IGF-I, IGF-I-binding proteins and GH-binding protein in malnourished elderly patients with inflammation receiving refeeding therapy.

OBJECTIVE: To investigate the mechanisms determining the success or failure of refeeding therapy in malnourished elderly patients with inflammation by studying changes in plasma IGF-I, GH-binding protein (GHBP) and IGF-binding protein (IGFBP) levels and IGFBP-3 proteolysis. DESIGN AND METHODS: We studied 15 severely malnourished hospitalized elderly patients. Weight, food intake, plasma albumin, transthyretin, C-reactive protein (CRP), orosomucoid, interleukin-6 (IL-6), IGF-I, intact and proteolytically degraded IGFBP-3 and GHBP levels were determined on admission and during refeeding therapy designed to increase food intake to 40 kcal/kg body weight per day (15% protein). RESULTS: Plasma IGF-I, IGFBP-3 and GHBP levels were significantly low for age on admission in all malnourished elderly patients. They increased in nine patients as nutritional status improved (albuminemia >30 g/l; transthyretinemia >200 mg/l or weight gain >5% of initial body weight) and levels of inflammation markers decreased (group 1). In contrast, plasma IGF-I, IGFBP-3 and GHBP levels remained low in six patients in whom nutritional status failed to improve and levels of inflammation markers increased (group 2). IGF-I showed greater variations than IGFBP-3 or GHBP with respect to nutritional status. High plasma CRP and IL-6 levels were associated with high levels of IGFBP-3 proteolysis. CONCLUSION: Efficient refeeding therapy was associated with a significant increase in IGF-I plasma levels. In patients with severe and persistent inflammation, high levels of proteolysis of IGFBP-3 may have contributed to the low plasma IGF-I levels, persistence of hypercatabolism and lack of improvement in nutritional status.

The thymus gland: a target organ for growth hormone.

Increasing evidence has placed hormones and neuropeptides among potent immunomodulators, in both health and disease. Herein, we focus on the effects of growth hormone (GH) upon the thymus. Exogenous GH enhances thymic microenvironmental cell-derived secretory products such as cytokines and thymic hormones. Moreover, GH increases thymic epithelial cell (TEC) proliferation in vitro, and exhibits a synergistic effect with anti-CD3 in stimulating thymocyte proliferation, which is in keeping with the data showing that transgenic mice overexpressing GH or GH-releasing hormone exhibit overgrowth of the thymus. GH also influences thymocyte traffic: it increases human T-cell progenitor engraftment into the thymus; augments TEC/thymocyte adhesion and the traffic of thymocytes in the lymphoepithelial complexes, the thymic nurse cells; modulates in vivo the homing of recent thymic emigrants, enhancing the numbers of fluroscein isothiocyanate (FITC)+ cells in the lymph nodes and diminishing them in the spleen. In keeping with the effects of GH upon thymic cells is the detection of GH receptors in both TEC and thymocytes. Additionally, data indicate that insulin-like growth factor (IGF)-1 is involved in several effects of GH in the thymus, including the modulation of thymulin secretion, TEC proliferation as well as thymocyte/TEC adhesion. This is in keeping with the demonstration of IGF-1 production and expression of IGF-1 by TEC and thymocytes. Also, it should be envisioned as an intrathymic circuitry, involving not only IGF-1, but also GH itself, as intrathymic GH expression is seen both in TEC and in thymocytes, and that thymocyte-derived GH could enhance thymocyte proliferation. Finally, the possibility that GH improve thymic functions, including thymocyte proliferation and migration, places this molecule as a potential therapeutic adjuvant in immunodeficiency conditions associated with thymocyte decrease and loss of peripheral T cells.

Generation of human soluble leptin receptor by proteolytic cleavage of membrane-anchored receptors.

The leptin receptor (ObR) exists in multiple isoforms. In rodents, a soluble isoform is generated by alternative splicing; but in humans, there is no mRNA encoding soluble receptor (leptin binding protein). We investigated the hypothesis that human leptin binding protein can be generated by proteolytic cleavage of membrane-anchored leptin receptors (ObRb and ObRa). Leptin binding protein of similar size to that previously detected in human serum was detected by HPLC in medium of cells transfected with ObRa. ObRa exhibited higher expression at the cell surface than ObRb and generated greater levels of leptin binding protein. Ligand-mediated immunofunctional and immunofluorometric assays revealed that the leptin binding protein in medium bound both leptin and an ObR-specific antibody and that the level of leptin binding protein correlated with receptor expression at the cell surface. Phorbol 12-myristate-13-acetate and N-ethylmaleimide increased the accumulation of leptin binding protein, an indication that the production of leptin binding protein was up-regulated by PKC and sulfhydryl group activation. The protease inhibitors, TNFalpha protease inhibitor 1 and Immunex compound 2, could inhibit the production of leptin binding protein, indicating that the enzyme responsible for leptin binding protein cleavage belongs to the metalloprotease family. In conclusion, human leptin binding protein is generated by proteolytic cleavage of membrane-anchored leptin receptor by a metalloprotease.

The proliferative and antiapoptotic actions of growth hormone and insulin-like growth factor-1 are mediated through distinct signaling pathways in the Pro-B Ba/F3 cell line.

Biological actions of GH can be direct or mediated through insulin-like growth factor I (IGF-I). In the interleukin-3 (IL-3)-dependent Ba/F3 cell line, IGF-I induces cell cycle entry and proliferation. Ba/F3 cells expressing the rat GH receptor (Ba/F3 GHR cells) have been shown to escape from apoptosis and to proliferate under GH stimulation. Using the Ba/F3 GHR cell model, we sought to dissect the signals elicited specifically by IGF-I or GH. In contrast to IGF-I or IL-3, GH is able to maintain cell cycle entry of Ba/F3 GHR cells cultured for 7 days in the absence of serum. The presence of IGF-I messenger RNA was not detected by RT-PCR, and by RIA, IGF-I was not found in culture medium of Ba/F3 GHR cells, unstimulated or stimulated by GH. Moreover, the addition of an anti-IGF-I antibody that blocks IGF-I effects suggests that the actions of GH are not mediated by IGF-I, but appear to be direct. GH or IGF-I stimulation increased expression of cyclins A and D(1) with comparable kinetics, whereas expression of p21(waf1/cip1) seemed delayed in IGF-I-stimulated cells compared with that in GH-stimulated cells. Contrary to GH or IL-3, IGF-I did not induce nuclear factor-kappaB DNA-binding activity in Ba/F3 cells. Inhibition of nuclear factor-kappaB through expression of the mutant IkappaBalpha (A32/36) abrogated the GH-mediated survival signal, but did not result in alterations of the cell cycle in Ba/F3 GHR cells treated with IGF-I. Phosphatidylinositol 3-kinase was required for both survival and proliferative responses to IGF-I. Transfection of a dominant negative form of AKT (AH-AKT) resulted in suppression of IGF-I-mediated cell survival, but not of the antiapoptotic effect of GH in Ba/F3 GHR cells. Thus, GH and IGF-I are able to promote cell survival and proliferation through independent and different pathways in Ba/F3 cells.

Growth hormone exerts antiapoptotic and proliferative effects through two different pathways involving nuclear factor-kappaB and phosphatidylinositol 3-kinase.

Dependence of murine pro-B Ba/F3 cells on interleukin-3 can be substituted by GH when cells are stably transfected with the GH receptor (GHR) complementary DNA. Recently, we demonstrated that Ba/F3 cells produce GH, which is responsible for the survival of cells expressing the GHR. This GH effect involves the activation of nuclear factor-kappaB (NF-kappaB). Here, we examined the signaling pathways mediating proliferation of growth factor-deprived Ba/F3 GHR cells. Exogenous GH stimulation of Ba/F3 GHR cells induced cyclins E and A and the cyclin-dependent kinase inhibitor p21(waf1/cip1) and repressed cyclin-dependent kinase inhibitor p27(kip1). The presence of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor Ly 294002 abolished proliferation induced by GH, arresting Ba/F3 GHR cells at the G(1)/S boundary, but did not promote apoptosis. Thus, the proliferative effect of GH is closely related to PI 3-kinase activation, whereas PI 3-kinase is not essential for GH-induced cell survival. Addition of Ly 294002 resulted in a moderate decrease in NF-kappaB activation by GH, suggesting a possible link between PI 3-kinase and NF-kappaB signaling by GH. Expression of c-myc was also induced by GH in Ba/F3 GHR cells, and inactivation of either PI 3-kinase or NF-kappaB reduced this induction. Overexpression of the dominant negative repressor mutant c-Myc-RX resulted in an inhibition of the GH proliferative effect, suggesting the involvement of c-myc in GH-induced proliferation. Taken together, these results suggest that the effects of GH on cell survival and proliferation are mediated through two different signaling pathways, NF-kappaB and PI 3-kinase, respectively; although cross-talk between them has not been excluded. NF-kappaB, which has been shown to be responsible for the antiapoptotic effect of GH, could also participate in GH-induced proliferation, as c-myc expression is promoted by PI 3-kinase, in an NF-kappaB-dependent and -independent manner.

Growth hormone prevents apoptosis through activation of nuclear factor-kappaB in interleukin-3-dependent Ba/F3 cell line.

The pro-B Ba/F3 cell line requires interleukin-3 and serum for growth, and their removal results in cell apoptosis. Ba/F3 cells transfected with the GH receptor (GHR) cDNA become able to proliferate in response to GH. To investigate the role of GH in the control of apoptosis, Ba/F3 cells expressing either the wild-type rat GHR (Ba/F3 GHR) or a mutated rat GHR (Ba/F3 ILV/T) were used. We show that Ba/F3 GHR cells, but not parental Ba/F3 or Ba/F3 ILV/T cells, were able to survive in the absence of growth factor. Furthermore, an autocrine/paracrine mode of GH action was suggested by the demonstration that Ba/F3 cells produce GH, and that addition of GH antagonists (B2036 and G120K) promotes apoptosis of Ba/F3 GHR cells. Consistent with survival, the levels of both antiapoptotic proteins Bcl-2 and Bag-1 were maintained in Ba/F3 GHR cells, but not in parental Ba/F3 cells upon growth factor deprivation. Constitutive activation of the transcription factor nuclear factor-kappaB (NF-kappaB), which has been shown to promote cell survival, was sustained in Ba/F3 GHR cells, whereas no NF-kappaB activation was detected in parental Ba/F3 cells in the absence of growth factor. Furthermore, addition of GH induced NF-kappaB DNA binding activity in Ba/F3 GHR cells. Overexpression of the mutated IkappaB alpha (A32/36) protein, known to inhibit NF-kappaB activity, resulted in death of growth factor-deprived Ba/F3 GHR cells, and addition of GH was no longer able to rescue these cells from apoptosis. Together, our results provide evidence for a new GH-mediated pathway that initiates a survival signal through activation of the transcription factor NF-kappaB and sustained levels of the antiapoptotic proteins Bcl-2 and Bag-1.

Responding to allegations of scientific misconduct: the procedure at the French National Medical and Health Research Institute.

Institutions in France are not yet well prepared to respond to allegations of scientific misconduct. Following a serious allegation in late 1997, INSERM, the primary organization for medical and health-related research in France, began to reflect on this subject, aided by scientists and jurists. The conclusions have resulted in establishing a procedure to be followed in cases of alleged misconduct, and also in reinforcing the application of good laboratory practices within each laboratory. Guidelines for authorship practices and scientific assessment must also be considered. Even though each institution must remain responsible for responding to allegations of scientific misconduct within its doors, INSERM would like to see national, European, and international co-ordination about the methods of such response.

Leptin binding activity changes with age: the link between leptin and puberty.

The timing of the physical transition from child to adult is determined by a biological clock that switches off the pituitary gonadal axis during infancy until puberty. Body composition (and in particular, fat mass), through leptin, are critical signals to this clock. However, no direct relationship between leptin and puberty has been demonstrated. Leptin is bound in the circulation by a high-affinity binding protein, which has been identified as a soluble leptin receptor. We found circulating levels of leptin binding activity (LBA) to be low at birth, to be high in the prepubertal years, to fall through puberty, and then to remain stable during adult life. LBA correlated with pubertal status in both boys and girls. We postulate that the fall in LBA, associated with increasing age and puberty, reflects a reduction in expression of truncated leptin receptors, and leptin is then available to the full-length receptor, which transmits the biological signal for leptin. The high levels of LBA occur during the years when the pituitary gonadal axis is quiescent. Thus, the change in LBA could explain how leptin regulates puberty.

Growth hormone (GH) and prolactin receptors in human peripheral blood mononuclear cells: relation with age and GH-binding protein.

GH receptors (GHRs) and PRL receptors (PRLRs) were studied in human peripheral blood mononuclear cells (PBMC) using flow cytometry, biotinylated anti-GH receptor monoclonal antibody 10B8, and biotinylated human PRL. Variations of GHR and PRLR expression and the relationship of plasma GHBP and GH receptor in PBMC subsets were examined as a function of age and sex. By double immunofluorescence staining, we show that about 30% of total cells express GH receptors, with a low expression in T cells, whereas almost all B cells and monocytes are GH receptor positive. Four age groups were defined among the 64 normal volunteers, aged 12 to 85 yr, who were included in the study. The percentage of PBMC expressing GH receptors is significantly lower in group 2 (20-40 yr) than in group 1 (12-20 yr) and group 4 (>60 yr). In T cells, monocytes and B cells, no significant changes are detected in either the percentage of GH receptor positive cells or in the GH receptor level per cell. The level of PRLRs expressed in PBMC is significantly higher in age group 2 than in age group 4. A negative correlation is observed between plasma GHBP and the percentage of PBMC expressing GH receptors. These results suggest that regulation of GH receptors in lymphocytes and in other target cells could be different.

Regulation of human growth hormone receptor gene transcription by triiodothyronine (T3).

In this study the hypothesis that triiodothyronine (T3) and growth hormone (GH) may have some direct or indirect effect on the regulation of GH-receptor/GH-binding protein (GHR/GHBP) gene transcription was tested. Different concentrations of T3 (0, 0.5, 2, 10 nmol/l) and GH (0, 10, 150 ng/ml) were added to human hepatoma (HuH7) cells cultured in serum-free hormonally-defined medium for 0, 1 and 2 h. Thereafter GHR/GHBP mRNA expression was quantitatively assessed by using PCR amplification. GH at a concentration of 10 ng/ml resulted in a significant increase of GHR/GHBP gene expression whereas a supraphysiological concentration of GH (150 ng/ml) caused a significant decrease of GHR/GHBP mRNA levels. The simultaneous addition of 0.5 nmol/l T3 to the variable concentrations of GH did not modify GHR/GHBP mRNA levels whereas the addition of 2 nmol/l up-regulated GHR/GHBP gene expression already after 1 h, an increase which was even more marked when 10 nmol/l of T3 was added. Interestingly, there was a positive correlation between the increase of GHR/GHBP mRNA levels and the T3 concentration used (r: 0.8). In addition, nuclear run-on experiments and GHBP determinations were performed which confirmed the changes in GHR/GHBP mRNA levels. Cycloheximide (10 microg/ml) did not alter transcription rate following GH addition but blocked GHR/GHBP gene transcription in T3 treated cells indicating that up-regulation of GHR/GHBP gene transcription caused by T3 requires new protein synthesis and is, therefore, dependent on indirect mechanisms. In conclusion, we present data showing that T3 on its own has a stimulatory effect on GHR/GHBP gene transcription which is indirect and additive to the GH-induced changes.

Four contiguous amino acid substitutions, identified in patients with Laron syndrome, differently affect the binding affinity and intracellular trafficking of the growth hormone receptor.

We have analyzed the GH receptor (GHR) gene in four individuals with Laron syndrome, and a missense mutation was identified for each patient in the extracellular domain of the GHR (D152H, I153T, Q154P, and V155G). The D152H mutation was previously reported. We have reproduced the three novel mutations in the GHR complementary DNA and analyzed their consequences in human 293 transfected cells. In cells expressing the I153T and V155G mutants, binding of [125I]human GH at the cell surface was very low, whereas binding to total membrane fractions was much less affected, suggesting impaired cell surface expression. Binding assays with cells expressing the Q154P mutant revealed severe defects both at the cell surface and in total particulate membrane fractions. Immunofluorescence experiments confirmed that cell surface expression of the three mutants was altered, and colocalization studies suggested that most of the mutant receptors are retained in the endoplasmic reticulum. Endoglycosidase H resistance tests also indicated that the majority of I153T and V155G GHRs are trapped in the endoplasmic reticulum. Thus, mutations on contiguous amino acids of the GHR result in various defects. The I153T, Q154P, and V155G mutations mainly affect intracellular trafficking and binding affinity of the receptor, whereas the D152H mutation affects receptor expression, dimerization, and signaling.

Role of prolactin and growth hormone on thymus physiology.

Intrathymic T-cell differentiation is under the control of the thymic microenvironment, which acts on maturing thymocytes via membrane as well as soluble products. Increasing data show that this process can be modulated by classical hormones, as exemplified herein by prolactin (PRL) and growth hormone (GH), largely secreted by the pituitary gland. Both PRL and GH stimulate the secretion of thymulin, a thymic hormone produced by thymic epithelial cells. Conversely, low levels of circulating thymulin parallel hypopituitary states. Interestingly, the enhancing effects of GH on thymulin seem to be mediated by insulinlike growth factor 1 (IGF-1) since they can be abrogated with anti-IGF-1 or anti-IGF-1-receptor antibodies. The influence of PRL and GH on the thymic epithelium is pleiotropic: PRL enhances in vivo the expression of high-molecular-weight cytokeratins and stimulates in vitro TEC proliferation, an effect that is shared by GH and IGF-1. Differentiating T cells are also targets for the intrathymic action of PRL and GH. In vivo inoculation of a rat pituitary cell line into old rats results in restoration of the thymus, including differentiation of CD4- CD8- thymocytes into CD4+ CD8+ cells. Furthermore, PRL may regulate the maintenance of thymocyte viability during the double-positive stage of thymocyte differentiation. Injections of GH into aging mice increase total thymocyte numbers and the percentage of CD3-bearing cells, as well as the Concanavalin-A mitogenic response and IL-6 production by thymocytes. Interestingly, similar findings are observed in animals treated with IGF-1. Lastly, the thymic hypoplasia observed in dwarf mice can be reversed with GH treatment. In keeping with the data summarized earlier is the detection of receptors for PRL and GH on both thymocytes and thymic epithelial cells. Importantly, recent studies indicate that both cell types can produce PRL and GH intrathymically. Similarly, production of IGF-1 and expression of a corresponding receptor has also been demonstrated. In conclusion, these data strongly indicate that the thymus is physiologically under control of pituitary hormones PRL and GH. In addition to the classical endocrine pathway, paracrine and autocrine circuits are probably implicated in such control.

Alternative splicing of the prolactin receptor gene generates a 1.7 kb RNA transcript that is linked to prolactin function in the red deer testis.

A cDNA encoding a putative non-membrane bound prolactin receptor was amplified by RT-PCR from red deer (Cervus elaphus) testis. Sequence analysis suggests that the testicular cDNA is generated by alternative splicing resulting in the deletion of exons 7 and 8, which code for: (a) the final 53 aa of the extracellular domain of the receptor including the fifth conserved cysteine residue and the WS x WS motif, (b) the entire transmembrane domain, (c) the first three cytoplasmic amino acid residues, and (d) two nucleotides of the fourth cytoplasmic amino acid codon. The resultant RNA would encode a putative protein of 174 aa due to a single bp frame shift and a premature stop codon. Northern blot analysis confirmed that the PCR-amplified cDNA is encoded by a specific 1.7 kb RNA transcript whereas the membrane bound receptor is encoded by transcripts of 3.5 and 2.5 kb. HPLC studies using media from 293 cells transfected with the 1.7 kb cDNA failed to detect any specific binding for prolactin. These data suggest that: (a) the deletion in the 1.7 kb transcript alters the structure of the prolactin binding domain in the putative protein encoded by the 1.7 kb transcript, and (b) alternative splicing of the prolactin receptor gene toward the 1.7 kb transcript is a means of down-regulating the expression of the full length prolactin receptor and hence may modify the role of prolactin in the testis of seasonally breeding mammals such as red deer. The sequence reported in this paper has been deposited in the Genbank/EMBL data base with accession number Y14753.

The D152H mutation found in growth hormone insensitivity syndrome impairs expression and function of human growth hormone receptor but is silent in rat receptor.

In two patients with growth hormone (GH) insensitivity syndrome (Laron syndrome), in whom the GH receptor is able to bind the hormone, the D152H mutation was identified, and lack of dimerization was proposed to explain GH resistance in these patients. To examine further the consequences of the substitution of conserved aspartate 152 on the function of the GH receptor (GHR), we reproduced the mutation in vitro on the full length GH receptor cDNA from man and rat. Effects of the mutation on expression and activity of the GHR were analyzed in 293 cells transfected with wild-type and mutant GHR cDNAs. Mutant human receptor protein was expressed at a lower level than wild-type receptor and its activity was reduced: GH-dependent signal transducer and activator of transcription 5 (Stat5)-mediated transactivation of a reporter gene was lower in 293 cells transfected with mutant GHR cDNA than in transfected cells expressing a comparable level of wild-type GHR. The membrane-bound form of the mutant and of the wild-type human GHR were able to homodimerize, as suggested by the size of the complexes detected in cross-linking experiments with 125I-human (h) GH, and also by the activity in the functional test. With the soluble GHR resulting from proteolysis of the wild-type membrane form, no dimeric complexes could be detected. However, when a soluble receptor lacking the transmembrane and cytoplasmic domains of the receptor was expressed, wild-type and not mutant GH binding protein (GHBP) was able to form dimers in the presence of hGH. The amino acid substitution has no effect on either expression or function of the rat receptor. Structural modeling of D152H soluble human and rat GHR (GHBP) supports the species-specific functional consequences of the mutation. Evaluation of the functional importance of the mutation strongly suggests that impairment in expression and activity of the mutant receptor, rather than complete lack of dimerization, explains the GH resistance of the patients.

Growth hormone and its receptor are expressed in human thymic cells.

GH has been shown to modulate various functions of the thymus. We now demonstrate the production of human GH (hGH) by human thymic cells, and the expression of GH receptors in thymic epithelial cells (TEC) and in thymocytes at different stages of differentiation. The presence of hGH messenger RNA was shown by RT-PCR in both human thymocytes and in primary cultures of TEC. Moreover, immunoreactive hGH material was detected in culture media of thymocytes and TEC with the use of a sensitive immunoradiometric assay. GH receptor gene expression was shown in TEC in primary cultures and in fetal and postnatal TEC lines as well as in thymocytes. By immunocytochemistry, the presence of GH receptors in the various TEC preparations was confirmed. In cytofluorometric studies with the use of a biotinylated anti-GH receptor monoclonal antibody, we could show that GH receptors are predominantly expressed by immature thymocytes: over 90% of CD3- CD4- CD8- CD19- CD34+ CD2- cells (a phenotype characterizing the most immature T cell progenitors in the thymus) were GH receptor positive. Our results provide a molecular basis for an autocrine/paracrine mode of action of GH in the human thymus.

Identification and modulation of a growth hormone-binding protein in rainbow trout (Oncorhynchus mykiss) plasma during seawater adaptation.

A soluble protein that specifically bound 125I-human growth hormone (hGH) was identified in rainbow trout plasma, using HPLC-gel filtration. The binding affinity of the protein for hGH was 1.2 x 10(9)M-1. 125I-rainbow trout GH (tGH) was also able to bind to the protein albeit with a lower affinity (6.6 x 10(7)M-1) than hGH. Crosslinking experiments using 125I-hGH revealed two specific bands of 150 and 130 kDa. The complex 125I-hGH-BP could be precipitated by a monoclonal anti-GH receptor antibody, suggesting a close relationship between the plasma GH-BP and the GH receptor. A fourfold increase in the hGH binding to the GH-BP was shown 48 h after transfer of the fishes from freshwater to seawater. The increase in binding was related to a high binding capacity without significant changes in binding affinity. These results suggest a potential role of this related GH-BP as an index of GH effects during seawater adaptation in salmonids.

Cirrhotic liver expresses low levels of the full-length and truncated growth hormone receptors.

In cirrhosis, as in other conditions of protein catabolism, there is a state of acquired GH resistance, as defined by high circulating GH levels with low insulin-like growth factor I levels. However, patients with end-stage liver failure respond to supraphysiological doses of GH with an increase in circulating insulin-like growth factor I levels. The present study represents a detailed analysis of GH receptor (GHR) expression in cirrhotic liver from 17 patients with end-stage liver disease. Specific binding of labeled GH was identified in all cirrhotic livers studied. The binding affinity for the GHR was similar in cirrhotic and normal livers, but the number of binding sites per mg protein of liver membrane was variable in both normal and cirrhotic liver, although it were generally lower in cirrhotic liver. GHR expression was identified in cirrhotic liver by Northern blotting, RT-PCR, and ribonuclease protection assay. On Northern blotting, a single transcript of 4.8 kb was identified in normal and cirrhotic tissues. RT-PCR identified expression of both full-length GHR and a truncated form of the GHR; this was confirmed by ribonuclease protection assay. In situ hybridization and immunohistochemistry confirmed the expression of GHR in regenerating hepatocytes and isolated cells in fibrous tissue. In conclusion, 1) the low level of GHR in cirrhotic liver may contribute to the acquired GH resistance found in cirrhotic patients; 2) the reduced expression of both full-length and truncated GHR is compatible with the low level of GH-binding protein found in cirrhosis, as this truncated receptor has previously been reported to generate large amounts of GH-binding protein; and 3) the demonstration of GH binding to cirrhotic liver explains why these patients with GH resistance may still respond to supraphysiological doses of GH.