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Studies of insulin-like growth factor I (IGF-I) gene knockout mice models have clearly shown that IGF-I is necessary for prenatal as well as postnatal body growth in mice. Clinical studies of a patient with an IGF-I gene defect which caused complete absence of IGF-I, verified that it is important for intrauterine and postnatal growth. Recent studies of mice with liver-specific and inducible IGF-I gene knockout indicated that liver-derived IGF-I is not necessary for postnatal body growth, although serum IGF-I levels are decreased by more than 80% in these mice. Therefore, extrahepatic IGF-I is sufficient for maintenance of postnatal body growth in mice. Further investigations are needed to assess whether liver-derived circulating IGF-I is essential for other biological functions.

Growth hormone secretagogues (GHSs) stimulate growth hormone (GH) secretion, which is lipolytic. Here we compared the effects of twice daily s.c. treatment of GH and the GHS, ipamorelin, on body fat in GH-deficient (lit/lit) and in GH-intact (+/lit and +/+) mice. In +/lit and lit/lit mice ipamorelin induced a small (15%) increase in body weight by 2 weeks, that was not further augmented by 9 weeks. GH treatment markedly enhanced body weight in both groups. Ipamorelin also increased fat pad weights relative to body weight in both lit/lit and +/lit mice. Two weeks GHS treatment (ipamorelin or GHRP-6) also increased relative body fat, quantified by in vivo dual energy X-ray absorpiometry (DEXA) in GH-intact mice. GH decreased relative fat mass in lit/lit mice and had no effect in GH-intact mice. Treatment with GHS, but not GH, increased serum leptin and food intake in GH-intact mice. Thus, GHSs increase body fat by GH-independent mechanisms that may include increased feeding., (Copyright 2001 Academic Press.)

Background/aims: In the absence of liver damage, rapid liver growth can be induced pharmacologically by so-called primary liver growth promoters. The importance of the acute-phase cytokines interleukin-6 and tumor necrosis factor-alpha for the actions of these compounds is not clear. This study aimed to investigate the importance of IL-6 and TNF-receptor-1 in pharmacologically-induced liver growth., Methods: IL-6 knockout (IL-6(-/-)), TNF-receptor-1 knockout (TNFR1(-/-)) and wild-type mice were treated with the peroxisome proliferator nafenopin and the anti-androgen cyproterone acetate (CPA) in one single injection or for 6 days with daily injections, and examined at 24 or 48 h after treatment. In a control experiment, IL-6(-/-) mice were subjected to two-thirds partial hepatectomy., Results: Nafenopin treatment increased relative liver weight and DNA synthesis similarly in IL-6(-/-), TNFR1(-/-) and wild-type mice. CPA increased liver weight similarly in all groups, but did not increase DNA synthesis. Expression of peroxisome proliferator activated receptor-alpha mRNA was increased in both IL-6(-/-) and wild-type mice by nafenopin treatment, but not by CPA treatment. After hepatectomy DNA synthesis was suppressed in IL-6(-/-) mice compared to wild-type mice., Conclusions: Liver growth induced by nafenopin and CPA was not dependent on the presence of IL-6 or TNF receptor-1, whereas liver regeneration was decreased in IL-6(-/-) mice.

Background/aims: The transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) is a transactivator of several genes in the liver, which are regulated by growth hormone., Methods: Growth hormone (100 ng/ml) was added to primary rat hepatocytes cultured on a laminin-rich matrix. C/EBP mRNA and protein levels were measured by RNase protection assay and Western blotting, respectively. DNA binding activity was measured by electrophoretic mobility shift assay (EMSA)., Results: Growth hormone treatment for 6 h to 3 days increased C/EBPalpha mRNA levels. Addition of growth hormone for 24 h and 4 days also enhanced the levels of the 42 and 30 kDa isoforms of immunoreactive C/EBPalpha. EMSA showed that addition of growth hormone for 24 h enhanced the abundance of a protein complex binding to a consensus C/EBP binding DNA oligonucleotide. This protein complex was supershifted by antibodies directed against C/EBPalpha but not against C/EBPbeta. There were no consistent effects on C/EBPbeta mRNA or protein at any timepoint. The growth hormone effect on C/EBPalpha expression was not affected by simultaneous incubation with insulin or glucocorticoids, two hormones that previously have been reported to affect C/EBPs., Conclusions: Growth hormone enhances the levels of C/EBPalpha mRNA and protein as well as the DNA binding activity of C/EBPalpha in cultured rat hepatocytes.

Enhanced hepatocyte growth factor (HGF) receptor (Met) signaling has been suggested to play an important role in the development and progression of various epithelial and nonepithelial tumors. N-terminally truncated forms of the HGF receptor have been shown to be constitutively activated and tumorigenic in animal experiments. In the present study, 102 benign and malignant human musculoskeletal tumors were examined for expression of the HGF receptor by Western blotting and/or immunohistochemistry. A clear predominance of HGF receptor expression was seen in malignant as compared to benign tumors (Western blotting, P < 0.001; immunohistochemistry, P < 0.02). For the first time we show HGF receptor expression in the following four tumor types: dermatofibrosarcoma protuberans, clear cell sarcoma of tendons, malignant primitive neuroectodermal tumor, and benign fibrous histiocytoma. In three cases of sarcoma with high HGF receptor expression by Western blotting, we found indications of a short 85-kd N-terminally truncated HGF receptor that was tyrosine phosphorylated and located in the cytoplasm. Although fragments of this length were seen in 18 of 65 tumors, most were not tyrosine-phosphorylated. Northern blotting revealed only the 7.5-kb full-length HGF receptor transcript, suggesting that the 85-kd fragment is generated by an alternative initiation of translation or by proteolytic cleavage. Southern blotting detected no amplification of the Hgfr/Met gene in the 35 tumors examined, in contrast to our recent report of Hgfr/Met gene amplification in 7, 12-dimethylbenz(a)anthracene (DMBA)-induced rat sarcomas. The present data suggest that the locally aggressive and malignant properties of human mesenchymal tumors maybe related, in part, to high levels of full-length HGF receptors, and in some cases to the occurrence of N-terminally truncated HGF receptors, activated independently of HGF.

Studies in cell lines have shown that Jak2 is the primary tyrosine kinase involved in signal transduction by the growth hormone receptor (GHR). In addition, growth hormone (GH) stimulates tyrosine phosphorylation of Jak1 and Jak3 in certain cell lines, while the effect on Tyk2 has not been analysed. We have investigated the expression of Jak proteins in human liver and analysed their interactions with the GHR. Using Western blot analysis and immunohistochemistry, we demonstrate that Jak1, Jak2, Jak3 and Tyk2 are present in human liver. Immunoprecipitation by an antibody against the GHR (Mab 263) followed by immunoblotting with specific antibodies against Jak proteins showed that Jak1, Jak2 and Tyk2 were associated with the GHR in this tissue. We conclude that the GHR associates with Jak1, Jak2 and Tyk2 in human liver. Although experiments in vitro indicate that Jak2 mediates GH signalling, our results open the possibility that other Jak proteins may influence GHR signalling in human liver., (Copyright 1999 Harcourt Publishers Ltd.)

Background/aims: Hepatic stellate cells appear to be the main producers of hepatocyte growth factor of the normal liver. Insulin-like growth factors in doses over 20 ng/ml have been reported to stimulate hepatocyte growth factor production in cultured hepatic stellate cells. The aim of the present study was to investigate whether parenchymal cell conditioned medium had insulin-like growth factor-independent effects on hepatic stellate cells., Methods: Primary rat hepatic stellate cells were cultured for 1-7 days. DNA synthesis was measured by 3H-thymidine incorporation. Hepatocyte growth factor and transforming growth factor beta1 immunoreactivity was quantified by ELISA. Hepatocyte growth factor mRNA levels were determined with gel RNase protection assay. Parenchymal cell conditioned medium was obtained from hepatocytes cultured for 2 days in medium without added serum or hormones., Results: Incubation of 1-7-day-old hepatic stellate cells for 2 days with parenchymal cell conditioned medium enhanced the medium content of hepatocyte growth factor. Parenchymal cell conditioned medium contained less than 5.0 ng/ml immunoreactive insulin-like growth factor-1 as measured by radio immunoassay. Parenchymal cell conditioned medium did not contain any insulin-like growth factor bioactivity measured as phosphorylation of type 1 insulin-like growth factor receptor beta subunit and a protein with a size consistent with that of insulin receptor substrate-1. The stimulatory effect of parenchymal cell conditioned medium on hepatocyte growth factor was time- and dose-dependent. The effects of a high dose of parenchymal cell conditioned medium (dilution 1:2 containing less than 2.5 ng/ml insulin-like growth factor-1) were additive to that of high doses (100 ng/ml) of insulin-like growth factor-1 or des (1-3) insulin-like growth factor-1, an analogue with low affinity to insulin-like growth factor binding proteins. Neither parenchymal cell conditioned medium nor insulin-like growth factor-1 enhanced transforming growth factor beta1 immunoreactivity in the medium. Both parenchymal cell conditioned medium and insulin-like growth factor-1 stimulated DNA synthesis in hepatic stellate cells, confirming previous reports., Conclusions: The present results indicate that both insulin-like growth factor-1 and insulin-like growth factor-1-independent factors from hepatocytes can stimulate hepatocyte growth factor production by hepatic stellate cells. Therefore, insulin-like growth factor-1 and other hepatocyte-derived factors may indirectly affect hepatocytes via a paracrine loop.

Background/aims: The proliferation rate of adult rat liver is normally very low. It is markedly enhanced during compensatory regeneration, e.g. after partial hepatectomy, or after administration of certain growth promoters, e.g. cyproterone acetate. These two types of liver cell proliferation appear to differ, since the expression of immediate early genes is induced during compensatory regeneration but not after cyproterone acetate treatment. The transcription factor C/EBP alpha, which has been associated with hepatocyte differentiation and growth arrest, is suppressed during compensatory regeneration. In contrast, C/EBP beta, associated with acute phase reaction, is increased during regeneration. We have investigated the effects of the liver growth promoter cyproterone acetate on the hepatic expression of C/EBP alpha and C/EBP beta., Methods: Adult male rats received either cyproterone acetate treatment or were subjected to partial hepatectomy. Livers were obtained at different time intervals for measurement of C/EBP alpha and C/EBP beta mRNA with solution hybridization/RNAse protection assay, and C/EBP alpha and C/EBP beta content with immunoblotting., Results: The levels of both C/EBP alpha and C/EBP beta mRNA and the corresponding immunoreactivities were unchanged 2-48 h after injection of cyproterone acetate. The levels of C/EBP alpha mRNA and immunoreactivity were significantly suppressed 10-18 h and 18-26 h after partial hepatectomy, respectively. The levels of C/EBP beta mRNA and immunoreactivity were enhanced during compensatory regeneration 2 h after partial hepatectomy., Conclusions: Liver cell proliferation during regeneration, but not in response to cyproterone acetate treatment, is associated with changes in C/EBP alpha and C/EBP beta expression. This further supports the notion that changes in expression of transcription factors during liver growth in vivo are dependent on the growth inducer.

The secretory pattern of growth hormone (GH) in female rats differs from that in males with respect to e.g. the inter-peak baseline levels being higher in females. In the present study the influence of sex steroids on plasma GH levels was investigated in male rats under various conditions. Administration of estradiol, but not testosterone, was found to increase GH release in rats with suppressed levels induced by exposure to swimming stress or by treatment with the monoamine depleting agent reserpine. In line with previous studies, administration of estradiol was found to increase also inter-peak GH levels in adult male rats; i.e. to cause a feminization of the secretory pattern. In stressed and in reserpinized animals as well as in normal male rats, the effect of estradiol is similar to that earlier demonstrated for somatostatin antiserum, and hence it is suggested that estradiol may act antagonistic to the GH inhibiting factor.