IGF-I Protein Levels Research Articles

Therapeutic Effects of IGF-1 on Stress Urinary Incontinence in Rats with Simulated Childbirth Trauma

We examined the effect of IGF-1 in a rat model of stress urinary incontinence induced by simulated childbirth trauma. Simulated birth trauma was induced by vaginal distension in female Sprague Dawley® rats. Four, 7, 14 and 28 days after distension we performed functional assessment by measuring leak point pressure, urethral baseline pressure and the urethral response during a passive increment in intravesical pressure. The expression of IGF-1 and IGF1R mRNA and protein in damaged tissues was examined by real-time reverse transcriptase-polymerase chain reaction and immunohistochemistry. Thereafter hrIGF-1 (50 and 150 μg/kg per day) was continuously delivered from 1 day before distension using subcutaneous osmotic pumps. Four and 7 days after distension the effect of hrIGF-1 treatment was examined by functional analysis of leak point pressure, urethral baseline pressure and the urethral response as well as Western blot and histological analysis. After 4 and 7 days rats with vaginal distension had significantly decreased leak point pressure, urethral baseline pressure and urethral responses. IGF-1 and IGF1R mRNA and protein levels were significantly increased in urethral and pudendal nerves 4 and 7 days after distension. IGF-1 treated groups showed significant improvement in leak point pressure, urethral baseline pressure and urethral responses 4 and 7 days after distension. Moreover, IGF-1 treatment increased Akt phosphorylation and induced cellular proliferation and antiapoptotic effects in the urethra. IGF-1 treatment accelerated recovery from stress urinary incontinence induced by simulated childbirth trauma in association with activation of the Akt signal transduction pathway in rats. This suggests that IGF-1 has therapeutic potential for stress urinary incontinence in women.

Role of the IGF-1/PI3K pathway and the molecular mechanism of Fuzhenghuayu therapy in a spontaneous recovery rat model of liver fibrosis

To determine the role of IGF-1/PI3K pathway and investigate the molecular mechanism of Fuzhenghuayu (FZHY) therapy in a spontaneous recovery rat model of liver fibrosis. The liver fibrosis model was induced in male Wistar rats by administering 8 weeks of twice weekly CCL4 intraperitoneal injections without (untreated model) or with once daily FZHY (treated model). Normal, untreated rats served as the control group. At weeks 4, 6 and 8 (fibrosis) and 10, 12 and 14 (spontaneous recovery) after modeling initiation, effects on protein (a-SMA, IGF-1, PI3K) and mRNA (IGF-1, PI3K) expression levels were evaluated by immunohistochemistry and RT-PCR, respectively. Serum markers of liver function (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) and liver cell damage (alkaline hydrolysis, HYP) were measured. Histology was performed to assess the degree of inflammation and fibrosis (Ishak scoring system). In the untreated model group, progression of liver fibrosis (weeks 4, 6 and 8) was accompanied by gradual increases in inflammation, necrosis, serum ALT and AST, and hepatic expression of a-SMA protein and IGF-1 and PI3K protein and mRNA; however, during the spontaneous recovery period (weeks 10, 12 and 14) the IGF-1 and PI3K protein and mRNA levels rapidly decreased and the HYP level, Ishak score, and a-SMA hepatic expression also decreased. The FZHY-treated model group showed significantly lower fibrosis-related up-regulation of IGF-1 and PI3K protein and mRNA expression, HYP level, Ishak score, and a-SMA hepatic expression at each time point (vs. untreated model group). The IGF-1/PI3K pathway may contribute to progression of liver fibrosis. The mechanism by which FZHY prevents liver fibrosis in a rat model may involve blocking of the IGF/PI3K pathway and inhibiting HSC activation.

High Prevalence of Cardiometabolic Risk Factors in Hispanic Adolescents: Correlations with Adipocytokines and Markers of Inflammation

This study assessed the association of cardiometabolic risk factors with systemic inflammation, insulin resistance, and adypocytokines in a Hispanic adolescent subgroup. A clinic-based sample of 101 Puerto Rican adolescents, 48 of whom were overweight or obese based on body mass index percentiles for age and sex, was recruited during 2010. Data were collected through interviews, blood pressure and anthropometric measurements, and blood drawing. Overall prevalence of the metabolic syndrome was 16.8 % and increased to 37.5 % among overweight/obese youth. The overweight/obese group exhibited significantly (p < 0.05) higher values for abdominal obesity measures, systolic blood pressure, triglycerides, insulin resistance, C peptide, high-sensitivity C reactive protein, fibrinogen, leptin, and IL-6 and lower levels of high density lipoprotein cholesterol, adiponectin, and IGF-1. Total adiponectin significantly correlated with most cardiovascular risk factors independent of sex, Tanner stage, and adiposity. Altered cardiometabolic and adipocytokine profiles were present in this Hispanic subgroup, reinforcing the need to strengthen strategies addressing childhood obesity.

Activated apoptotic and anti‐survival effects on rat hearts with fructose induced metabolic syndrome

Consumption of fructose has been linked to the development of metabolic syndrome, whereas the cardiomyopathic changes and cardiac apoptosis of dietary high-fructose intake have not yet been clarified. The purpose of this study was to evaluate the effects of high-fructose on cardiac apoptotic and survival pathways. Thirty-two Wistar rats were randomly divided into a control group (CON), which received a standard chow diet, and a fructose-induced metabolic syndrome group (FIMS), which received a 50% fructose-content diet for 13 weeks. Histopathological analysis, TUNEL assays and Western blotting were performed on the excised hearts from both groups. The blood pressure, glucose, insulin, triglyceride and cholesterol levels were significantly increased in the FIMS group, compared with the CON group. The abnormal myocardial architecture, enlarged interstitial space and increased cardiac TUNEL-positive apoptotic cells were observed in the FIMS group. The TNF-α, TNF receptor 1, Fas ligand, Fas receptor, FADD, and activated caspase-3 and 8 protein levels (Fas pathway) and the Bax, Bak, Bax/Bcl-2, Bak/Bcl-xL, cytosolic cytochrome c, and activated caspase-3 and nine protein levels (mitochondria pathway) were increased in the FIMS group compared with those in the CON group. The IGFI, IGFI-R, p-PI3K, p-Akt, Bcl-2 and Bcl-xL protein levels (survival pathway) were all significantly decreased in the FIMS group compared with those in the CON group. High-fructose intake elevated blood pressure and glucose levels; moreover, high-fructose diet activated cardiac Fas-dependent and mitochondria-dependent apoptotic pathways and suppressed the survival pathway, which might provide one possible mechanism for developing heart failure in patients with metabolic syndrome.

Down-regulation of programmed cell death 5 by insulin-like growth factor 1 in osteoarthritis chondrocytes

The aim of this study was to investigate the expression of insulin-like growth factor (IGF)-1 and programmed cell death 5 (PDCD5) in osteoarthritis chondrocytes, and to explore the potential correlation between them in the apoptosis process of osteoarthritis chondrocytes. Patients with knee osteoarthritis were placed into four categories according to radiological staging. The mRNA and protein levels of IGF-1 and PDCD5 in osteoarthritis chondrocytes were respectively detected by quantitative reverse transcriptase polymerase chain reaction (qPCR) and western blotting. In addition, IGF-1 and PDCD5 protein expression in chondrocytes were also measured by immunohistochemistry. Apoptotic cells were measured by TUNEL staining. Both the mRNA and protein levels of IGF-1 were down-regulated, while the levels of PDCD5 were up-regulated, and the mRNA and protein levels of IGF-1 were negatively correlated with those of PDCD5, respectively. The apoptotic cell was significantly increased in osteoarthritis chondrocytes compared with control. Importantly, the apoptosis rate was positively correlated with PDCD5 protein expression and negatively correlated with IGF-1 protein expression We concluded that IGF-1 may down-regulate the expression of PDCD5 and thus inhibit the apoptosis of osteoarthritis chondrocytes.

Expression of the IGF-1, IGFBP-3 and IGF-1 receptors in dental pulp stem cells and impacted third molars

IGF-1 regulates the metabolism of hard dental tissue through binding to the IGF-1 receptor on target cells. Furthermore, IGF-binding-protein-3 promotes the accessibility of IGF-1. The aim of this study was to investigate the expression of IGF-1, IGFBP-3 and IGF-1R in STRO-1-positive dental pulp stem cells (DPSCs) and fully impacted wisdom teeth in relation to tooth development. Third molars were surgically removed from 60 patients and classified into two groups: teeth showing ongoing development (group 1) and teeth that had completed root shaping (group 2). The transcript and protein levels of IGF-1, IGFBP-3 and IGF-1R were investigated using RT-PCR and immunohistochemistry. The expression of the same proteins was also analyzed in DPSCs. The teeth from group 1 showed significantly stronger expression of IGF-1 and IGF-1R. The major sources of all of the proteins investigated immunohistochemically in sections of wisdom teeth were odontoblasts, cementoblasts and cell colonies in the pulpal mesenchyme. These colonies were identified as stem cells in view of their positivity for STRO-1, and the cells were subsequently sorted by flow cytometry. These DPSCs demonstrated high levels of pluripotency markers and IGF-1 and IGF-1R. We conclude that members of the IGF-1 family are involved in the late stage of tooth development and the process of pulpal differentiation.

Relaxed imprinting of IGF2 in peripheral blood cells of patients with a history of prostate cancer

BackgroundInsulin-like growth factor 2 (IGF2) is the predominant IGF in adults and regulates cell growth. In contrast to normal tissues, where IGF2 is imprinted and only expressed from the paternal allele, loss of imprinting (LOI) and biallelic IGF2 expression are observed in many cancers including prostate cancer (PCa). We here studied whether LOI of IGF2 in normal circulating peripheral blood lymphocytes can predict increased PCa risk.Samples and methodsWe analyzed IGF2 protein levels, IGF2 820G/A genotype and imprinting status, as well as methylation status of the IGF2 imprinting control region (ICR) in 113 blood samples of patients with a history of radical prostatectomy (RPE) for PCa by ELISA, restriction-fragment length polymorphism, and bisulfite-DNA sequencing. Results were compared to 249 male blood donors with unknown prostate specific antigen (PSA) status.ResultsThe 820G/A genotype was enriched in the RPE group and was associated with younger age at cancer diagnosis. LOI in patients was only slightly more frequent than in controls, but IGF2 levels were significantly higher and uncoupled from the imprinting status. Analysis of the IGF2/H19 ICR revealed marked hypermethylation.ConclusionsThe IGF 820G/A genotype is associated with PCa diagnosis at younger age. Increased IGF2 in patients with PCa appears to be the result of impaired imprinting in non-neoplastic cells rather than a paracrine tumor product. Uncoupling of IGF2 protein levels from imprinting status (not LOI alone) and hypermethylation of the ICR characterized PCa patients and could have the potential to indicate persons at risk in screening programs.

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.

Decline in Circulating Insulin-Like Growth Factors and Mortality in Older Adults: Cardiovascular Health Study All-Stars Study

The association between changes in IGF-I and IGF binding protein (IGFBP) levels and mortality in older adults is unknown. Participants were 997 persons 77 to 100 yr old enrolled in the Cardiovascular Health Study All Stars Study. Plasma levels of IGF-I, IGFBP-1, and IGFBP-3 were assessed at two study examinations (1996-1997 and 2005-2006). Mortality was assessed between 2006 and 2010. Cumulative mortality (CM) was similar among individuals who had at least 10% decreases over time in IGF-I levels (CM = 29.6%), individuals who had at least 10% increases over time in IGF-I levels (CM = 24.7%), and individuals who had IGF-I levels remaining within ±10% over time (CM = 23.5%). Adjusted for age, sex, race, diabetes, body mass index, creatinine, albumin, and C-reactive protein, decreasing IGF-I level had no significant association with overall cancer mortality or noncancer mortality. Levels of IGFBP-1 increased markedly over time by 38% (median). Individuals with the largest increases in IGFBP-1 level over time had significantly increased risk of mortality. The adjusted hazard ratio per sd of IGFBP-1 change was 1.40 for overall cancer mortality (95% confidence interval = 1.10, 1.77; P = 0.01) and 1.14 for noncancer mortality (95% confidence interval = 1.02, 1.27; P = 0.02). Changes in IGFBP-3 levels were not significantly associated with mortality. Among older adults, decreasing IGF-I level over time does not predict subsequent all-cause mortality, whereas increasing IGFBP-1 predicts increased risk of mortality.

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).

Anti-apoptotic and pro-survival effects of exercise training on hypertensive hearts

Activated cardiac apoptosis was found in hearts from hypertensive animals, but little information regarding the effects of exercise training on cardiac apoptosis in hypertension is available. The purpose of this study was to evaluate the anti-apoptotic and pro-survival effects of exercise training on hypertensive hearts. 28 spontaneously hypertensive rats were divided into sedentary group (SHR) or underwent running exercise on treadmill for 1 h/day, 5 sessions/wk, for 12 wk (SHR-EX). Fourteen age-matched Wistar Kyoto rats served as a sedentary normotensive group (WKY). After exercise training or sedentary status, the excised hearts were measured by hemotoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) assay, and Western blotting. Fewer TUNEL-positive apoptotic cells were in SHR-EX groups than those in SHR. Protein levels of Fas ligand, Fas death receptor, tumor necrosis factor (TNF)-α, TNF receptor 1, Fas-associated death domain (FADD), activated caspase-8, and activated caspase-3 (Fas-dependent apoptotic pathways), as well as Bid, t-Bid, Bad, p-Bad, Bak, cytochrome c, activated caspase 9, and activated caspase-3 (mitochondria-dependent apoptotic pathways) were decreased in the SHR-EX group compared with the SHR group. Protein levels of IGF-1, IGF-1R, p-PI3K, p-Akt, p-Bad, and Bcl2 (cardiac pro-survival pathway) become more activated in SHR-EX groups than SHR and WKY. Exercise training prevented hypertension-enhanced cardiac Fas-dependent and mitochondria-dependent apoptotic pathways and enhanced cardiac pro-survival pathway in rat models. Our findings demonstrate new therapeutic effects of exercise training on hypertensive hearts for preventing apoptosis and enhancing survival.

The functional deficiency of bone marrow mesenchymal stromal cells in ALS patients is proportional to disease progression rate

Amyotrophic lateral sclerosis (ALS) is caused by motor neuron death. The relationship between the prognosis of ALS patients and the function of their bone marrow mesenchymal stromal cells (BM-MSCs) is unclear. We designed this study to assess the correlation between the progression rate of the ALS Functional Rating Scale-revised version (ΔFS), which is reported to predict prognosis, and the pluripotency and trophic factor secreting capacity of ALS patients' BM-MSCs. We evaluated ΔFS in 23 ALS patients and isolated BM-MSCs from those patients and five healthy people. Levels of Nanog, Oct-4, and Nestin mRNA were examined to evaluate pluripotency, and levels of BDNF, ECGF1, bFGF-2, HGF, IGF-1, PGF, TGF-1β, SDF-1α, GDNF, VEGF, and ANG mRNA were examined to assess trophic factor secreting capacity. In addition, we measured the protein levels of Nanog, Oct-4, Nestin, SDF-1α, ANG, bFGF-2, VEGF, IGF-1, GDNF, and BDNF. mRNA levels of Nanog, Oct-4, ECGF1, bFGF-2, HGF, IGF-1, PGF, TGF-1β, SDF-1α, GDNF, VEGF, and ANG were negatively correlations with ΔFS. However, those of Nestin and BDNF were not significantly correlated with ΔFS. Similarly, Nanog, Oct-4, SDF-1α, ANG, bFGF-2, VEGF, IGF-1, and GDNF protein levels had a significant negative correlation with ΔFS. Results indicate that the pluripotency and trophic factor secreting capacity of the BM-MSCs of ALS patients are reduced in proportion to a poorer prognosis. We therefore suggest that healthy allogeneic BM-MSCs might be a better option for cell therapy in ALS patients.

Insulin Growth Factor-2 Binding Protein 3 (IGF2BP3) Is a Glioblastoma-specific Marker That Activates Phosphatidylinositol 3-Kinase/Mitogen-activated Protein Kinase (PI3K/MAPK) Pathways by Modulating IGF-2

Glioblastoma is the most common and malignant form of primary astrocytoma. Upon investigation of the insulin-like growth factor (IGF) pathway, we found the IGF2BP3/IMP3 transcript and protein to be up-regulated in GBMs but not in lower grade astrocytomas (p < 0.0001). IMP3 is an RNA binding protein known to bind to the 5′-untranslated region of IGF-2 mRNA, thereby activating its translation. Overexpression- and knockdown-based studies establish a role for IMP3 in promoting proliferation, anchorage-independent growth, invasion, and chemoresistance. IMP3 overexpressing B16F10 cells also showed increased tumor growth, angiogenesis, and metastasis, resulting in poor survival in a mouse model. Additionally, the infiltrating front, perivascular, and subpial regions in a majority of the GBMs stained positive for IMP3. Furthermore, two different murine glioma models were used to substantiate the above findings. In agreement with the translation activation functions of IMP3, we also found increased IGF-2 protein in the GBM tumor samples without a corresponding increase in its transcript levels. Also, in vitro IMP3 overexpression/knockdown modulated the IGF-2 protein levels without altering its transcript levels. Additionally, IGF-2 neutralization and supplementation studies established that the proproliferative effects of IMP3 were indeed mediated through IGF-2. Concordantly, PI3K and MAPK, the downstream effectors of IGF-2, are activated by IMP3 and are found to be essential for IMP3-induced cell proliferation. Thus, we have identified IMP3 as a GBM-specific proproliferative and proinvasive marker acting through IGF-2 resulting in the activation of oncogenic PI3K and MAPK pathways.

Does insulin-like growth factor 1 genotype influence muscle power response to strength training in older men and women?

The CA-repeat polymorphism in the insulin-like growth factor 1 (IGF1) gene promoter region has been associated with strength and circulating IGF-I protein levels. The purpose of the study was to determine if the IGF1 CA-repeat polymorphism influences muscle power at baseline and in response to ST in older adults. Knee extensor peak power (PP) was measured at 50, 60, and 70% of 1-RM strength before and after 10 weeks of unilateral knee extensor ST in older adults, aged 50-85 years, to determine the changes in absolute and relative PP with ST. Subjects (N = 114) were genotyped for the IGF1 CA-repeat polymorphism and grouped as homozygous for the 192 allele, heterozygous, or non-carriers of the 192 allele. The 192 homozygotes had significantly lower baseline PP at 50, 60, and 70% of 1-RM strength than the non-carriers when age, sex, and baseline fat-free mass were covaried (all P < 0.05). This same relationship was observed when the highest PP within these ranges was compared (e.g., 317.6 ± 13.5 for 192 homozygotes and 380.2 ± 16.3 for non-carriers of the 192 allele, P < 0.05). Both absolute and relative PP increased significantly with ST in all genotype groups as expected, but there were no significant relationships among IGF1 genotypes and any of the PP changes. Despite a significant relationship between IGF1 genotype and knee extensor peak power at baseline, IGF1 genotype does not appear to influence changes in knee extensor peak power with ST.

Placental IGF‐I, IGFBP‐1, zinc, and iron, and maternal and infant anthropometry at birth

To correlate placental protein levels of insulin-like growth factor (IGF)-I and insulin-like growth factor binding protein (IGFBP)-1, with previously determined levels of IGF-I and IGF-II mRNA expression, and the micronutrients zinc and iron, and maternal and newborn anthropometry. Placental samples were collected from rural field sites in Pakistan. Samples were divided into small and large for gestational age groups (SGA and LGA, respectively). IGFBP-1 levels were assessed using Western immunoblotting. IGF-I protein levels were assessed using ELISA techniques. IGF mRNA expression, zinc, and iron, were quantified as previously described and were used for comparative purposes only. Thirty-three subjects were included (SGA, n = 12; LGA n = 21). Higher levels of IGFBP-1 were seen in the SGA group (p < 0.01). IGFBP-1 correlated positively with maternal and infant triceps skin-fold thickness in the LGA and SGA groups, respectively (p < 0.05). Significantly lower IGF-I protein levels were seen in the SGA group. IGF-I levels correlated significantly with maternal and newborn anthropometry. IGFBP-1 correlated significantly with IGF-II mRNA expression (p < 0.05). Placental protein levels of IGF-I and IGFBP-1 appear to be associated with maternal anthropometry. Maternal anthropometry may thus influence IGFBP-1 and IGF-I levels and may possibly be used for screening of pregnancies, with the potential for timely identification of these high-risk pregnancies.

Trans-10,cis-12 Conjugated Linoleic Acid Inhibits Insulin-Like Growth Factor-I Receptor Signaling in TSU-Pr1 Human Bladder Cancer Cells

Conjugated linoleic acid (CLA) is a mixture of positional and geometric isomers of linoleic acid; the major isomers are trans-10,cis-12 CLA (t10c12) and cis-9,trans-11 CLA (c9t11). CLA has been demonstrated to exert strong anticarcinogenic effects in a variety of experimental cancer models. We previously observed that CLA (a mixture of isomers) and t10c12 decreased the growth of TSU-Pr1 cells, whereas linoleic acid and c9t11 exerted no effects. In the current study, the mechanisms underlying the t10c12-mediated regulation of the growth of these bladder cancer cells were evaluated. TSU-Pr1 cells were incubated in serum-free medium with various concentrations of t10c12 or c9t11 in the presence or absence of insulin-like growth factor (IGF)-I. The incorporation of [(3)H]thymidine into DNA was decreased, and the number of annexin V-stained cells was increased after t10c12 treatment, whereas c9t11 had no effect on apoptosis or [(3)H]thymidine incorporation. Treatment with exogenous IGF-I alone increased the numbers of viable cells but did not counteract the t10c12-induced growth inhibition of TSU-Pr1 cells. t10c12 effected a dose-dependent reduction in IGF-I receptor (IGF-IR) transcripts and protein levels, whereas c9t11 exerted no effects. Additionally, t10c12 inhibited the IGF-I-induced phosphorylation of IGF-IR, the recruitment of the p85 regulatory subunit of phosphoinositide 3-kinase to IGF-IR, and the phosphorylation of Akt and extracellular signal-regulated kinase (ERK)-1/2. These results indicate that the inhibition of IGF-IR signaling and the activation of Akt and ERK-1/2 contributed to decreased cell proliferation and increased apoptosis in TSU-Pr1 cells treated with t10c12.

Effect of pulmonary rehabilitation on muscle remodelling in cachectic patients with COPD

It is known that non-cachectic patients with chronic obstructive pulmonary disease (COPD) respond well to pulmonary rehabilitation, but whether cachectic COPD patients are capable of adaptive responses is both important and unknown. 10 cachectic and 19 non-cachectic COPD patients undertook high-intensity cycling training, at the same relative intensity, for 45 min x day(-1), 3 days x week(-1) for 10 weeks. Before and after rehabilitation vastus lateralis muscle biopsies were analysed morphologically and for the expression of muscle remodelling factors (insulin-like growth factor (IGF)-I, myogenic differentiation factor D (MyoD), tumour necrosis factor (TNF)-alpha, nuclear factor (NF)-kappaB and myostatin) and key components of ubiquitin-mediated proteolytic systems (muscle ring finger protein (MURF)-1 and Atrogin-1). Rehabilitation improved peak work-rate and the 6-min walk distance similarly in non-cachectic (18+/-3% and 42+/-13 m, respectively) and cachectic (16+/-2% and 53+/-16 m, respectively) patients, but quality of life only improved in non-cachectic COPD. Mean muscle fibre cross-sectional area increased in both groups, but significantly less in cachectic (7+/-2%) than in non-cachectic (11+/-2%) patients. Both groups equally decreased the proportion of type IIb fibres and increased muscle capillary/fibre ratio. IGF-I mRNA expression increased in both groups, but IGF-I protein levels increased more in non-cachectic COPD. MyoD was upregulated, whereas myostatin was downregulated at the mRNA and protein level only in non-cachectic patients. Whilst rehabilitation had no effect on TNF-alpha expression, it decreased the activation of the transcription factor NF-kappaB in both groups by the same amount. Atrogin-1 and MURF-1 expression were increased in cachectic COPD, but it was decreased in non-cachectic patients. Cachectic COPD patients partially retain the capacity for peripheral muscle remodelling in response to rehabilitation and are able to increase exercise capacity as much as those without cachexia, even if they exhibit both quantitative and qualitative differences in the type of muscle fibre remodelling in response to exercise training.

Atrogin‐1, MuRF1, and FoXO, as well as phosphorylated GSK‐3β and 4E‐BP1 are reduced in skeletal muscle of chronic spinal cord–injured patients

Chronic complete spinal cord injury (SCI) is associated with severe skeletal muscle atrophy as well several atrophy and physical-inactivity-related comorbidity factors such as diabetes, obesity, lipid disorders, and cardiovascular diseases. Intracellular mechanisms associated with chronic complete SCI-related muscle atrophy are not well understood, and thus their characterization may assist with developing strategies to reduce the risk of comorbidity factors. Therefore, the aim of this study was to determine whether there was an increase in catabolic signaling targets, such as atrogin-1, muscle ring finger-1 (MuRF1), forkhead transcription factor (FoXO), and myostatin, and decreases in anabolic signaling targets, such as insulin-like growth factor (IGF), v-akt murine thymoma viral oncogene (Akt), glycogen synthase kinase-beta (GSK-3beta), mammalian target of rapamycin (mTOR), eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and p70(s6kinase) in chronic complete SCI patients. In SCI patients, when compared with controls, there was a significant reduction in mRNA levels of atrogin-1 (59%; P < 0.05), MuRF1 (55%; P < 0.05), and myostatin (46%; P < 0.01), and in protein levels of FoXO1 (72%; P < 0.05), FoXO3a (60%; P < 0.05), and atrogin-1 (36%; P < 0.05). Decreases in the protein levels of IGF-1 (48%; P < 0.001) and phosphorylated GSK-3beta (54%; P < 0.05), 4E-BP1 (48%; P < 0.05), and p70(s6kinase) (60%; P = 0.1) were also observed, the latter three in an Akt- and mTOR-independent manner. Reductions in atrogin-1, MuRF1, FoXO, and myostatin suggest the existence of an internal mechanism aimed at reducing further loss of muscle proteins during chronic SCI. The downregulation of signaling proteins that regulate anabolism, such as IGF, GSK-3beta, and 4E-BP1, would reduce the ability to increase protein synthesis rates.

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.

Dynamic regulation of estrogen receptor-α isoform expression in the mouse fallopian tube: mechanistic insight into estrogen-dependent production and secretion of insulin-like growth factors

Estrogen receptors (ERs) are members of the nuclear receptor superfamily and are involved in regulation of fallopian tube functions (i.e., enhancement of protein secretion, formation of tubal fluid, and regulation of gamete transport). However, the ER subtype-mediated mechanisms underlying these processes have not been completely clarified. Recently, we identified ERbeta expression and localization in rat fallopian tubes, suggesting a potential biological function of ERbeta related to calcium-dependent ciliated beating. Here we provide for the first time insight into the less studied ERalpha isoforms, which mediate estrogen-dependent production and secretion of IGFs in vivo. First, Western blot studies revealed that three ERalpha isoforms were expressed in mouse fallopian tubes. Subsequent immunohistochemical analysis showed that ERalpha was detected in all cell types, whereas ERbeta was mainly localized in ciliated epithelial cells. Second, ERalpha isoform levels were dramatically downregulated in mouse fallopian tubes by treatment with E(2) or PPT, an ERalpha agonist, in a time-dependent manner. Third, the presence of ICI 182,780, an ER antagonist, blocked the E(2)- or PPT-induced downregulation of tubal ERalpha isoform expression in mice. However, alteration of ERalpha immunoreactivity following ICI 182,780 treatment was only detected in epithelial cells of the ampullary region. Fourth, changes in ERalpha isoform expression were found to be coupled to multiple E(2) effects on tubal growth, protein synthesis, and secretion in mouse fallopian tube tissues and fluid. In particular, E(2) exhibited positive regulation of IGF-I and IGF-II protein levels. Finally, using growth hormone receptor (GHR) gene-disrupted mice, we showed that regulation by E(2) of IGF production was independent of GH-induced GHR signaling in mouse fallopian tubes in vivo. These data, together with previous studies from our laboratory, suggest that the long-term effects of estrogen agonist promote IGF synthesis and secretion in mouse tubal epithelial cells and fallopian tube fluid via stimulation of ERalpha.