IGF-I mRNA Levels Research Articles

Effects of naringin on proliferation, differentiation and maturation of rat calvarial osteoblasts in vitro

To investigate the effects of naringin on the proliferation, differention and maturaion of rat calvarial osteoblasts (ROB). Segregated neonatal SD rat skull, enzyme digestion to obtain ROB. The culture medium was replaced every three days. Serial subcultivation proceeded when cells covered with 80% culture dish. Naringin supplemented into the culture at 1 x 10(-4), 1 x 10(-5), 1 x 10(-6), 1 x 10(-7) mol x L(-1) respectively. MTT method was adopted in proliferation analysis and the activity of ALP was examined after induced 9 days. Search the best concentration and supplemented into the medium, then the osteogenic differentiation markers including the secretion amount of osteocalcin, osteopontin and bone morphogenetic protein-2 were compared between the naringin-supplemented group and the control. Total RNA was isolated and the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERa and ERbeta was investigated by Real time RT-PCR. Total protein also was isolated and the expression ERa, ERbeta and collagen I was examined by Western blot. After the addition of ICI 182.780, an inhibitor of the estrogen signal pathway, these index also was examined and the changes were compared. The ROB proliferation was motivated by naringin dose-dependently. And it evidently leads to osteogenic process and maturation. 1 x 10(-5) mol x L(-1) is the best concentration. Naringin improved the secretion of osteocalcin, osteopontin, bone morphogenetic protein-2 and collagen I significantly. Besides, it can also enhanced the mRNA level of bFGF, IGF-1, Runx-2, Osterix, ERalpha and ERbeta. While all these effects can be restrained by ICI 182.780. The naringin with final concentration of 1 x 10(-5) mol x L(-1) enhances the osteogenic differentiation and maturation of ROB significantly, while the promoting effects vanished after the addition of ICI 182.780. These results suggesting that naringin is one of the phytoestrogens and have the activity of bone formation may via estrogen signal pathway, it can be developed into a new drug for osteoporosis therapy.

Tissue-specific relationship of S-adenosylhomocysteine with allele-specific H19/Igf2 methylation and imprinting in mice with hyperhomocysteinemia

DNA methylation is linked to homocysteine metabolism through the generation of S-adenosylmethionine (AdoMet) and S-Adenosylhomocysteine (AdoHcy). The ratio of AdoMet/AdoHcy is often considered an indicator of tissue methylation capacity. The goal of this study is to determine the relationship of tissue AdoMet and AdoHcy concentrations to allele-specific methylation and expression of genomically imprinted H19/Igf2. Expression of H19/Igf2 is regulated by a differentially methylated domain (DMD), with H19 paternally imprinted and Igf2 maternally imprinted. F1 hybrid C57BL/6J x Castaneous/EiJ (Cast) mice with (+/−), and without (+/+), heterozygous disruption of cystathionine-β-synthase (Cbs) were fed a control diet or a diet (called HH) to induce hyperhomocysteinemia and changes in tissue AdoMet and AdoHcy. F1 Cast x Cbs+/− mice fed the HH diet had significantly higher plasma total homocysteine concentrations, higher liver AdoHcy, and lower AdoMet/AdoHcy ratios and this was accompanied by lower liver maternal H19 DMD allele methylation, lower liver Igf2 mRNA levels, and loss of Igf2 maternal imprinting. In contrast, we found no significant differences in AdoMet and AdoHcy in brain between the diet groups but F1 Cast x Cbs+/− mice fed the HH diet had higher maternal H19 DMD methylation and lower H19 mRNA levels in brain. A significant negative relationship between AdoHcy and maternal H19 DMD allele methylation was found in liver but not in brain. These findings suggest the relationship of AdoMet and AdoHcy to gene-specific DNA methylation is tissue-specific and that changes in DNA methylation can occur without changes in AdoMet and AdoHcy.

Insulin-like Growth Factor 1 mRNA Expression in the Uterus of Streptozotocin-treated Diabetic Mice

Reproductive functions decline with the onset of diabetes in female mice. Diabetic mice have smaller uteri with an underdeveloped endometrium, suggesting diminished estrogen-induced growth. We aimed to clarify the changes in the estrous cycle and in insulin-like growth factor 1 (IGF1) expression in the uteri of streptozotocin (STZ)-treated diabetic mice, because IGF1 is one of the main growth factors involved in estrogen-induced uterine growth. ICR female mice were intraperitoneally administered STZ (10 mg/100 g BW), and blood glucose levels were determined. Mice with blood glucose levels > 200 mg/dl were classified as diabetic mice. The onset of diabetes was associated with acyclic estrous cycles. Diabetes was also induced with STZ in ovariectomized mice. Uterine Igf1 mRNA levels were reduced in ovariectomized STZ-treated diabetic mice. Estrogen is known to stimulate Igf1 mRNA expression in the uterus, but estrogen action was abolished in the uteri of STZ-treated diabetic mice. mRNA expressions of estrogen receptor α (ERα) and steroid hormone receptor coactivators (SRC-1/Ncoa1, SRC-2/Ncoa2, SRC-3/Ncoa3 and CBP/p300/Crebbp) were reduced in the uteri of ovariectomized STZ-treated diabetic mice. The present study demonstrates that diabetes induces a decline in female reproductive functions in mice. Igf1 expression in ovariectomized diabetic female mice was decreased, and decreased responsiveness to estrogen in the uteri of diabetic mice is probably associated with a reduction in ERα and steroid receptor coactivator mRNA expression.

Expression patterns of insulin-like growth factor system members and their correlations with growth and carcass traits in Landrace and Lantang pigs during postnatal development

The IGF system plays important roles in growth. Nevertheless, few data have been reported so far on the expression of IGF system members and their relationship with growth in domestic animals, especially pigs. In this study, hepatic transcript level of IGF1, IGF2, IGF binding protein 2 (IGFBP2), IGFBP3 and IGF 1 receptor (IGF1R), plasma protein level of IGF1 and IGFBP3, and eight growth or carcass traits, including chest circumference, body length, body height (BH), body weight, carcass weight, loin muscle area (LMA), back fat thickness and average daily gain, were measured in fast-growing Landrace and slow-growing Lantang pigs at the age of 1, 27, 90, 150 and 180 days. The results showed that liver mRNA level of IGF1, IGF2 and IGF1R, and blood protein level of IGF1 have a similar developmental profile in both Landrace and Lantang pigs. Their levels were higher at the early age than that at other older ages. Hepatic transcript abundances of the two growth inhibitors, IGFBP2 and IGFBP3, were mostly higher in Lantang pigs than that in Landrace pigs, at 5 examined postnatal stages. The IGF system members' liver mRNA level and/or serum protein level have significant correlation with each other in different age of Landrace or Lantang pigs. Hepatic mRNA level or serum protein level of IGF system members also has significant correlation with investigated traits, especially with BH and LMA, in different age of Lantang or Landrace pigs. Our results revealed the change profiles of porcine IGF system members' liver transcript level and plasma protein level between different pig breeds and different postnatal developmental ages. Moreover, the correlation analysis results suggest that the IGF system members act coordinately to regulate the growth performance and carcass composition in pigs. The information obtained from the present study is important for elucidation of the regulatory mechanism of IGF system underlying growth, and for genetic improvement in pigs.

IGF2 mRNA binding protein p62/IMP2-2 in hepatocellular carcinoma: antiapoptotic action is independent of IGF2/PI3K signaling

The insulin-like growth factor II (IGF2) mRNA binding protein (IMP) p62/IMP2-2, originally isolated from a hepatocellular carcinoma (HCC) patient, induces a steatotic phenotype when overexpressed in mouse livers. Still, p62 transgenic livers do not show liver cell damage but exhibit a pronounced induction of Igf2 and activation of the downstream survival kinase AKT. The aim of this study was to investigate the relation between p62 and IGF2 expression in the human system and to study potential antiapoptotic actions of p62. p62 and IGF2 mRNA levels were assessed by real-time RT-PCR. For knockdown and overexpression experiments, human hepatoma HepG2 and PLC/PRF/5 cells were transfected with siRNA or plasmid DNA. Phosphorylated AKT and ERK1/2 were analyzed by Western blot. Investigations of 32 human HCC tissues showed a strong correlation between p62 and IGF2 expression. Of note, p62 expression was increased markedly in patients with poor outcome. In hepatoma cells overexpression of p62 lowered levels of doxorubicin-induced caspase-3-like activity. Vice versa, knockdown of p62 resulted in increased doxorubicin-induced apoptosis. However, neither PI3K inhibitors nor a neutralizing IGF2 antibody showed any effects. Western blot analysis revealed increased levels of phosphorylated ERK1/2 in hepatoma cells overexpressing p62 and decreased levels in p62 knockdown experiments. When p62-overexpressing cells were treated with ERK1/2 inhibitors, the apoptosis-protecting effect of p62 was completely abrogated. Our data demonstrate that p62 exerts IGF2-independent antiapoptotic action, which is facilitated via phosphorylation of ERK1/2. Furthermore, p62 might serve as a new prognostic marker in HCC.

IGF-I 3' untranslated region: strain-specific polymorphisms and motifs regulating IGF-I in osteoblasts.

Reduced IGF-I is associated with low bone mass in humans and mice. C3H/He/J (C3H) mice have higher skeletal IGF-I and greater bone mass than C57BL/6J (B6). We hypothesized that strain-related genotypic differences in Igf1 affected skeletal function. The Igf1 coding region is nonpolymorphic, but its 3' untranslated region (UTR) is polymorphic between C3H and B6. Luciferase-Igf1 3' UTR reporter constructs showed that these polymorphic regions did not affect UTR function. IGF-I splice variants give rise to a common mature IGF-I peptide, but different E peptides. We identified two splice products, exon 4+6 (Ea) and exon 4+5+6 (Eb, mechano-growth factor) and found that their abundance was unchanged during osteoblastic differentiation. The Igf1 3' UTR encoded by exon 6 contains alternative polyadenylation sites. Proximal site use produces a short 3' UTR of approximately 195 bases, whereas distal site usage results in an approximately 6300-base UTR. Although Igf1 mRNA levels did not change during osteoblastic differentiation, distal polyadenylation site usage was increased in B6 cells but not in C3H. The resulting long Igf1 RNA isoform is less stable and has decreased translation efficiency, which may be one mechanism contributing to decreased IGF-I in B6 vs. C3H mice. Although the long UTR contains a conserved [GU](18) repeat, which is a positive regulator of UTR activity, it is also targeted by negative regulators, miR-29 and miR-365. These microRNAs are increased in B6 and C3H cells during osteoblastic differentiation. Differential expression of the long Igf1 3' UTR isoform may be a possible mechanism for enhanced IGF-I regulation in B6 vs. C3H mice.

The production and regulation of IGF and IGFBPs in human adipose tissue cultures

BackgroundAdipocytes express and secrete IGFs and IGFBPs; proteins with important effects on adipocyte homeostasis. However, the factors that control adipocyte generation of IGFs and IGFBPs are not clarified. AimTo identify regulators of the synthesis of IGFs and IGFBs in adipose tissue. MethodsSubcutaneous adipose tissue fragments (500mg) from 7 healthy lean women were incubated for 48h following addition of GH (50μg/l), dexamethasone (DXM, 20nM), insulin (100nM), interleukin (IL)-1β (50ng/l), IL-6 (50ng/l) and tumor-necrosis factor (TNF)-α (10ng/l). Outcome parameters included tissue mRNA and culture media IGF and IGFBP levels. ResultsAdipose tissue cultures secreted more IGF-II than IGF-I protein (1.14±0.41 vs. 0.26±0.09μg/l [mean±SEM]; P<0.02). IGF-I mRNA and protein levels were stimulated by GH (to 340% [153; 477] (median [interquartiles]) and 270±26%, respectively; P<0.003), and inhibited by IL-1β (to 28% [21; 77] and 68±11%, respectively; P<0.003). TNF-α reduced IGF-I and IGF-II protein levels to 51±8% and 69±8%, respectively (P≤0.002), without affecting mRNA levels. IGF protein levels were unaffected by DXM, insulin and IL-6. All IGFBPs IGFBP-1 were expressed. IGFBP-4 was by far the most predominant IGFBP by immunoassay and WLB revealed two bands at 28 and 24kDa, most likely representing glycosylated and non-glycosylated IGFBP-4. ConclusionAdipose tissue cultures secrete more IGF-II than IGF-I, and predominantly IGFBP-4. The secretion of IGF-I is affected by GH, IL-1β and TNF-α, whereas IGF-II is affected by TNF-α only. Hence, cytokines may control adipocyte homeostasis by affecting local IGF-generation.

GH–IGF system regulation of attenuated muscle growth and lipolysis in Atlantic salmon reared at elevated sea temperatures

Growth regulation in adult Atlantic salmon (1.6 kg) was investigated during 45 days in seawater at 13, 15, 17, and 19 °C. We focused on feed intake, nutrient uptake, nutrient utilization, and endocrine regulation through growth hormone (GH), insulin-like growth factors (IGF), and IGF-binding proteins (IGFBP). During prolonged thermal exposure, salmon reduced feed intake and growth. Feed utilization was reduced at 19 °C after 45 days compared with fish at lower temperatures, and body lipid storage was depleted with increasing water temperature. Although plasma IGF-1 concentrations did not change, 32-Da and 43-kDa IGFBP increased in fish reared at ≤17 °C, and dropped in fish reared at 19 °C. Muscle igf1 mRNA levels were reduced at 15 and 45 days in fish reared at 15, 17, and 19 °C. Muscle igf2 mRNA levels did not change after 15 days in response to increasing temperature, but were reduced after 45 days. Although liver igf2 mRNA levels were reduced with increasing temperatures after 15 and 45 days, temperature had no effect on igf1 mRNA levels. The liver igfbp2b mRNA level, which corresponds to circulating 43-kDa IGFBP, exhibited similar responses after 45 days. IGFBP of 23 kDa was only detected in plasma in fish reared at 17 °C, and up-regulation of the corresponding igfbp1b gene indicated a time-dependent catabolic response, which was not observed in fish reared at 19 °C. However, higher muscle ghr mRNA levels were detected in fish at 17 and 19 °C than in fish at lower temperatures, indicating lipolytic regulation in muscle. These results show that the reduction of muscle growth in large salmon is mediated by decreased igf1 and igf2 mRNA levels in addition to GH-associated lipolytic action to cope with prolonged thermal exposure. Accordingly, 13 °C appears to be a more optimal temperature for the growth of adult Atlantic salmon at sea.

Experimental alcohol-related peripheral neuropathy: role of insulin/IGF resistance.

The mechanisms of alcohol-related peripheral neuropathy (ALPN) are poorly understood. We hypothesize that, like alcohol-related liver and brain degeneration, ALPN may be mediated by combined effects of insulin/IGF resistance and oxidative stress. Adult male Long Evans rats were chronically pair-fed with diets containing 0% or 37% ethanol (caloric), and subjected to nerve conduction studies. Chronic ethanol feeding slowed nerve conduction in the tibial (p = 0.0021) motor nerve, and not plantar sensory nerve, but it did not affect amplitude. Histological studies of the sciatic nerve revealed reduced nerve fiber diameters with increased regenerative sprouts, and denervation myopathy in ethanol-fed rats. qRT-PCR analysis demonstrated reduced mRNA levels of insulin, IGF-1, and IGF-2 polypeptides, IGF-1 receptor, and IRS2, and ELISAs revealed reduced immunoreactivity for insulin and IGF-1 receptors, IRS-1, IRS-4, myelin-associated glycoprotein, and tau in sciatic nerves of ethanol-fed rats (all p < 0.05 or better). The findings suggest that ALPN is characterized by (1) slowed conduction velocity with demyelination, and a small component of axonal degeneration; (2) impaired trophic factor signaling due to insulin and IGF resistance; and (3) degeneration of myelin and axonal cytoskeletal proteins. Therefore, ALPN is likely mediated by molecular and signal transduction abnormalities similar to those identified in alcoholic liver and brain degeneration.

Effects of dietary arginine levels and carbohydrate-to-lipid ratios on mRNA expression of growth-related hormones in largemouth bass, Micropterus salmoides

Utilizing the tissue samples and growth data collected from our two preceding researches in largemouth bass (LMB), we have investigated effects of dietary arginine (Arg) levels and carbohydrate-to-lipid (CHO/LIP) ratios on the GH, IGF-I and insulin expression in related tissues to find possible relationships between the nutrient intake, growth performance and transcript level. Hepatic IGF-I and pituitary GH mRNA levels were significantly up-regulated by lower dietary Arg levels from 1.94% to 3.01% and by higher levels from 2.76% to 3.01%, respectively, while Brockmann body (BB)-containing tissue insulin mRNA expression was not affected. Dietary CHO/LIP ratios ranging from 0.32 to 5.17 (w/w) affected pituitary GH, liver IGF-I and BB-containing tissue insulin mRNA expression in a ratio-specific pattern. The lower ratios from 0.32 to 2.36 significantly up-regulated GH and insulin transcript levels, but significantly down-regulated IGF-I transcript levels; the higher ratios did no longer exert any further effects on them. Meanwhile, two strong positive correlations (r=0.892, r=0.885) between hepatic IGF-I transcript levels and specific growth rates of tested fish were observed with varying dietary Arg levels and CHO/LIP ratios, respectively. These findings indicate that in LMB dietary Arg levels and CHO/LIP ratios regulate differentially the endocrine system of GH, IGF-I and insulin at transcription level; this system, in turn, plays a fundamental role in the regulation of the nutrient metabolism and somatic growth; and that hepatic IGF-I mRNA abundance should be a more reliable index to assess growth and nutritional fitness than the others, at least in LMB.

Relationships between gill Na+,K+-ATPase activity and endocrine and local insulin-like growth factor-I levels during smoltification of masu salmon (Oncorhynchus masou)

We established profiles of insulin-like growth factor (IGF)-I mRNA in the liver, gill and white muscle and circulating IGF-I during smoltification of hatchery-reared masu salmon, and compared with that of gill Na+,K+-ATPase (NKA) activity. Gill NKA activity peaked in May and dropped in June. Liver igf1 mRNA was high in March and decreased to low levels thereafter. Gill igf1 increased from March, maintained its high levels during April and May and decreased in June. Muscle igf1 mRNA levels were relatively high during January and April when water temperature was low. Serum IGF-I continuously increased from March through June. Serum IGF-I during March and May showed a positive correlation with NKA activity, although both were also related to fish size. These parameters were standardized with fork length and re-analyzed. As a result, serum IGF-I and gill igf1 were correlated with NKA activity. On the other hand, samples from desmoltification period (June) that had high serum IGF-I levels and low NKA activity disrupted the relationship. Expression of two IGF-I receptor (igf1r) subtypes in the gill decreased in June, which could account for the disruption by preventing circulating IGF-I from acting on the gill and retaining it in the blood. The present study suggests that the increase in gill NKA activity in the course of smoltification of masu salmon was supported by both endocrine and local IGF-I, and the decrease during desmoltification in freshwater was due at least in part to the down-regulation of gill IGF-I receptors.

Transforming Growth Factor-α mRNA Expression and Its Possible Roles in Mouse Endometrial Stromal Cells

Transforming growth factor-α (TGFα) is thought to be involved in the regulation of endometrial cells. We investigated Tgfa mRNA expression, and the effects of TGFα on DNA-synthesis and gene expression of insulin-like growth factor 1 (IGF1), IGF binding protein-3 (IGFBP3) and IGF1 receptor in the mouse endometrial cells, because IGF1 is involved in estrogen-induced growth of endometrial cells. We also investigated the role of TGFα on matrix metalloproteinase (MMP) expression, as MMPs are involved both in tissue remodeling during cell proliferation and in enhancement of IGF1 signaling through the degradation of IGFBP3. Tgfa mRNA expression was detected in endometrial luminal and glandular epithelial cells, and stromal cells. Tgfa mRNA signals did not appear to change in endometrial luminal epithelial cells, but signals in glandular epithelial cells were higher at diestrus 1, 2 and proestrus, and the number of stromal cells showing strong signals appeared to increase at diestrus 1 and 2. Endometrial epithelial and stromal cells were treated with estradiol-17β (E2) or progesterone (P4). E2 or P4 stimulated Tgfa mRNA expression in stromal cells. TGFα stimulated DNA synthesis in endometrial epithelial and stromal cells, while E2 and P4 stimulated DNA synthesis in stromal cells. In stromal cells, TGFα, at as low as 1 ng/ml, decreased Igfbp3 and Mmp9 mRNA levels, while high dose (10 ng/ml) of TGFα decreased Igf1 mRNA level and increased Mmp3 mRNA level. These results imply that TGFα stimulates proliferation of endometrial stromal cells through multiple mechanisms, including its regulation of Igfbp3 and Mmp3 transcription.

Regulation of IGF-1 but not TGF-β1 by NGF in the smooth muscle of the inflamed urinary bladder

Intraperitoneal injection of cyclophosphamide (CYP) causes hemorrhagic cystitis with excess growth of muscular layer leading to bladder hypertrophy; this could be attributable to changes in the expression profiles of growth factors in the inflamed urinary bladder. The growth factors characterized in the current study include nerve growth factor (NGF), insulin-like growth factor (IGF)-1, and transforming growth factor (TGF)-β1. We found that following CYP injection for 8h and 48h, the mRNA levels of all three factors were increased in the inflamed bladder when compared to control. The level of NGF mRNA was mainly increased in the urothelium layer while the levels of IGF-1 mRNA and TGF-β1 mRNA were increased in the smooth muscle layer. The level of NGF high affinity receptor TrkA mRNA was also increased in both the urothelium and the smooth muscle layers during bladder inflammation. When we blocked NGF action with NGF neutralizing antibody in vivo, we found that the up-regulation of IGF-1 in the inflamed bladder was reversed while the up-regulation of TGF-β1 was not affected by NGF neutralization. The effect of NGF on regulating IGF-1 expression was further confirmed in bladder smooth muscle culture showing that exogenous NGF increased the mRNA level of IGF-1 after 30min to 1h stimulation. These results suggested that bladder inflammation induced region-specific changes in the expression profiles of NGF, IGF-1 and TGF-β1. The up-regulation of NGF in the urothelium may have a role in affecting bladder smooth muscle cell physiology by regulating IGF-1 expression.

Regulation of H19 and its encoded microRNA-675 in osteoarthritis and under anabolic and catabolic in vitro conditions

Cartilage degeneration in the course of osteoarthritis (OA) is associated with an alteration in chondrocyte metabolism. In order to identify molecules representing putative key regulators for diagnosis and therapeutic intervention, we analyzed gene expression and microRNA (miR) levels in OA and normal knee cartilage using a customized cartilage cDNA array and quantitative RT-PCR. Among newly identified candidate molecules, H19, IGF2, and ITM2A were significantly elevated in OA compared to normal cartilage. H19 is an imprinted maternally expressed gene influencing IGF2 expression, whose transcript is a long noncoding (lnc) RNA of unknown biological function harboring the miR-675. H19 and IGF2 mRNA levels did not correlate significantly within cartilage samples suggesting that deregulation by imprinting effects are unlikely. A significant correlation was, however, observed for H19, COL2A1, and miR-675 expression levels in OA tissue, and functional regulation of these candidate molecules was assessed under anabolic and catabolic conditions. Culture of chondrocytes under hypoxic signaling showed co-upregulation of H19, COL2A1, and miRNA-675 levels in close correlation. Proinflammatory cytokines IL-1β and TNF-α downregulated COL2A1, H19, and miR-675 significantly without close statistical correlation. In conclusion, this is the first report demonstrating deregulation of an lncRNA and its encoded miR in the context of OA-affected cartilage. Stress-induced regulation of H19 expression by hypoxic signaling and inflammation suggests that lncRNA H19 acts as a metabolic correlate in cartilage and cultured chondrocytes, while the miR-675 may indirectly influence COL2A1 levels. H19 may not only be an attractive marker for cell anabolism but also a potential target to stimulate cartilage recovery.

Abstract 22: Differential IGF-II response of AA and CA normal breast cell lines to prolactin treatment

Abstract African-American (AA) breast cancer patients have lower survival compared with Caucasian-American (CA) patients. This survival disparity is significant in view of the lower incidence of breast cancer among AA. Lower incidence of breast cancer is associated with parity and lactation in young women. Lactation's protective effect is mediated by fatty-acid binding protein 3 (FABP3), a protein that promotes differentiation of breast epithelial cells. We have demonstrated that AA normal women below 50 years have lower FABP3 levels as compared to CA women (p = 0.030). FABP3 is a protective tumor suppressor that increases during pregnancy and lactation, in response to the lactation hormone prolactin (PRL). PRL effects on the breast are mediated by IGF-II. Thus, we propose that PRL treatment of normal epithelial breast cell lines would increase IGF-II to promote differentiation, increase FABP3, and decrease FABP5. FABP5 is associated with metastasis and may compensate for FABP3 decrease. We treated MCF10A (CA) and AG11132 (AA) normal breast epithelial cell lines with recombinant human PRL (0-200 ng/ml) for 24 hours. Cell lysates were examined for protein and mRNA levels of IGF-II, FABP3, and FABP5. In MCF10A cells, PRL stimulated an increase in protein levels of IGF-II (p=0.044), and a decrease in protein levels of FABP5 (p=0.044). Protein levels of FABP3 significantly decreased with increasing PRL concentration (p=0.030). IGF-II mRNA levels also increased at 50 and 200 ng/ml PRL (2.59 and 2.58 fold, respectively). In AG11132 cells, protein levels of FABP3 significantly increased with increasing PRL concentration (p=0.016). IGF-II and FABP5 mRNA levels increased significantly at 10 ng/ml PRL (49.91 and 2.13 fold, respectively); however, no corresponding changes were seen in protein levels. Though both cell lines respond to PRL treatment, only the MCF10A line displays increased IGF-II and decreased FABP5, which are protective changes in normal mammary cells. FABP3 levels decreased in MCF10A cells at higher PRL concentrations; this may be due to untreated MCF10A cells expressing significantly more FABP3 and prolactin, relative to AG11132 cells. The higher levels of PRL and FABP3 in untreated MCF10A cells indicate that they are more differentiated and thus require less PRL stimulation, relative to the AG11132 cells, to achieve a differentiated state. The dissimilar responsiveness of these cell lines to PRL may suggest decreased protection against breast carcinogenesis in AA patients, and may help us to further understand this health disparity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 22. doi:1538-7445.AM2012-22

Abstract 3131: Higher IGF-II expression in Vietnamese triple negative breast cancer tumors is dependent on Biallelic or Monoallelic IGF-II with ApaI SNP

Abstract Triple negative breast cancer (TNBC) is more likely to be advanced, poorly differentiated, larger, and present in women of younger age and of lower socioeconomic status. Moreover, TNBC patients have worse outcomes when treated with currently available adjuvant chemotherapy. In the United States, African American (AA) women have a lower incidence of breast cancer compared with Caucasian women (CA), but higher overall mortality. Our lab have demonstrated that BC tumors from AA patients express significantly higher levels of IGF-II and show a higher activation of the IGF signaling pathways (Kalla et.al., 2010a, 2010b). IGF-II levels regulate survival proteins and activate the ER signaling pathway promoting an aggressive tumor development. Thus we designed a study to determine if IGF-II regulates the progression of TNBC tumors. TNBC incidence is increasing in younger Vietnamese women. Thus, we designed a study to analyze the IGF-II expression in paired normal-tumor BC tissues from Vietnamese TNBC patients. Furthermore, we decided to characterize the IGF-II genomic imprinting status, to correlate IGF-II levels with Monoallelic or Biallelic IGF-II gene expression. A total of 24 paired BC samples obtained from ILSbio were analyzed by Western Blot, RT-PCR and gDNA PCR with Apa I restriction digestion. Our results showed that the IGF-II genomic imprinting status in all 24 pairs was the same in normal and tumor tissues from the same patient. The levels of IGF-II correlated with the imprinting status, i.e., Biallelic (BA; n=14)≫, Monoallelic with SNP (MAS; n=5)&amp;gt;, and Monoallelic non-ApaI SNP (MA; n=5)&amp;gt;. Higher IGF-II levels were expressed in tumor as compared to normal tissue from the same patient. The free proIGF-II protein levels were 6.5-15.22 fold higher in biallelic tumor tissues as compared to the proIGF-II levels detected in monoallelic tumor samples. The IGF-II mRNA levels were 0.4-1 fold higher in biallelic tumor tissues as compared to the proIGF-II levels detected in monoallelic tumor samples. We also assessed growth factor receptors; Insulin receptor A (IRA), Insulin receptor B (IRB), Vascular endothelial growth factor receptor (VEGFR), Estrogen receptor-1 (ESR1), Progesterone receptor (PGR) and Human epidermal growth factor receptor (ERBB2) status to determine if IGF-II monoallelic or biallelic dosage regulated the receptor levels. We conclude that biallelic IGF-II expression in TNBC tumors results in higher IGF-II mRNA and protein levels than in monoallelic samples. Furthermore, higher IGF-II resulted in increased expression and signaling activation of IRA, IRB, VEGFR, ESR1, PGR and ERBB2 growth factor receptors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3131. doi:1538-7445.AM2012-3131

Androgens Affect Myogenesis In Vitro and Increase Local IGF-1 Expression

The mechanism whereby anabolic androgens are associated with hypertrophy of skeletal muscle is incompletely understood but may involve an interaction with locally generated insulin-like growth factor (IGF) 1. The present investigation utilized a cell culture model of human skeletal muscle-derived cell maturation to test the hypothesis that androgens increase differentiation of human muscle precursor cells in vitro and to assess effects of androgen with or without IGF-1 on IGF-1 messenger RNA (mRNA) expression in human muscle precursor cells. Differentiation of muscle-derived cells was induced under standard low-serum conditions. Cultures were then exposed to androgen (testosterone (T)) at 50, 100, and 500 nM or IGF-1 (10-50 ng·mL⁻¹). Immunocytochemistry and real-time polymerase chain reaction (RT-PCR) were used to assess effects of androgens and IGF-1 after 3- (early) or 7-d (late) muscle differentiation, respectively; RT-PCR was used to quantify the effects on androgen receptor expression. Under low-serum conditions, 3-d exposure to androgens or IGF-1 or both resulted in no significant increase in cellular myogenic commitment. After 7-d exposure, however, T and IGF-1 were both found to increase fusion index with no observable synergistic effect. T also increased IGF-1 mRNA generation (P < 0.0001), whereas exogenous IGF-1 (P < 0.001) reduced IGF-1 mRNA transcription relative to control. The T effect was reversible after treatment with flutamide, an androgen receptor antagonist. Both T and IGF-1 increase myogenic commitment after 7-d exposure to a differentiation medium. With T causing a concomitant increase in IGF-1 mRNA underpinning IGF-1 as a central mediator in the cellular pathways associated with muscle hypertrophy, including those affected by androgens. The novel system described has the potential for elucidating the pattern of growth factor effects associated with androgens in skeletal muscle.

Pubertal effects of 17α-methyltestosterone on GH–IGF-related genes of the hypothalamic–pituitary–liver–gonadal axis and other biological parameters in male, female and sex-reversed Nile tilapia

The influence of 17α-methyltestosterone (MT) on growth responses, biological parameters and the expression of genes involved in the GH–IGF pathway of the hypothalamic–pituitary–liver–gonadal axis were investigated in female, male, and sex-reversed Nile tilapia to evaluate the relationship between sex and MT-induced changes in these parameters. Female fish had a lower growth rate than male and sex-reversed fish, and MT increased growth performance and duodenal villi in females. Most but not all biological parameters of sex-reversed fish were similar to those of male fish. Male fish had higher red blood cell counts and hemoglobin levels than female and sex-reversed fish, suggesting that these hematological indices reflect a higher metabolic rate in male fish. Greater blood triglyceride levels indicated the vitellogenin process in female fish. MT increased the alternative complement activity in female fish (P<0.05). Sex and MT had no significant effects on the hypothalamic mRNAs of GHRH and PACAP. Although not statistically significant, females tended to have higher GH mRNA levels than male and sex-reversed fish. Additionally, MT tended to decrease and increase GH mRNA levels in female and male fish, respectively. There were significant differences among sexes in the expression of GHR, and IGF mRNAs at the peripheral level in the liver and gonads. Females had lower hepatic GHRs and higher ovarian GHRs than male and sex-reversed fish. While the mRNA levels of IGF-1 were lower in the ovary, the levels of IGF-2 were higher compared with those in testes. A significant correlation between GHRs and IGFs was demonstrated in the liver and gonad (except for IGF-1). Multiple regression analysis showed a significant relationship between GH mRNA and both GHRs and IGFs in the liver and gonad. MT exerted androgenic and, to some extent, estrogenic effects on several physiological parameters and GH–IGF action.

Growth and endocrine effects of recombinant bovine growth hormone treatment in non-transgenic and growth hormone transgenic coho salmon

To examine the relative growth, endocrine, and gene expression effects of growth hormone (GH) transgenesis vs. GH protein treatment, wild-type non-transgenic and GH transgenic coho salmon were treated with a sustained-release formulation of recombinant bovine GH (bGH; Posilac™). Fish size, specific growth rate (SGR), and condition factor (CF) were monitored for 14weeks, after which endocrine parameters were measured. Transgenic fish had much higher growth, SGR and CF than non-transgenic fish, and bGH injection significantly increased weight and SGR in non-transgenic but not transgenic fish. Plasma salmon GH concentrations decreased with bGH treatment in non-transgenic but not in transgenic fish where levels were similar to controls. Higher GH mRNA levels were detected in transgenic muscle and liver but no differences were observed in GH receptor (GHR) mRNA levels. In non-transgenic pituitary, GH and GHR mRNA levels per mg pituitary decreased with bGH dose to levels seen in transgenic salmon. Plasma IGF-I was elevated with bGH dose only in non-transgenic fish, while transgenic fish maintained an elevated level of IGF-I with or without bGH treatment. A similar trend was seen for liver IGF-I mRNA levels. Thus, bGH treatment increased fish growth and influenced feedback on endocrine parameters in non-transgenic but not in transgenic fish. A lack of further growth stimulation of GH transgenic fish suggests that these fish are experiencing maximal growth stimulation via GH pathways.

Sodium arsenite represses the expression of myogenin in C2C12 mouse myoblast cells through histone modifications and altered expression of Ezh2, Glp, and Igf-1

Arsenic is a toxicant commonly found in water systems and chronic exposure can result in adverse developmental effects including increased neonatal death, stillbirths, and miscarriages, low birth weight, and altered locomotor activity. Previous studies indicate that 20nM sodium arsenite exposure to C2C12 mouse myocyte cells delayed myoblast differentiation due to reduced myogenin expression, the transcription factor that differentiates myoblasts into myotubes. In this study, several mechanisms by which arsenic could alter myogenin expression were examined. Exposing differentiating C2C12 cells to 20nM arsenic increased H3K9 dimethylation (H3K9me2) and H3K9 trimethylation (H3K9me3) by 3-fold near the transcription start site of myogenin, which is indicative of increased repressive marks, and reduced H3K9 acetylation (H3K9Ac) by 0.5-fold, indicative of reduced permissive marks. Protein expression of Glp or Ehmt1, a H3-K9 methyltransferase, was also increased by 1.6-fold in arsenic-exposed cells. In addition to the altered histone remodeling status on the myogenin promoter, protein and mRNA levels of Igf-1, a myogenic growth factor, were significantly repressed by arsenic exposure. Moreover, a 2-fold induction of Ezh2 expression, and an increased recruitment of Ezh2 (3.3-fold) and Dnmt3a (~2-fold) to the myogenin promoter at the transcription start site (−40 to +42), were detected in the arsenic-treated cells. Together, we conclude that the repressed myogenin expression in arsenic-exposed C2C12 cells was likely due to a combination of reduced expression of Igf-1, enhanced nuclear expression and promoter recruitment of Ezh2, and altered histone remodeling status on myogenin promoter (−40 to +42).