48 hours of transfection were assessed by real-time PCR, western blot and flow cytometry (FCM) assay respectively. The cell growth was detected by cell counting kit-8 (CCK-8) at 24, 48, 72, 96 hours of transfection. After 24 hours of transfection, the cells were cultured with different concentrations of cisplatin (DDP) for 24 hours, the cell growth inhibition rate was evaluated by CCK-8. Following incubation with 10 microg/ml DDP for 24 hours after 24 hours of transfection, the apoptosis cells and the protein expression level of apoptosis-related gene, B cell leukemia/lymphoma 2 (Bcl-2), were identified by FCM and western blot respectively. (1) Expression levels of IGF1R mRNA and protein were markedly decreased respectively at 48 hours of transfection IGF1R siRNA. (2) Suppression of IGF1R accompanied the reduction of cell growth at 48, 72, 96 hours of transfection with IGF1R siRNA, absorbance were 1.71+/-0.13, 2.32+/-0.23, 2.79+/-0.28 respectively (P<0.01). (3) IGF1R siRNA induces arrest of G2 phase, the G2 phase rate of cells were 24.37% (P<0.05). (4) Following treatment with 2.5, 5, 10, 20 microg/ml DDP for 24 hours after 24 hours of transfection, the cell growth inhibition rates were (25.94+/-0.08)%, (40.25+/-0.05)%, (59.48+/-0.03)% and (74.18+/-0.08)% respectively (P<0.01). (5) Treatment with 10 microg/ml DDP for 24 hours after 24 hours of transfection, induces 17.95% of cells apoptosis (P<0.05), and decreases Bcl-2 protein level. RNA interference of IGF1R gene induces the IGF1R silence in HO8910PM cell line significantly, inhibits cell growth in vitro, arrests the G2 phase, and enhances the chemosensitization to DDP.

Disruption of Growth Hormone Signaling Retards Prostate Carcinogenesis in the Probasin/TAg Rat

We asked whether down-regulation of GH signaling could block carcinogenesis in the Probasin/TAg rat, a model of aggressive prostate cancer. The Spontaneous Dwarf rat, which lacks GH due to a mutation (dr) in its GH gene, was crossed with the Probasin/TAg rat, which develops prostate carcinomas at 100% incidence by 15 wk of age. Progeny were heterozygous for the TAg oncogene and homozygous for either the wild-type GH gene (TAg/Gh(+/+)) or the dr mutation (TAg/Gh(dr/dr)). Prostate tumor incidence and burden were significantly reduced, and tumor latency was delayed in TAg/Gh(dr/dr) rats relative to TAg/Gh(+/+) controls. At 25 wk of age, loss of GH resulted in a 20 and 80% decrease in the area of microinvasive carcinoma in the dorsal and lateral lobes, respectively. By 52 wk of age, invasive prostate adenocarcinomas were observed in all TAg/Gh(+/+) rats, whereas the majority of TAg/Gh(dr/dr) did not develop invasive tumors. Suppression of carcinogenesis could not be attributed to alterations in prostate expression of TAg or androgen receptor or changes in serum testosterone levels. As carcinogenesis progressed in TAg/Gh(+/+) rats, prostate GHR mRNA and protein expression increased significantly, but prostate IGF-I receptor mRNA and protein levels dropped. Furthermore, serum IGF-I and prostate IGF-I levels did not change significantly over the course of carcinogenesis. These findings suggest that GH plays a dominant role in progression from latent to malignant prostate cancer driven by the powerful probasin/TAg fusion gene in rats and suggest that GH antagonists may be effective at treating human prostate cancer.

Mechanism involved in genistein activation of insulin-like growth factor 1 receptor expression in human breast cancer cells

Our previous studies have shown that genistein can enhance the insulin-like growth factor (IGF)-1 receptor signalling pathway via an oestrogen receptor (ER) in human breast cancer MCF-7 cells. The present study aims to investigate how genistein regulates IGF-1 receptor expression in human MCF-7 cells. Genistein at 1 microm stimulated the growth of MCF-7 cells and this effect could be completely blocked by the IGF-1 receptor antagonist JB-1, suggesting that IGF-1 receptor is essential for mediating the proliferative effects of genistein in MCF-7 cells. Genistein increased IGF-1 receptor promoter activity. This effect could be completely abolished by co-treatment of MCF-7 cells with ICI 182,780 (10- 6 m). Genistein increased IGF-1 receptor gene expression and this effect could be completely blocked by the IGF-1 receptor antagonist JB-1. Co-treatment of MCF-7 cells with cycloheximide (5 microg/ml) completely blocked the induction of IGF-1 receptor protein and mRNA expression by genistein. The results indicated that the induction of IGF-1 receptor promoter activity by genistein required the action of ER while the stimulatory actions of genistein on IGF-1 receptor expression required the activity of the IGF-1 receptor and de novo protein synthesis. These data provide evidence to support the hypothesis that the inductive effects of genistein on IGF-1 receptor expression require the cross-talk between IGF-1 receptor and the ER-dependent pathways.

Effects of insulin‐like growth factor‐1 on cellular and molecular characteristics of bovine blastocysts produced in vitro

Addition of insulin-like growth factor-1 (IGF-1) to culture medium increases the proportion of bovine embryos that develop to the blastocyst stage and increases embryo survival following transfer to heat-stressed, lactating dairy cows. The objective of the present study was to determine molecular and cellular correlates of these actions of IGF-1. Embryos were produced in vitro and cultured for 7 days with or without 100 ng/ml IGF-1. On d 7 after insemination, grade 1 expanded blastocysts were harvested and used to determine total cell number, percent apoptosis, cell allocation to the inner cell mass and trophectoderm, and the relative abundance of several developmentally important gene transcripts. There was no significant effect of IGF-1 treatment on blastocyst cell number, the proportion of blastomeres that were apoptotic, or the number of cells in the inner cell mass and trophectoderm. However, differences in the relative abundance of several mRNA transcripts were observed between control and IGF-1 treated embryos. Addition of IGF-1 increased (P < 0.02) amounts of mRNA for IGF binding protein-3 and desmocollin II and tended (P < 0.08) to increase amounts of mRNA for Na/K ATPase and Bax. Moreover, IGF-1 treatment decreased (P < 0.05) steady-state amounts of transcripts for heat shock protein 70 and tended (P < 0.08) to reduce amounts of IGF-1 receptor mRNA. In conclusion, increased survival of embryos treated with IGF-1 does not appear due to effects on cell number, percent apoptosis, or cell allocation. Addition of IGF-1 to culture can, however, alter expression of several transcripts which may be important for embryo development and survival following transfer.

Fetal programing of colon cancer in adult rats: correlations with altered neonatal growth trajectory, circulating IGF-I and IGF binding proteins, and testosterone

We examined effects of dietary soy protein isolate (SPI) or genistein (GEN; soy isoflavone) during pregnancy on development of colon cancer in male progeny Sprague-Dawley rats. Four groups of rats were used: a lifetime casein-fed group (CAS; control diet), a lifetime SPI-fed group (positive control for protective effect of diet on colon carcinogenesis), a group whose dams received SPI only during pregnancy and CAS thereafter (SPI/CAS), and a group whose dams received CAS+GEN only during pregnancy and CAS thereafter (GEN/CAS). At 47 and 55 days of age, male progeny were administered the intestinal carcinogen azoxymethane (AOM). Tumors, endocrine status, and colon gene expression were evaluated at 20 week post-AOM. The SPI group had 47% decreased colon tumor incidence compared with the CAS group (P<0.05), whereas SPI/CAS, GEN/CAS, and CAS groups did not differ in this regard. Maternal-only SPI increased the percentage of animals bearing multiple colon tumors (P<0.05), an effect not mimicked by GEN. Serum insulin and leptin concentrations were decreased by lifetime SPI (P<0.05), whereas serum IGF-I was elevated in the SPI/CAS group (P<0.05). The SPI/CAS group had reduced serum testosterone levels (P<0.05) and exhibited a tendency for increased mucosal expression of IGF-I receptor and glucose transporter-1 mRNAs. Results indicate an effect of dietary protein type during pregnancy on colon tumor multiplicity and colon tissue gene expression, and serum IGF-I and testosterone in progeny rats as later adults.

Highlights From The Literature

Highlights From The Literature

Somatostatin inhibits insulin-like growth factor-I receptor expression in the gill of a teleost fish ( Oncorhynchus mykiss)

Rainbow trout gill tissue was used to examine the role of somatostatin (SS) on insulin-like growth factor-I (IGF-I) receptor expression. In vivo implantation of fish with somatostatin-14 (SS-14) reduced expression of IGF-I receptor mRNAs as well as [ 125I]-IGF-I binding. In vitro incubation of gill filaments with SS-14 or various SS isoforms, including SS-28 and [Tyr 7, Gly 10]-SS-14-containing peptides, directly inhibited IGF-I receptor mRNA expression. SS-14 also inhibited [ 125I]-IGF-I binding in vitro. These data indicate that SSs inhibit the mRNA and functional expression of IGF-I receptors in gill, and suggest that SSs regulate growth in an extrapituitary manner by reducing sensitivity to IGF-I.

Endocrine systems in juvenile anadromous and landlocked Atlantic salmon ( Salmo salar): Seasonal development and seawater acclimation

The present study compares developmental changes in plasma levels of growth hormone (GH), insulin-like growth factor I (IGF-I) and cortisol, and mRNA levels of their receptors and the prolactin receptor (PRLR) in the gill of anadromous and landlocked Atlantic salmon during the spring parr-smolt transformation (smoltification) period and following four days and one month seawater (SW) acclimation. Plasma GH and gill GH receptor (GHR) mRNA levels increased continuously during the spring smoltification period in the anadromous, but not in landlocked salmon. There were no differences in plasma IGF-I levels between strains, or any increase during smoltification. Gill IGF-I and IGF-I receptor (IGF-IR) mRNA levels increased in anadromous salmon during smoltification, with no changes observed in landlocked fish. Gill PRLR mRNA levels remained stable in both strains during spring. Plasma cortisol levels in anadromous salmon increased 5-fold in May and June, but not in landlocked salmon. Gill glucocorticoid receptor (GR) mRNA levels were elevated in both strains at the time of peak smoltification in anadromous salmon, while mineralocorticoid receptor (MR) mRNA levels remained stable. Only anadromous salmon showed an increase of gill 11β-hydroxysteroid dehydrogenase type-2 (11β-HSD2) mRNA levels in May. GH and gill GHR mRNA levels increased in both strains following four days of SW exposure in mid-May, whereas only the anadromous salmon displayed elevated plasma GH and GHR mRNA after one month in SW. Plasma IGF-I increased after four days in SW in both strains, decreasing in both strains after one month in SW. Gill IGF-I mRNA levels were only increased in landlocked salmon after 4 days in SW. Gill IGF-IR mRNA levels in SW did not differ from FW levels in either strain. Gill PRLR mRNA did not change after four days of SW exposure, and decreased in both strains after one month in SW. Plasma cortisol levels did not change following SW exposure in either strain. Gill GR, 11β-HSD2 and MR mRNA levels increased after four days in SW in both strains, whereas only the anadromous strain maintained elevated gill GR and 11β-HSD2 mRNA levels after one month in SW. The results indicate that hormones and receptors of the GH and cortisol axes are present at significantly lower levels during spring development and SW acclimation in landlocked relative to anadromous salmon. These findings suggest that attenuation of GH and cortisol axes may, at least partially, result in reduced preparatory upregulation of key gill ion-secretory proteins, possibly a result of reduced selection pressure for marine adaptations in landlocked salmon.

Insulin-like growth factor system gene expression in cervical scrapes from women with squamous intraepithelial lesions and cervical cancer

Background There is ample evidence that the insulin-like growth factors (IGF) system is involved in the development of several types of cancer. The aim of this study was to evaluate the expression levels of IGF-I, IGF-II, IGF binding protein 3 (IGFBP-3) and IGF-I receptor (IGF-IR) in exfoliated cervical cells in cervical carcinogenesis. Methods mRNA levels of IGF-I, IGF-II, IGFBP-3 and IGF-IR were assessed by real-time PCR in 105 cervical scrapes obtained from 16 patients diagnosed with low-grade squamous intraepithelial lesions (LSIL), 24 with high-grade SIL (HSIL), 23 with cervical cancer, and 42 from controls with normal Papanicolau (Pap) test. Results IGF-I mRNA levels were very low and no significant differences were seen between control and other groups. IGF-II mRNA levels were significantly lower in LSIL than in control group (median [arbitrary units]: 0.38 vs. 2.42, P = 0.006) but its expression in HSIL and cervical cancer was similar to the one observed in controls. IGFBP-3 mRNA levels were significantly lower in cancer than in controls (median [arbitrary units]: 0.43 vs. 0.73, P = 0.03). We observed a decrease in IGF-IR gene expression as the SIL degree increased (median for controls, LSIL, HSIL, and cervical carcinoma [arbitrary units]: 31.24, 9.08, 8.95, and 3.56, respectively). IGF-IR mRNA levels were significantly lower in HSIL and cervical cancer in comparison with controls ( P = 0.043 and P < 0.001, respectively). Conclusions The present observations suggest that a reduced expression of IGFBP-3 and IGF-IR can be associated with progression to cervical cancer; the specific role played by the IGF-IR in this process remains unclear.

Insights into a role of GH secretagogues in reversing the age-related decline in the GH/IGF-I axis

Growth hormone (GH) secretion and serum insulin-like growth factor-I (IGF-I) decline with aging. This study addresses the role played by the hypothalamic regulators in the aging GH decline and investigates the mechanisms through which growth hormone secretagogues (GHS) activate GH secretion in the aging rats. Two groups of male Wistar rats were studied: young-adult (3 mo) and old (24 mo). Hypothalamic growth hormone-releasing hormone (GHRH) mRNA and immunoreactive (IR) GHRH dramatically decreased (P < 0.01 and P < 0.001) in the old rats, as did median eminence IR-GHRH. Decreases of hypothalamic IR-somatostatin (SS; P < 0.001) and SS mRNA (P < 0.01), and median eminence IR-SS were found in old rats as were GHS receptor and IGF-I mRNA (P < 0.01 and P < 0.05). Hypothalamic IGF-I receptor mRNA and protein were unmodified. Both young and old pituitary cells, cultured alone or cocultured with fetal hypothalamic cells, responded to ghrelin. Only in the presence of fetal hypothalamic cells did ghrelin elevate the age-related decrease of GH secretion to within normal adult range. In old rats, growth hormone-releasing peptide-6 returned the levels of GH and IGF-I secretion and liver IGF-I mRNA, and partially restored the lower pituitary IR-GH and GH mRNA levels to those of young untreated rats. These results suggest that the aging GH decline may result from decreased GHRH function rather than from increased SS action. The reduction of hypothalamic GHS-R gene expression might impair the action of ghrelin on GH release. The role of IGF-I is not altered. The aging GH/IGF-I axis decline could be rejuvenated by GHS treatment.

Aging Impairs IGF-I Receptor Activation and Induces Skeletal Resistance to IGF-I

IGF-I plays an important anabolic role in stimulating bone formation and maintaining bone mass. We show that the pro-proliferative, anti-apoptotic, and functional responses to IGF-I in bone and BMSCs decrease with aging. These changes are associated with impaired receptor activation and signal transduction through the MAPK and PI3K pathways. IGF-I is a potent anabolic agent having effects across diverse tissues and cell types. With aging, bone becomes resistant to the anabolic actions of IGF-I. To examine the effects of aging on bone responsiveness to IGF-I, we measured the pro-proliferative, anti-apoptotic, and functional responses of bone and bone marrow stromal cells (BMSCs) to IGF-I and evaluated IGF-I signal transduction in young, adult, and old mice. Male C57BL/6 mice 6 wk (young), 6 mo (adult), and 24 mo (old) were treated with IGF-I for 2 wk using osmotic minipumps, and osteoblast proliferation (BrdU labeling) in vivo, and osteoprogenitor number (BMSC culture and calcium nodule formation) were measured. Proliferation, apoptosis, and expression of key osteoblast factors (alkaline phosphatase, collagen, osteocalcin, RANKL, osteoprotegerin (OPG), macrophage-colony stimulating factor [M-CSF]) and IGF-I signaling elements and their activation in IGF-I-treated cells were studied using QRT-PCR and Western blot analysis. Data were analyzed using ANOVA. Aging decreased the basal and IGF-I-stimulated number of BrdU-labeled osteoblasts and reduced the ability of IGF-I to stimulate osteoprogenitor formation (calcium nodule number) by 50%. The pro-proliferative and anti-apoptotic actions of IGF-I were blunted in cells from old animals. These changes were accompanied by age-related alterations in the ability of IGF-I to regulate alkaline phosphatase, collagen, osteocalcin, RANKL, OPG, and M-CSF expression. IGF-I binding was normal, but IGF-I receptor mRNA and protein expression was increased in aged animals by 2- and 10-fold, respectively. The age-related changes in proliferation, apoptosis, and function were accompanied by loss of IGF-I-induced signaling at the receptor level and at key regulatory sites along the MAPK (ERK1/2) and PI3K (AKT) pathways. Our data show that aging is accompanied by loss of bone and BMSC/osteoblast responsiveness to IGF-I and that these changes are associated with resistance to IGF-I signaling that involve receptor activation and downstream signaling events.

Insulin-like growth factor (IGF) family genes are aberrantly expressed in bovine conceptuses produced in vitro or by nuclear transfer

Embryos produced through somatic cell nuclear transfer (NT) or in vitro production (IVP) are often associated with increased abortion and abnormalities thought to arise from disruptions in normal gene expression. The insulin-like growth factor (IGF) family has a major influence on embryonic, fetal and placental development; differences in IGF expression in NT- and IVP-derived embryos may account for embryonic losses during placental attachment. In the present study, expression of IGF-I, IGF-II, IGF-I receptor (IGF-IR), and IGF-IIR mRNAs was quantitated in Day 7 and 25 bovine embryos produced in vivo, by NT, IVP, or parthenogenesis, to further understand divergent changes occurring during development. Expression of the IGF-I gene was not detected in Day 7 blastocysts for any treatment. However, there were no differences ( P > 0.10) among Day 7 treatments in the amounts of IGF-IR, IGF-II, and IGF-IIR mRNA. For Day 25 conceptuses, there was higher expression of IGF-I mRNA for NT and IVP embryonic tissues than for in vivo embryonic tissues ( P < 0.05). Furthermore, embryonic tissues from NT-derived embryos had higher expression of IGF-II mRNA than IVP embryonic tissues ( P < 0.05). Placental expression of IGF-IIR mRNA was greater for NT-derived than in vivo-derived embryos ( P < 0.05). There were no differences in IGF-IR mRNA across all treatments and tissues ( P > 0.10). In conclusion, these differences in growth factor gene expression during early placental attachment and rapid embryonic growth may directly or indirectly contribute to increased losses and abnormalities in IVP- and NT-derived embryos.

Radiohybridization PET imaging of KRAS G12D mRNA expression in human pancreas cancer xenografts with [64Cu]DO3A-peptide nucleic acid-peptide nanoparticles

There is a compelling need to image pancreas cancer at an early stage. Human pancreas cancer cells display elevated levels of KRAS protein due to high copy numbers of KRAS mRNA, and elevated levels of insulin-like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. Therefore we hypothesized that pancreas cancer could be detected in vivo with a single probe that targets both KRAS mRNA and IGF1R. Because positron emission tomography (PET) is a sensitive imaging technique, we designed a probe incorporating the positron-emitting nuclide 64Cu. The KRAS-specific hybridization probe consisted of 1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetyl (DO3A) on the N-terminus of a peptide nucleic acid (PNA) hybridization sequence (GCCATCAGCTCC) linked to a cyclized IGF1 peptide analog (D-Cys-Ser-Lys-Cys) on the C-terminus, for IGF1R-mediated endocytosis. A series of such KRAS radiohybridization probes with 0, 1, 2, or 3 mismatches to KRAS G12D mRNA, including exact matches to wild type KRAS mRNA and KRAS G12V mRNA, along with a double D(Ala) replacement IGF1 peptide control, were assembled by continuous solid phase synthesis. To test the hypothesis that KRAS-IGF1 dual probes could specifically image KRAS mRNA expression noninvasively in human IGF1R-overexpressing AsPC1 pancreas cancer xenografts in immunocompromised mice, [64Cu]PNA radiohybridization probes and controls were administered by tail vein. The [64Cu]KRAS¬-IGF1 radiohybridization probe yielded strong tumor contrast in PET images, 8.6±1.4-fold more intense in the center of human pancreas cancer xenografts than in the contralateral muscle at 4 h post-injection. Control experiments with single base KRAS¬ mismatches, an IGF1 peptide mismatch, and a breast cancer xenograft lacking KRAS activation yielded weak tumor contrast images. These experiments are consistent with our hypothesis for noninvasive PET imaging of KRAS oncogene expression in pancreas cancer xenografts. Imaging oncogene mRNAs with radiolabel-PNA-peptide nanoparticles might provide specific genetic characterization of pre-invasive and invasive pancreas cancers for staging and choice of therapy.

The relationship of blood glucose and the expression of insulin-like growth factor-1 receptor mRNA in diabetic cardiomyopathy: a preliminary study with rats

To investigate the role of insulin-like growth factor-1 receptor mRNA (IGF-1R mRNA) in diabetic cardiomyopathy. Alloxan of the doses of 150 and 200 mg/kg was injected intraperitoneally into 20 male Wistar rats respectively so as to establish type 1 diabetes mellitus models (Groups B and C). The rats were used as normal controls (Groups A). Three months later the rats were killed and blood samples were collected to undergo examination of plasma glucose, insulin, glycated hemoglobin (HbA1c), and C peptide. The hearts were taken out to undergo light and transmission electron microscopy and in situ hybridization to detect the expression of IGF-1R mRNA in the myocardium. The levels of plasma glucose, HbA1c, insulin and C peptide of Groups B and C were all significantly higher than those of Group A (all P < 0.01), and the levels of plasma glucose, HbA1c, insulin and C peptide of Group C being significantly higher than those of Group B too (all P < 0.01). Expression of IGF-1R mRNA was found in the myocardium of Groups A, B and C with the distribution areas of 20%, 30%, and 38% respectively. The expression of IGF-1R mRNA in myocardium was positively correlated with blood glucose and HbA1c (r = 0.869, 0.865, both P < 0.01). The increased expression of IGF-1R mRNA in the myocardium may play an important role in the progression of diabetic cardiomyopathy.

Antisense treatment of IGF-IR induces apoptosis and enhances chemosensitivity in central nervous system atypical teratoid/rhabdoid tumours cells

Central nervous system (CNS) atypical teratoid/rhabdoid tumours (AT/RT) are among the paediatric malignant tumours with the worst prognosis and fatal outcome. Insulin-like growth factor I receptor (IGF-IR) protects cancer cells from apoptosis induced by a variety of anticancer drugs and radiation. In the present study, IGF-IR was expressed in 8/8 primary AT/RT as detected by immunohistochemistry. Moreover, we found IGF-I and IGF-II mRNA in BT-16 CNS AT/RT cells and IGF-II mRNA in BT-12 CNS AT/RT cells, and autophosphorylated IGF-IR in both cell lines, indicating the potential presence of an autocrine/paracrine IGF-I/II/IGF-IR loop in CNS AT/RT. IGF-IR antisense oligonucleotide treatment of human CNS AT/RT cells resulted in significant down-regulation of IGF-IR mRNA and protein expression, induction of apoptosis, and chemosensitisation to doxorubicin and cisplatin. These studies provide evidence for the influence of IGF-IR on cellular responses to chemotherapy and raise the possibility that curability of selected CNS AT/RT may be improved by pharmaceutical strategies directed towards the IGF-IR.

IGF-1 receptor signaling pathways and effects of AMPK activation on IGF-1-induced progesterone secretion in hen granulosa cells

IGF-1 plays a key role in the proliferation and differentiation of granulosa cells. However, the molecular mechanism of IGF-1 action in avian granulosa cells during follicle maturation is unclear. Here, we first studied IGF-1 receptor (IGF-1R) expression, IGF-1-induced progesterone production and some IGF-1R signaling pathways in granulosa cells from different follicles. IGF-1R (mRNA and protein) was higher in fresh or cultured granulosa cells from the largest follicles (F1 or F2) than in those from smaller follicles (F3 or F4). In vitro, IGF-1 treatment (10 −8 M, 36 h) increased progesterone secretion by four-fold in mixed F3 and F4 (F3/4) granulosa cells and by 1.5-fold in F1 granulosa cells. IGF-1 (10 −8 M, 30 min)-induced increases in tyrosine phosphorylation of IGF-1R beta subunit and phosphorylation of ERK were higher in F1 than in F3/4 granulosa cells. Interestingly, IGF-1 stimulation (10 −8 M, 10 min) decreased the level of AMPK Thr172 phosphorylation in F1 and F3/4 granulosa cells. We have recently showed that AMPK (AMP-activated protein kinase) is a protein kinase involved in the steroidogenesis in chicken granulosa cells. We then studied the effects of AMPK activation by AICAR (5-aminoimidazole-4-carboxamide ribonucleoside), an activator of AMPK, on IGF-1-induced progesterone secretion by F3/4 and F1 granulosa cells. AICAR treatment (1 mM, 36 h) increased IGF-1-induced progesterone secretion, StAR protein levels and decreased ERK phosphorylation in F1 granulosa cells. Opposite data were observed in F3/4 granulosa cells. Adenovirus-mediated expression of dominant negative AMPK totally reversed the effects of AICAR on IGF-1-induced progesterone secretion, StAR protein production and ERK phosphorylation in both F3/4 and F1 granulosa cells. Thus, a variation of energy metabolism through AMPK activation could modulate differently IGF-1-induced progesterone production in F1 and F3/4 granulosa cells.

Effects of Homologous Ghrelins on the Growth Hormone/Insulin-like Growth Factor-I Axis in the Tilapia, Oreochromis mossambicus

Ghrelin is a gut-brain peptide synthesized mainly in the oxyntic mucosal cells of the stomach, and has potent growth hormone (GH)-releasing and orexigenic activities. Recently, two forms of ghrelin, ghrelin-C8 and -C10, were identified in the Mozambique tilapia (Oreochromis mossambicus). The present study describes in vitro and in vivo effects of these endogenous ghrelins on the GH/insulin-like growth factor-I (IGF-I) axis. Ghrelin-C8 (100 nM) stimulated GH release from primary cultures of pituitary cells after 4 and 8 h of incubation, whereas no effect was seen on prolactin (PRL) release. Stimulatory effects of ghrelin-C8 and -C10 (100 nM) on GH release during 6 h of incubation were blocked by pre-incubation with GHS receptor antagonist, [D-Lys(3)]-GHRP-6 (10 microM). Intraperitoneal injection of ghrelin-C8 (1 ng/g body weight) and -C10 (0.1 and 1 ng/g body weight) significantly increased plasma GH levels after 5 h. Significant increases were observed also in hepatic expression of IGF-I and GH receptor (GHR) mRNA following injections of both forms of ghrelin (0.1 and 1 ng/g body weight), although there was no effect on plasma levels of IGF-I. In the next experiment, both forms of ghrelin (1 ng/g body weight) significantly increased plasma IGF-I levels 10 h after the injection. No significant effect of either ghrelin was observed on plasma PRL levels. Both forms of GHS receptor (GHSR-1a and -1b) were found in the pituitary, clearly indicating that tilapia ghrelins stimulate primarily GH release through the GHS receptor. Stimulation of hepatic expression of IGF-I and GHR suggests metabolic roles of ghrelin in tilapia.