Determination of the pharmacokinetic profiles of insulin-like growth factor binding proteins-1 and -2 in rats.

The insulin-like growth factor binding proteins (IGFBPs) are present in serum and alter the half-life of IGF-I and II in the vascular compartment. Because both IGFBP-1 and 2 have been proposed as regulators of IGF transport, we directly determined their distribution and elimination characteristics in rats. 125I-IGFBP-1 and 2 were injected into anesthetized normal rats. Multiple blood samples were drawn over time and the trichloroacetic acid precipitable radioactivity used to determine their pharmacokinetic profiles. The mean residence time for IGFBP-1 was 129 +/- 31 min and for IGFBP-2 116 +/- 24 min. The volume of distribution at steady state was 300 +/- 87 ml/kg for IGFBP-1 and 242 +/- 32 ml/kg for IGFBP-2. The elimination half-life was 120 +/- 26 min for IGFBP-1 and 144 +/- 32 for IGFBP-2. The results show that two separate methods of analysis give equivalent results for estimation of half-lives of these forms of IGFBPs and that their half-lives are substantially greater than that reported for free IGF-I, but less than that reported for the 150-kilodalton IGF complex. The findings suggest that these proteins equilibrate with extravascular compartments and may have a role in modulating transvascular transport of IGF-I and II.     

Depolarization induces insulin-like growth factor binding protein-2 expression in vivo via NMDA receptor stimulation

The effect of depolarization and N-methyl-D-aspartate (NMDA) receptor blockade on insulin-like growth factor-I (IGF-I), IGF binding protein-2 (IGFBP-2) and IGFBP-4 expression was analysed in vivo. Depolarization was induced in adult rat brains by applying 3 M KCl to the exposed cortex for 10 min. A subgroup of animals also received daily injections of MK-801. Four days after KCl exposure, the brains were analysed by in situ hybridization, immunohistochemistry and TUNEL. A significant upregulation of IGFBP-2 mRNA and protein was detected in astrocytes after KCl exposure This upregulation was reduced by MK-801 treatment. No alterations in IGF-I or IGFBP-4 mRNA levels were noted. We did not detect TUNEL positive cells, morphological signs of necrosis or apoptosis, or neuronal loss in the depolarized zone. Taken together, these findings indicate that upregulation of IGFBP-2 by depolarization is mediated by NMDA receptors, and, as no neuronal damage was detected, astrocytic NMDA receptors may be responsible for this upregulation.     

Dopamine D2 receptor gene expression in human adenohypophysial adenomas

The inhibitory effects of dopamine on adenohypophysial cells are mediated via dopamine subtype 2 receptor (D2R). Dopamine agonists inhibit hormone release and induce tumor shrinkage in most prolactin-secreting adenomas, whereas in other adenoma types such effects are sporadic. We investigated D2R gene expression by in situ hybridization (ISH) and immunocytochemistry in different types of pituitary adenomas. By ISH, a variable D2R signal was detected in 79 of 89 cases: 4 of 6 densely granulated and 8 of 8 sparsely granulated somatotroph, 4 of 4 mammosomatotroph, 7 of 7 mixed somatotroph-lactotroph, 4 of 4 acidophil stem cell, 16 of 16 sparsely granulated lactotroph, 11 of 16 corticotroph (functioning and silent), 3 of 4 silent subtype 3, 5 of 5 thyrotroph, 5 of 6 gonadotroph, 5 of 6 null cell, and 7 of 7 oncocytic adenomas. By immunocytochemistry, D2R protein was localized in cytoplasm and nuclei of 60 of 62 adenomas. In lactotroph adenomas, long-acting bromocriptine (BEC-LAR) induced a major increase in D2R mRNA, which was not accompanied by increased D2R immunoreactivity, suggesting mRNA stabilization. In conclusion, D2R gene is expressed in the majority of pituitary adenomas representing all tumor types. The significance of nuclear localization of D2R protein remains to be clarified.     

Regulation of insulin-like growth factor-I and of insulin-like growth factor binding protein-1, -3 and -4 in cocultures of rat hepatocytes and Kupffer cells by interleukin-6.

BACKGROUND/AIMS: Catabolism is associated with decreased serum concentrations of insulin-like growth factor (IGF)-I and insulin-like growth factor binding protein (IGFBP)-3 associated with elevated IGFBP-3 protease activity and increased concentrations of IGFBP-1 and -4. The effects of the acute phase mediators interleukin (IL)-6, IL-1beta and tumor necrosis factor alpha (TNFalpha) on the biosynthesis of IGF-I and IGFBPs were studied in primary rat liver cells. METHODS: mRNA levels of IGF-I and of IGFBPs were analyzed by Northern blotting, secretion of IGFBPs by [(125)I]IGF-I ligand blotting. Proteolytic activity was measured using iodinated recombinant IGFBP-3 as the substrate. RESULTS: In hepatocytes, Kupffer cells (KC) and cocultures of hepatocytes with KC, IL-6 reduced IGF-I biosynthesis dose-dependently. IL-6 stimulated mRNA expression and protein secretion of IGFBP-1 and -4 in hepatocytes and that of IGFBP-3 in KC, respectively. In cocultures, biosynthesis of IGFBP-1, -3 and -4 was increased dose-dependently by IL-6, while the effects of IL-1beta or TNFalpha were less prominent. At neutral pH, proteolytic activity against IGFBP-3 was not detected in media of cocultures treated with IL-6. CONCLUSIONS: The alterations of IGF-I, IGFBP-1 and -4 observed in catabolism correlate with the effects of IL-6 on the biosynthesis of these components in primary rat liver cells, while a neutral IGFBP-3 protease was not detectable.     

The expression of transforming growth factor-beta and interleukin-1beta mRNA and the response to 1,25(OH)2D3' 17 beta-estradiol, and testosterone is age dependent in primary cultures of mouse-derived osteoblasts in vitro

The aim of the present study was to examine the hypothesis that primary cultures of osteoblasts obtained from bones of young animals respond to hormones better than cell cultures obtained from old animals. We studied in cultured osteoblastic cells the effects of 1,25(OH)2D3 and sex steroid hormones on several mouse osteoblastic phenotypic expressions including transforming growth factor-beta (TGF-beta) and interleukin-1beta (IL-1beta) mRNAs. Second passages of long bone-derived osteoblastic cells from young donors (5-12 wk) and old donors (10-12 mo old) were used for this study. The cells obtained from old animals had decreased ALP activity and cAMP compared with cells obtained from young animals with no change in collagen production and mineralization. The addition of 17beta-estradiol and testosterone increased ALP activity and mineralization in the cultured cells from both age groups and collagen production in cells obtained from old mice. Using in situ hybridization IL-1beta and TGF-beta mRNA expression was observed to be higher in the osteoblasts from young than from old donors. 1,25(OH)2D3 increased IL-1beta mRNA expression in the cells derived from young mice. Testosterone and 17beta-estradiol inhibited IL-1beta mRNA expression only in cells derived from young mice. Sex steroid hormones did not change TGF-beta mRNA expression in any of the cell lines, but 1,25(OH)2D3 increased its expression in cells derived from old donors. The results of the present study indicate that cells obtained from old mice are generally less active than those obtained from young animals.     

Insulin-like growth factor-I (IGF-I) colocalizes with IGF-binding proteins (IGFBPs) in mouse and rat ovary

The cellular localization of insulin-like growth factor (IGF)-I mRNA, IGF-I peptide, and IGF-binding proteins (IGFBPs) was examined in mouse and rat ovaries through use ofin situ hybridization and immunohistochemical methods. IGF-I mRNA was found to be most abundant in granulosa cells, although lower levels were also detected in cells of the theca interna, stroma, and corpus luteum. In contrast, IGF-I immunoreactivity was undetectable or low in granulosa cells, weak and variable in oocytes, high in theca interna and the corpus luteum, and highest in the stroma. Antibodies directed against IGFBP-2, 3, and 5 yielded similar patterns of immunoreactivity to that observed for IGF-I peptide. The results indicate that IGF-I is synthesized in ovarian follicles, and that IGF-I of ovarian or systemic origin becomes localized to sites containing IGFBPs in the ovary.     

Human epidermal growth factor receptor 2 expression in mixed gastric carcinoma

AIM: To investigate human epidermal growth factor receptor 2 (HER2) amplification and protein expression in mixed gastric carcinoma.METHODS: Fluorescence in situ hybridization and immunohistochemistry were used to detect HER2 amplification and protein expression in 277 cases of mixed gastric carcinoma. Protein staining intensity was rate as 1+, 2+, or 3+.RESULTS: Of the 277 cases, 114 (41.2%) expressed HER2 protein. HER2 3+ staining was observed in 28/277 (10.1%) cases, 2+ in 37/277 (13.4%) cases, and 1+ in 49/277 (17.7%) cases. A HER2 amplification rate of 17% was detected, of which 25/28 (89.3%) were observed in the HER2 3+ staining group, 17/37 (45.9%) in 2+, and 5/49 (10.2%) in 1+. Of the 47 patients with HER2 amplification who received chemotherapy plus trastuzumab, 22 demonstrated median progression-free and overall survivals of 9.1 mo and 16.7 mo, respectively, which were significantly better than those achieved with chemotherapy alone (5.6 mo and 12.1 mo, respectively) in 19 previously treated patients (Ps < 0.05).CONCLUSION: HER2 detection in mixed gastric carcinoma displays high heterogeneity. Relatively quantitative parameters are needed for assessing the level of HER2 amplification and protein expression.     

Vascular endothelial growth factor and transforming growth factor-beta1 regulate the expression of insulin-like growth factor-binding protein-3, -4, and -5 in large vessel endothelial cells

We investigated the effect of diabetes-associated growth factors on the expression of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs) in cultured endothelial cells from bovine aorta. Gene expression was measured by solution hybridization, and proteins were measured by enzyme-linked immunosorbent assay, RIA, or Western blot. The cells expressed messenger RNA (mRNA) for IGFBP-2 through -6 and IGFBP-2 through -5 proteins were detected in conditioned medium. Vascular endothelial growth factor inhibited IGFBP-3 mRNA (P < 0.01) and protein expression and increased IGFBP-5 mRNA (P < 0.001) and protein. Transforming growth factor-beta1 inhibited IGFBP-3 (P < 0.01), IGFBP-4 (P < 0.01), and IGF-I mRNA expression, whereas at the protein level only IGFBP-3 was significantly decreased. IGF-I, insulin, or angiotensin II did not affect IGF-I or IGFBP mRNA expression. At the protein level, IGF-I clearly increased IGFBP-5 levels in conditioned medium. In conclusion, vascular endothelial growth factor and transforming growth factor-beta1 regulate IGFBP expression in bovine aortic endothelial cells. These observations provide a new aspect of regulation for the IGF-system in macrovascular endothelium, with possible implications for subendothelial smooth muscle cells and development of diabetic angiopathy.     

1,25-Dihydroxyvitamin D3 stimulates the production of insulin-like growth factor-binding proteins-2, -3 and -4 in human bone marrow stromal cells

BACKGROUND: 1,25-Dihydroxyvitamin D3 (calcitriol) inhibits proliferation and stimulates differentiation of multiple cell types, including osteoblasts. Human (h) bone marrow stromal cells (MSCs) are a homogenous non-hematopoietic population of cells present in the bone marrow and exhibit a less differentiated osteoblastic phenotype. The IGF system, including IGFs-I, and -II and IGF binding proteins (IGFBPs), plays an important role in osteoblast cell proliferation and differentiation. OBJECTIVE: To examine the pattern of expression of the IGF system in hMSCss and its regulation by calcitriol. METHODS AND RESULTS: hMSCs express mRNA of both IGFs-I, and -II and IGFBPs-1 to -6 as shown by RT-PCR and northern blot analysis. As assessed by western ligand blotting (WLB) and western immmunoblot analysis, hMSCs secrete 38-42 kDa IGFBP-3, 24-28 kDa IGFBP-4 and a 33 kDa IGFBP-2. Calcitriol (dose range 10-10 mol/l) exerted no consistent dose-dependent effects on either IGF-I or IGF-II mRNA levels. In contrast, calcitriol treatment increased steady-state mRNA levels of IGFBPs-2, -3 and -4, but had no effect on IGFBP-5 or -6. Similarly, calcitriol increased the secretion of IGFBPs-2, -3 and -4 as determined by WLB. We found no detectable basal IGFBP-3 or IGFBP-4 protease activities in the absence or presence of calcitriol treatment. CONCLUSIONS: Our results demonstrate that hMSCs expressed a distinct pattern of IGFs and IGFBPs that may be related to their stage of differentiation. The observed increase in production of IGFBPs-2, -3 and -4 by hMSCs upon treatment with calcitriol may be an important mechanism mediating the effects of calcitriol on MSC proliferation and differentiation.     

Dehydroepiandrosterone administration reverses the inhibitory influence of aging on gonadotrophin-releasing hormone gene expression in the male and female rat brain

Dehydroepiandrosterone (DHEA) has been shown to exert a beneficial influence on some aging-associated deficits in rodents. It is well documented that in the rat, aging is associated with a decline in reproductive functions. In order to evaluate the effect of DHEA on GnRH gene expression in aged animals, we have studied the effect of 2.5-d administration of DHEA to young (50–54 d of age) and aged (18 mo of age) rats of both sexes. In the young males, DHEA induced an 18% reduction in the hybridization signal. In the aged animals, the mRNA levels were 10% lower than those observed in the young rats. DHEA completely restored the mRNA levels when compared to those detected in young male animals. In the young female, DHEA produced a 11% increase in GnRH mRNA, whereas, in the aged animals, hybridization signal was decreased by 28%. DHEA administration to aged females induced a 33% increase in the amount of mRNA, thus completely reversing the influence of aging. These results indicate that the decrease in GnRH gene expression which is likely involved in the loss of reproductive functions in aged rats can be totally reversed by a short term administration of DHEA which restored the GnRH neuronal activity. They also suggest that DHEA might play a role in the prevention and/or improvement of some deficits associated with aging through stimulation of GnRH biosynthesis.     

Total and free insulin-like growth factor I, insulin-like growth factor binding protein 3 and acid-labile subunit reflect clinical activity in acromegaly

The aim was to evaluate, markers of disease activity in acromegaly in relation to perceived disease activity. Thirty-seven consecutively treated, acromegalic patients, classified by clinical symptoms as inactive (n=16), slightly active (n=10) and active (n=11), entered the study. When evaluating the inactive and the active groups, we found that positive and negative predictive values (PV(pos), PV(neg)) for clinical disease activity of total and free insulin-like growth factor-I (IGF-I) were 0.59, 0.90 and 1.00, 0.82 respectively. Acid-labile subunit (ALS) showed diagnostic merit similar to insulin-like growth factor binding protein-3 (IGFBP-3) with PV(pos) of 0.69 and 0.71 and PV(neg) of 0.91 and 0.92 respectively. We conclude that free IGF-I is more closely related than total IGF-I to perceived disease activity and is as such useful when evaluating previously treated acromegaly for disease activity. Total IGF-I, IGFBP-3 and ALS possess a higher PV(neg) for the clinical disease activity. None of the parameters can at present be claimed to be superior to the others and thus all the measured parameters are recommended to be part of the evaluation of acromegalic patients.     

Role of epidermal growth factor in peptic ulcer healing

In recent years, increasing interest has been focused on peptide growth factors, and impressive progress has been made in the understanding of their role in tumor development and progression. However, evidence is mounting that peptides such as epidermal growth factor and transforming growth factor-alpha may be of much more physiological than pathological importance. This brief article is intended to give a rapid overview of the available data supporting a role for epidermal growth factor and its human homologue urogastrone in peptic ulcer healing.     

Time dependent impact of perinatal hypoxia on growth hormone,insulin-like growth factor 1 and insulin-like growth factor binding protein-3

Hypoxic-ischemia (HI) is a widely used animal model to mimic the preterm or perinatal sublethal hypoxia, including hypoxic-ischemic encephalopathy. It causes diffuse neurodegeneration in the brain and results in mental retardation, hyperactivity, cerebral palsy, epilepsy and neuroendocrine disturbances. Herein, we examined acute and subacute correlations between neuronal degeneration and serum growth factor changes, including growth hormone (GH), insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3) after hypoxic-ischemia (HI) in neonatal rats. In the acute phase of hypoxia, brain volume was increased significantly as compared with control animals, which was associated with reduced GH and IGF-1 secretions. Reduced neuronal survival and increased DNA fragmentation were also noticed in these animals. However, in the subacute phase of hypoxia, neuronal survival and brain volume were significantly decreased, accompanied by increased apoptotic cell death in the hippocampus and cortex. Serum GH, IGF-1, and IGFBP-3 levels were significantly reduced in the subacute phase of HI. Significant retardation in the brain and body development were noted in the subacute phase of hypoxia. Here, we provide evidence that serum levels of growth-hormone and factors were decreased in the acute and subacute phase of hypoxia, which was associated with increased DNA fragmentation and decreased neuronal survival.     

Growth selection in mice reveals conserved and redundant expression patterns of the insulin-like growth factor system

Transgenic and knockout models have been used successfully in order to attribute specific functions to distinct growth factors. However, it is not clear which from the different IGF-components are actually altered when growth is affected. Furthermore it is not clear if unique or redundant patterns of IGF-component expression are present under conditions of elevated or reduced growth. To address these questions we have used a unique set of mouse models generated by divergent selection for high and low body growth. The set of mouse models consisted of eight mouse lines established in different laboratories. We have studied systemic and local expression of growth relevant genes in these mouse lines highly diverging for body and carcass weights but also for nose-rump lengths. As a strictly conserved pattern, serum IGF-I levels were dramatically increased in all H-lines if compared with the respective L-lines. By contrast serum IGFBP concentrations did not reveal clear patterns of expression in response to growth selection: IGFBP-3 was elevated in some H-lines, IGFBP-2 was increased in H- or L-lines and IGFBP-4 was similar in H- and L-lines. The fact that IGFBP-2 was the only IGFBP elevated in part of the L-lines, identifies IGFBP-2 as an exclusive although facultative negative effector for growth in the circulation among all other IGFBPs. In muscle tissue from selected breeding groups characterized by specific increases of the carcass weights we found redundant patterns of gene expression indicating the absence of tissue-specific or uniquely fixed expression patterns during growth selection within muscle tissue. The finding that serum but not tissue IGF-I levels were strictly positively correlated with growth during growth selection argues for an important role of endocrine IGF-I for postnatal growth in mice.     

Expression of Tissue Inhibitors of Metalloproteinases (TIMPs) in Gastric Cancer

Matrix metalloproteinases (MMPs) are one of the major classes of proteolytic enzymes involved in tumor invasion and metastasis, being inhibited by naturally occurring tissue inhibitors of metalloproteinases (TIMPs). Sixty-five patients who underwent surgery for gastric cancer in 1992 at Chonnam National University Hospital were selected for this study. The primary selection criteria were the availability of formalin-fixed and paraffin-embedded blocks and sufficient clinical follow-up for tumor-specific survival analysis. In this study, we examined the expression of TIMP-1 and TIMP-2 in human gastric cancer tissue by in situ hybridization and immunohistochemistry, and the correlation between their expression and clinicopathological parameters. TIMP-1 and TIMP-2 expressions were detected predominantly in the peritumor stromal cells rather than tumor cells themselves. Immunohistochemical stainings were concordant with the result obtained by in situ hybridization. The intensity of TIMP-1 immunohistochemical stromal staining correlated with tumor stage (P = 0.009) and patient survival (P = 0.025). However, the intensity of TIMP-2 immunohistochemical stromal staining did not correlate with tumor stage (P = 0.339) and patient survival (P = 0.474). The correlation between the increased TIMP-1 expression and cancer stage noted in this study reflects a role of TIMP-1 in predicting the aggressive behavior of gastric cancer. TIMP-2 expression did not correlate with clinicopathological parameters. However, expression of TIMP-1 and the possible additional value of TIMP-2 should be further explored in determining the prognosis of gastric cancer.     

Estrous cycle specific immunolocalization of different domains of the epidermal growth factor receptor in the porcine oviduct

Although porcine uterus is known to contain active and inactive forms of epidermal growth factor receptor (EGF-R), the latter consist of the extracellular domain only; it is currently unknown whether different EGF-R isoforms are expressed in the porcine oviduct during estrous cycle. Therefore, we used two different monoclonal antibodies, one against the extracellular and the other against the cytoplasmic domain of the EGF-R, to investigate cycle-dependent and cell-type-specific expression of full-size and truncated receptor forms. At metestrus, the majority of epithelial cells of the oviduct were strongly immunopositive for both antibodies, indicating the presence of the full-size receptor. In diestrous and proestrous stages, we found a low level of cytoplasmic but no extracellular EGF-R staining in epithelial cells. While the staining intensity of cytoplasmic domain of the EGF-R was only faint or absent in muscular tissue and blood vessels throughout the estrous cycle, extracellular domain of the EGF-R exhibited a strong immunostaining of smooth muscle cells and vascular smooth muscle cells, especially in diestrous and proestrous stages. There was no significant difference between the oviductal ampulla and isthmus in either the intensity or the pattern of both cytoplasmic and extracellular EGF-R immunostaining. We conclude that the restricted presence of the functional full-size receptor to the epithelial layer indicates a specific role during early embryonic development, whereas truncated EGF-R forms may potentially regulate contractions and blood flow in the oviduct.     

Estrogens decrease expression of the corticotropin-releasing factor gene in the hypothalamic paraventricular nucleus and of the proopiomelanocortin gene in the anterior pituitary of ovariectomized rats

It is known that estrogens modulate the hypothalamopituitary—adrenal (HPA) axis both under resting conditions and during exposure to stress. Nevertheless, the site of action of estrogens is not still fully elucidated. We sought to determine if estrogens could act on the major hypothalamic ACTH secretagogue: corticotropin-releasing factor (CRF). Mature rats were ovariectomized (OVX) and 2 weeks later implanted with silastic capsules containing 17β-estradiol (E₂). Animals were sacrificed 7 days later. CRF mRNA in the hypothalamic paraventricular nucleus (PVN) and proopiomelanocortin (POMC) mRNA in the anterior pituitary were measured byin situ hybridization. CRF content in the median eminence was measured by semiquantitative immunocytochemistry. E₂ treatment induced a significant decrease of CRF mRNA levels in the PVN (3.70±0.14vs 4.79±0.15 copies of probe×10⁻³/μm³ of tissue in OVX rats,P<0.05), an accumulation of immunoreactive CRF in the zona externa of the median eminence (207±36vs 100±15% in OVX rats,P<0.05), and a decrease of POMC mRNA levels in the anterior pituitary (4.6±0.6vs 6.9±0.6 copies of probe ×10⁻²/μm³ of tissue in OVX rats,P<0.05). These results demonstrate that estrogens have a negative effect on CRF gene expression and secretion and on POMC gene expression. Whether estrogens modulate directly the CRF-synthesizing cells or act through an increase of the glucocorticoid negative feedback remains to be determined.