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Impairment of Liver GH Receptor Signaling by Fasting

Unité de Diabétologie et Nutrition, Université Catholique de Louvain (V.B., B.W., V.d.C., J.-P.T.), B-1200 Brussels, Belgium; Unité d’Endocrinologie Moléculaire, INSERM, U-344, Faculté de Médecine Necker-Enfants Malades (V.B., M.E.), 75730 Paris, France; Department of Cell Biology, University of Alabama (S.J.F.), Birmingham, Alabama 35294

Address all correspondence and requests for reprints to: Jean-Paul Thisssen, M.D., Ph.D., Unité de Diabétologie et Nutrition, UCL/DIAB 5474, Avenue Hippocrate 54, B-1200 Brussels, Belgium. E-mail: . thissen{at}diab.ucl.ac.be

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fasting causes a state of GH resistance responsible for low circulating IGF-I levels. To investigate whether this resistance may result from alterations in the GH signaling pathway, we determined the effects of fasting on the GH transduction pathway in rat liver. Forty-eight-hour fasted or fed male rats were injected with recombinant rat GH via the portal vein. Liver was removed 0 and 15 min after injection. Although GH stimulated Janus kinase 2 (JAK2) phosphorylation in all animals, this was severely blunted in fasted animals. Similarly, the phosphorylation of the GH receptor, although observed in both fasted and fed rats after GH injection, was markedly reduced in fasted rats. A rapid signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation was also induced in the liver of fed animals in response to GH. In contrast, in fasted rats only a slight phosphorylated STAT5 signal was observed. The inhibitory effect of fasting on these GH signaling molecules occurred without changes in their protein content. Furthermore, the impairment of the JAK-STAT pathway in fasted animals was associated with increased liver suppressor of cytokine signaling 3 mRNA levels. Although glucocorticoids, which are increased by fasting, may cause GH resistance, adrenalectomy failed to prevent alterations in the JAK-STAT pathway caused by fasting. In conclusion, the GH resistance induced by fasting is associated with impairment of the JAK-STAT signaling pathway. This might contribute to the decrease in liver IGF-I production observed in fasting.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
MALNUTRITION RETARDS growth. In malnourished patients, plasma IGF-I concentrations are decreased despite elevated GH secretion. Furthermore, administration of GH to fasted rats fails to increase circulating IGF-I concentrations (1). These observations indicate that nutritional deprivation induces a resistance to the action of GH. Because the decrease in liver GH-binding sites parallels the fall in serum IGF-I during fasting, the GH resistance caused by fasting has been related to decreased GH binding (2, 3). The consequences of decreased liver GH binding on its intracellular signaling remain unsettled.

The immediate events following GH binding to its receptor in target tissues are now well defined (4) and consist in dimerization of the GH receptor (GHR), followed by activation of Janus kinase 2 (JAK2). Subsequently, JAK2 phosphorylates on tyrosine itself, the GHR and several members of the signal transducer and activator of transcription (STAT) protein family. Phosphorylation of the STAT proteins leads to their dimerization and translocation to the nucleus, where they modulate gene transcription. Although GH has been shown to activate other signaling molecules, such as STAT1 and -3, insulin receptor substrate 1 and 2, the SHC protein, and MAPK, STAT5 appears to be a key mediator of GH action in male rat liver (5). GH stimulates IGF-I gene expression in liver and other cell types, but the GH-regulated transcription factors involved are still not well identified. There is, however, evidence that STAT5 plays a role in this stimulatory effect. Knockout STAT5 mice are characterized by growth retardation together with low circulating IGF-I concentrations (5). In Hep3B cells expressing GHR, a synergistic action of STAT5 and hepatocyte nuclear factor 1 has been involved in the GH-dependent activation of the salmon IGF-I promoter (6).

Recent studies suggest that nutrients may regulate the functional activity of some liver transcription factors in rat (7). Whether GH resistance induced by fasting may result from alterations in the activation of STAT5 by GH is unknown.

A family of suppressors of cytokine signaling (SOCS) molecules has been recently identified. These SOCS have been shown to act as an intracellular negative feedback loop. Overexpression of SOCS3, one member of this family, has indeed been reported to inhibit GH action (8) by decreasing JAK-STAT pathway activation (9). Increased liver SOCS3 gene expression by fasting could therefore result in resistance to GH.

Glucocorticoids in excess cause GH resistance. They indeed inhibit GH induction of IGF-I gene expression in rat primary cultured hepatocytes (10) and decrease GH activation of the JAK-STAT pathway in 3T3-F442A cells (11) through a diminution of the levels of GHR, as seen in the liver of fasted rats. Furthermore, adrenalectomy has been shown to reverse the GH resistance caused by streptozotocin-induced diabetes (12). The role of the physiological up-regulation of glucocorticoids by fasting in fasting-induced GH resistance has not yet been investigated.

In this study we first investigated whether GH resistance may result from alterations in the GH signaling pathway by determining the effects of fasting on the GH transduction pathway in rat liver. In addition, we analyzed the effects of fasting on SOCS3 mRNA levels. As part of these investigations, we also studied whether adrenalectomy could prevent the effects of fasting on the early steps of GH action.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Recombinant rat GH (NIDDK rGH-13; AFP-87401) was provided by Dr. Parlow, National Hormone and Pituitary Program. Anti-GHRcyt-AL47 (AL47) (13) is a rabbit antiserum raised against a bacterially-expressed N-terminally His-tagged fusion protein incorporating human GHR residues 271–620 (the entire cytoplasmic domain). The cDNA encoding this fusion was created by PCR in the pET vector system (Novagen, San Diego, CA; PCR primers available upon request). Although AL47 is more potent, it exhibited otherwise very similar characteristics for immunoprecipitation and immunoblotting of GHRs than the anti-GHRcyt-AL37, another anti-GHR cytoplasmic domain antibody described previously (14) (Beauloye, V., unpublished observations). A second antibody to GHR (GHBP), provided by W. R. Baumbach (American Cyanamid, Princeton, NJ), was raised in rabbits against recombinant rat GH-binding protein produced in Escherichia coli (15). Thus, AL47 recognized the cytoplasmic domain of the GHR, whereas GHBP recognized the extracellular domain of the GHR. Antibodies directed against other GH-signaling proteins were obtained from the following commercial sources: JAK2 (antibody 06-255) and antiphosphotyrosine antibody 4G10 from Upstate Biotechnology, Inc. (Lake Placid, NY), and STAT5b (C-17 sc-835) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). This antibody also partially recognized STAT5a.

Animals
All experimental procedures were carried out in compliance with the appropriate institutional and national ethical guidelines for work with laboratory animals. Six-week-old male Wistar rats (KUL, Leuven, Belgium), weighing 180–200 g, were acclimated under standardized conditions of light (12 h of light, from 0700–1900 h) and temperature (22 ± 2 C), with free access to standard rodent chow and water. Rats were housed two or three individuals per cage in an isolated room, separated from the conventional animal room. After a 3-d adaptation period, food was withdrawn for 48 h in the fasted groups, whereas fed groups were kept on the standard diet.

Experimental design and surgery
Rats (n = 3/group) were anesthetized with pentobarbital (60 mg/kg, ip) and were incised 15–20 min later, i.e. as soon as anesthesia was assured by the loss of pedal reflexes. The abdominal cavity was opened, the portal vein was exposed, and vehicle or recombinant rat GH was injected at a dose of 1.5 mg/kg BW. At 0 or 15 min (unless otherwise mentioned) after the injection, rats were killed by decapitation. Blood was collected into glass tubes and centrifuged (2800 rpm, 10 min, 4 C), and serum was stored at -20 C until analysis. Liver was removed, frozen in liquid nitrogen, and stored at -80 C for subsequent analysis. This procedure was carried out between 1000–1100 h because endogenous GH concentrations have been demonstrated previously (16) to be low (<10 ng/ml) 3–4 h after lights on. Based on these observations, we decided to inject GH at this time, when the liver sensitivity to GH is expected to be the highest. Previous studies in vivo also showed that GH stimulation resulted in almost maximal tyrosine phosphorylation of major GH signaling proteins 15 min after injection (17, 18).

Five-week-old male Wistar rats (n = 3/group) were adrenalectomized (adx) bilaterally by dorsal incision and were given a 0.9% NaCl/10% sucrose solution to drink. After 5-d recuperation, adx rats were either kept on rat chow pellets or fasted for 2 d, allowing ad libitum saline but without sucrose drinking water. The same experimental design as that described above was then performed. Serum corticosterone levels in adx and non-adx rats were measured by RIA (ICN Biomedicals, Inc., Costa Mesa, CA) after affinity chromatography to separate corticosterone from 11-deoxycortisol (18A ).

Immunoprecipitation and immunoblotting
Livers were homogenized with an Ultraturrax T25 (IKA, Staufen, Germany) in ice-cold lysis buffer [10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 1 mM Na₃ VO₄, 10% glycerol, and 0.5% Nonidet P-40] containing protease inhibitors (1 mM phenylmethylsulfonylfluoride, 1 µg/ml pepstatin A, 2 µg/ml leupeptin, and 5 µg/ml aprotinin). To remove insoluble material, lysates were centrifuged at 15,000 x g at 4 C for 15 min. The supernatants were collected and stored in aliquots at -80 C. The protein concentrations were determined with a Bradford dye-binding assay kit (Bio-Rad Laboratories, Inc., Hercules, CA), using BSA as a standard.

Equal amounts of liver lysate protein (3 mg for JAK2, 4 mg for GHR, 1 mg for STAT5) were incubated in the above-described buffer with 25 µl protein A-agarose (Santa Cruz Biotechnology, Inc.) and specific antibodies (JAK2, 1:400; STAT5, 1:250) for 2 h at room temperature or with anti-GHR antibodies (AL47 or GHBP, 1:500) overnight at 4 C. Antibody complexes were washed three times in lysis buffer and boiled for 5 min in sample buffer [125 mM Tris (pH 6.8), 4.6% SDS, 10% ß-mercaptoethanol, and 20% glycerol]. Proteins were separated on SDS-PAGE (7.5%), transferred (Mini Trans-blot Cell, Bio-Rad Laboratories, Inc.) onto polyvinylidene difluoride transfer membrane (Polyscreen, NEN Life Science Products), and immunodetected with antiphosphotyrosine (1:10,000) or antibodies to JAK2 (1:1,000), AL47 or GHBP (1:1,000), and STAT5 (1:1,000) for 2 h at room temperature. The membranes were then incubated with an antimouse or antirabbit IgG-conjugated horseradish peroxidase (1:5,000 and 1:4,000, respectively) for 1 h at room temperature and revealed by the ECL detection system (Amersham Pharmacia Biotech, Little Chalfont, UK). To reprobe the blot with an other antibody, the blot was rehydrated in methanol, rinsed, and incubated with stripping buffer [65 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM] for 30 min at 50 C. The signals were quantified by densitometric scanning (Ultroscan XL laser densitometry, LKB, Bromma, Sweden) using Gel Scan software (Amersham Pharmacia Biotech). Data are expressed in arbitrary densitometric units (ADU). For blots with darker background, a lighter exposure was used for densitometric analysis or background was subtracted. For graphic representation of Figs. 1C, 2C, and 3C, tyrosine-phosphorylated protein readings were corrected for the respective protein levels present in the immunoprecipitates, and the ratios were normalized to the GH-stimulated fed mean, which was assigned a value of 100.

View larger version (40K): [in this window] [in a new window]   Figure 1. A, Effects of fasting on GH-stimulated JAK2 tyrosine phosphorylation in rat liver. Liver extracts from rats killed at 0 or 15 min after injection of saline (-) or GH (+; 1.5 mg/kg) were prepared as described in Materials and Methods. Liver extracts were immunoprecipitated with JAK2 antibody (1:400 dilution) and immunoblotted with an antiphosphotyrosine antibody (4G10; 1:10,000 dilution). The same blot was then stripped and incubated with JAK2 antibody (1:1,000) to check the protein amount. B, GH-stimulated JAK2 tyrosine phosphorylation in fasted or fed rat livers of three independent experiments. Tissue extracts were immunoprecipitated with JAK2 antibody and immunoblotted first with 4G10, then with JAK2 as described above. C, Densitometric analysis of P-JAK2 (top) and total JAK2 protein content (bottom) in the livers of GH-treated fasted () and fed () rats. Phosphoprotein signals were corrected for the specific protein levels present in the immunoprecipitate, and the ratios were normalized to the GH-stimulated fed mean, which was assigned a value of 100. Each data point represents the mean ± SEM for three rats expressed as a percentage of the stimulated fed mean (top) or as arbitrary ADU (bottom). ***, P < 0.001 vs. fed rats.

View larger version (45K): [in this window] [in a new window]   Figure 2. A, Effects of fasting on GH-stimulated GHR tyrosine phosphorylation in rat liver. Liver extracts from rats injected with saline (-) or GH (+; 1.5 mg/kg) for 15 min were prepared as described in Materials and Methods. Tissue extracts were immunoprecipitated with GHR antibodies AL47 or GHBP (1:500 dilution) and immunoblotted with an antiphosphotyrosine antibody (4G10; 1:10,000 dilution). The same blot was then stripped and incubated with same GHR antibodies (AL47 or GHBP; 1:1,000) to check protein amount. B, GH-stimulated GHR tyrosine phosphorylation in fasted or fed rat livers of three independent experiments. Tissue extracts were immunoprecipitated with GHR antibody AL47 and immunoblotted with 4G10 as described above. C, Densitometric analysis of P-GHR (top) and total GHR protein content (bottom) in the livers of GH-treated fasted () and fed () rats. Phosphoprotein signals were corrected for the specific protein levels present in the immunoprecipitate, and the ratios were normalized to the GH-stimulated fed mean, which was assigned a value of 100. Each data point represents the mean ± SEM for three rats, expressed as a percentage of the stimulated fed mean (top) or as ADU (bottom). *, P < 0.05 vs. fed rats.

View larger version (38K): [in this window] [in a new window]   Figure 3. A, Effects of fasting on GH-stimulated STAT5 tyrosine phosphorylation in rat liver. Liver extracts from rats killed at 0 or 15 min after injection of saline (-) or GH (+; 1.5 mg/kg) were prepared as described in Materials and Methods. Tissue extracts were immunoprecipitated with STAT5 antibody (1:250 dilution) and immunoblotted with an antiphosphotyrosine antibody (4G10; 1:10,000 dilution). The same blot was then stripped and incubated with STAT5 antibody (1:1,000) to check protein amount. B, GH-stimulated STAT5 tyrosine phosphorylation in fasted or fed rat livers of three independent experiments. Tissue extracts were immunoprecipitated with STAT5 antibody and immunoblotted first with 4G10, then with STAT5 as described above. C, Densitometric analysis of P-STAT5 (top) and total STAT5 protein content (bottom) in the livers of GH-treated fasted () and fed () rats. Phosphoprotein signals were corrected for the specific protein levels present in the immunoprecipitate, and the ratios were normalized to the GH-stimulated fed mean, which was assigned a value of 100. Each data point represents the mean ± SEM for three rats expressed as a percentage of the stimulated fed mean (top) or as ADU (bottom). *, P < 0.05 vs. fed rats.

Statistical analysis
Data are presented as the mean ± SEM and were analyzed by unpaired two-tailed t test. Data on SOCS-3 mRNA were analyzed by Mann-Whitney test, as SD values were not equal. P < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
To determine whether fasting leads to GH resistance by decreasing GH signaling in vivo, we first studied the effects of fasting on GH-stimulated tyrosine phosphorylation of JAK2, which plays a central role in mediating GH action (4) (Fig. 1). Liver lysates were analyzed using anti-JAK2 immunoprecipitation (IP), followed by antiphosphotyrosine (4G10) immunoblotting. As shown in Fig. 1A, no JAK2 tyrosine phosphorylation could be detected at 0 and 15 min after vehicle injection. In contrast, 15 min after its injection, GH stimulated tyrosine phosphorylation of JAK2 in both fasted and fed rats. However, fasting markedly reduced JAK2 phosphorylation in response to GH. After stripping and reprobing the blot with an anti-JAK2 antibody, JAK2 protein content was comparable in the fasted and fed groups. These results were confirmed in three rats in independent experiments (Fig. 1B) and by densitometric analysis (Fig. 1C), showing a 73% (P < 0.001) decrease in GH-induced JAK2 phosphorylation in the liver of fasted rats without any change in JAK2 protein content between GH-treated fasted and fed rats (GH-treated fasted, 812 ± 145 ADU; GH-treated fed, 798 ± 118 ADU; P = NS; n = 3).

To assess the functional significance of the diminished JAK2 tyrosine phosphorylation by fasting, we examined the effects of fasting on the GH-induced signal immediately downstream from JAK2, i.e. GHR phosphorylation. Liver proteins were immunoprecipitated with two different antibodies, AL47 or GHBP, and immunoblotted with an antiphosphotyrosine antibody (4G10; Fig. 2A). In fed rats, 15 min of GH treatment resulted in a marked increase in the tyrosine phosphorylation of a single dominant approximately 120-kDa protein band, the expected size for the rat GHR previously described by VanderKuur et al. (21). The two antibodies used for immunoprecipitation (AL47 or GHBP) displayed the same expected band and were able to demonstrate GH-stimulated GHR tyrosine phosphorylation. Fasting had no effect on the basal level of GHR phosphorylation in the absence of GH. However, after GH treatment, fasted rats exhibited a marked attenuation of GHR phosphorylation. Protein content was similar in the different lanes. The impairment by fasting of the GH stimulatory effect on GHR phosphorylation was confirmed in three different animals as shown in Fig. 2B and was statistically significant by densitometric analysis (Fig. 2C).

STAT5 tyrosine phosphorylation, one signal immediately downstream from GHR activation, was studied next. A rapid STAT5 tyrosine phosphorylation was observed in the liver of fed rats 15 min after GH injection (Fig. 3A). In contrast, in fasted rats only a weak level of STAT5 tyrosine phosphorylation could be detected in response to GH. These results were reproduced in three different rats (Fig. 3, B and C). This reduction of STAT5 phosphorylation was not due to a decrease in the levels of STAT5 protein.

Immunoblotting analysis (without IP) of our lysates confirmed that JAK2 and STAT5 protein contents were similar in the liver of fasted and fed rats (JAK2, 304 ± 36 vs. 349 ± 60 ADU; STAT5, 2775 ± 89 vs. 2603 ± 131 ADU, respectively, in fasted and fed rats; both P = NS; n = 4/group). This immunoblotting analysis (without IP) also showed that 15 min of GH treatment did not affect liver JAK2 and STAT5 protein contents [JAK2: without GH, 317 ± 49; with GH, 336 ± 53 ADU (P = NS; n = 4/group); STAT5: without GH, 2791 ± 40; with GH, 2586 ± 124 ADU (P = NS; n = 4/group)].

The effect of fasting on GH-induced GHR and STAT5 phosphorylation was detectable as early as 3 min after GH treatment and persisted, albeit less markedly, at least until 30 min after GH injection, as evidenced by our time-course study (Fig. 4). These data suggested that the effect of fasting on the GHR signaling pathway is not due to a delayed GH activation induced by fasting.

View larger version (44K): [in this window] [in a new window]   Figure 4. Time course of the effect of fasting on GH-stimulated STAT5 and GHR tyrosine phosphorylation. Liver extracts from rats killed at time zero or injected with GH (1.5 mg/kg) for 3, 15, or 30 min were prepared as described in Materials and Methods. Tissue extracts were immunoprecipitated with STAT5 or AL47 antibody (1:250 and 1:500 dilutions) and immunoblotted with an antiphosphotyrosine antibody (4G10; 1:10,000 dilution).

View larger version (36K): [in this window] [in a new window]   Figure 5. Effects of fasting on SOCS3 mRNA levels. A, Representative Northern blot of SOCS3 mRNA (top row) and 18S RNA (bottom row) in the livers of 48-h fasted and fed rats. B, Densitometric analysis of SOCS3 mRNA in the liver of 48-h fasted () and fed () rats. Each data point represents the mean ± SEM for five rats expressed as ADU. *, P < 0.05 vs. fed rats.

View larger version (55K): [in this window] [in a new window]   Figure 6. Effects of adrenalectomy on the fasting-induced impairment of the GH-stimulated signaling pathway. Left panel, Adrenalectomized rats were either normally fed or 48-h fasted and then injected with GH for 15 min. Liver extracts and Western blotting were performed as described above. Right panel, Nonadrenalectomized rats were either normally fed or fasted for 48 h and then injected with GH for 15 min. Liver extracts and Western blotting were performed as described. Pairs of lanes have been included for comparison. A, GH-induced JAK2 tyrosine phosphorylation and protein content in the livers of fasted and fed rats. B, GH-induced GHR tyrosine phosphorylation and protein content in the livers of fasted and fed rats. C, GH-induced STAT5 tyrosine phosphorylation and protein content in the livers of fasted and fed rats.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The mechanisms by which GH regulates IGF-I gene transcription remain poorly understood. However, current evidence strongly supports the role of JAK2, GHR, and STAT5 phosphorylation. Activation of JAK2 via association with the box-1 membrane-proximal region of the GHR (21, 24) is indeed required for most responses to GH, including GHR (24), SHC, MAPK, insulin receptor substrate, and STAT activation as well as induction of c-fos and spi-2.1 gene transcription and stimulation of glucose uptake (25). Mutational analysis of tyrosine residues of the GHR indicates that GHR phosphorylation also is essential for the biological response to GH. For example, phosphorylation of tyrosine Y333 and Y338 in the rat GHR is required for GH-stimulated lipid and protein synthesis (26) and maximal activation of STAT 5 (27). Finally, phosphorylation of STAT5b is crucial for the physiological effects of GH pulses on body growth and liver gene expression in male rats (5). Knockout STAT5 mice are characterized by growth retardation together with low circulating IGF-I concentrations (5). Convincing evidence that STAT5b is required for GH to stimulate IGF-I gene expression was provided by a recent study in which GH was shown to fail to induce liver IGF-I mRNA in hypox male STAT5b^-/- mice, although these mice exhibit both normal liver GHR expression and strong induction of cytokine-inductible SH2-containing protein mRNA levels (28). Taken together, these observations suggest that phosphorylation of JAK2, GHR, and STAT5 is essential for GH to exert its biological effects on growth and IGF-I gene expression.

The observed decrease in the activation of the JAK-STAT pathway by GH provides a potential mechanism to explain GH resistance in fasting. This inhibition does not seem specific to fasting, as similar alterations in JAK2 and STAT5 phosphorylation have recently also been described in the endotoxin-induced GH resistance state (22). The role of these signaling alterations in the decreased IGF-I production by the liver is supported by several data. First, decreased JAK2 phosphorylation has been reported to be correlated to the decrease in circulating IGF-I levels occurring with age. This does not seem to be the case for other GH-activated pathways, such as the MAPK pathway (29). Second, GH resistance in GH-binding protein-positive Laron syndrome children has been shown to be caused by dysfunction in GH-induced activation of STAT, as evidenced by a study performed in fibroblasts isolated from some affected individuals (30).

The mechanisms by which fasting alters the JAK-STAT pathway remain to be determined. Doses of GH reported to maximally stimulate JAK-STAT phosphorylation (18) in our present work fail to induce comparable response in fasted and fed rats. This observation suggests that the fasting-induced inhibition of the JAK-STAT pathway may result from decreased GH-binding sites below the number of GHR critical for GH action or from a postreceptor defect (31, 32). In a similar model we demonstrated that fasting decreases in parallel both GH binding on liver homogenates and binding of GH to the hepatocyte cell surface (33). As, to our knowledge, spare receptors for GH have never been described, it seems that any reduction in GH binding may result in decrease in GH intracellular action. This conclusion is further supported by in vitro studies showing a tight relationship between GH binding and GH transduction or GH biological effects. King et al. (34) showed that dexamethasone induces a closely correlated decrease in GH-stimulated JAK2 and GHR tyrosine phosphorylation and in the number of GH-binding sites in the plasma membrane of 3T3-F442A fibroblasts. Recent studies also showed that certain growth factors, e.g. platelet-derived growth factor, can down-regulate in parallel both GHR abundance and GHR signaling (35, 36). Moreover, a linear increase in STAT5 phosphorylation in response to GH has been demonstrated in mouse L cell lines expressing increasing (0–150,000 GHR/cell) amounts of mouse GHR (36A ). It is therefore likely that the defect in the JAK-STAT pathway activation observed in fasting mainly results from the decrease in liver GH binding. Although the decrease in liver GH binding is clearly established in the fasting state by numerous studies (2, 3, 33, 37), in the present study no difference was found in the GHR protein content between fasted and fed rats, either with an antibody that recognizes the extracellular domain of the GHR or with an antibody raised against the intracellular part of the GHR. Such a discrepancy between GH-binding sites and GHR amount has also been described in the GH resistance state caused by endotoxin. Although Defalque et al. (38) observed a decreased number of GH-binding sites in the liver of endotoxin-treated rats, the GHR protein content assessed by Mao et al. (22) was found to be identical in the livers of control and endotoxin-treated rats. As GHR could be localized in cytosol (39), we hypothesize that GH binding studies analyze GH-binding sites on cell membrane, whereas the Western blot technique, performed on whole cell lysates, could detect all of the GHRs present in the cell. It suggests that fasting induces a change in the cellular distribution of the GHRs. Supporting this hypothesis, King et al. (34) demonstrated that dexamethasone antagonizes GH action in 3T3-F442A fibroblasts by decreasing GH binding without altering the total amount of cellular GHR protein measured by GHR Western blotting. The same mechanism was reported by Leung et al. (40) for insulin-induced GHR down-regulation in osteoblasts.

The ability of overexpressed SOCS3 to blunt JAK-STAT activation in transfected cells (9, 41) and our present observation of increased SOCS3 expression by fasting suggest a role for SOCS3 in the fasting-induced JAK-STAT alterations. A similar mechanism has been proposed to explain the JAK-STAT alterations observed in two other models of GH resistance [chronic renal failure (42) and sepsis (22)] also associated with increased gene expression of several SOCSs in rat liver. As in fasting, in the endotoxin model SOCS3 up-regulation is the strongest among SOCS family members and is temporally correlated to inhibition of the GH-stimulated JAK-STAT pathway by endotoxin (22). Furthermore, SOCS3 has been shown to inhibit the transcriptional activation of a GH-responsive element and suppress JAK2 tyrosine kinase activity (9). As a good correlation was found between levels of SOCS3 mRNA and protein (23), induction of the SOCS3 gene, in addition to diminished cell surface GHR, could thus be one of the mechanisms involved in fasting-induced GH resistance. However, the mechanisms by which fasting induces SOCS3 remain to be determined. Changes in hormones such as leptin, glucocorticoids, or insulin are probably not involved. Although leptin and insulin increase SOCS3 gene expression (43, 44), glucocorticoids inhibit it (45). Because leptin (46) and insulin levels are down-regulated in fasting, and glucocorticoids are elevated, their role is unlikely.

The possibility for nutrition to modulate the GH activation of transcription factors is supported by a study indicating changes in rat liver transcription factors activity in response to dietary protein restriction (7). However, to our knowledge, ours is the first study in which the effects of fasting on JAK2 and STAT5 activation and content have been studied. Changes in hormonal status during fasting might be responsible for the inhibition of GH signaling. It is difficult to define the role of low insulin levels in the inhibition of the JAK-STAT pathway observed in fasting. Although a large body of evidence indicates that insulin plays a positive role in GH action (47, 48), it has been demonstrated in a hepatoma cell line that GH signaling through the GHR-JAK2-STAT5 pathway is inhibited by insulin (49). However, the consequences of insulin deficiency on the activation of the JAK-STAT pathway by GH have never been investigated in vivo and remain to be determined. Pharmacological doses of glucocorticoids have been shown to inhibit the GH-induced JAK-STAT signaling (11). In diabetic rats, which are also GH resistant, adrenalectomy was indeed shown to restore the GH-induced IGF-I response (12). However, in contrast to what has been observed in diabetic rats, adrenalectomy in fasted animals failed to reverse the fasting-induced inhibition of the early steps of the liver GHR signaling pathway. Taken together, these observations suggest that other hormones or nutrient availability by itself might regulate GH signaling.

In conclusion, the GH resistance induced by fasting is associated with impairment of the JAK-STAT signaling pathway. This could occur through a mechanism involving SOCS3 induction, but is not explained by fasting-induced glucocorticoid up-regulation. Decreased GH activation of the JAK-STAT pathway during fasting might contribute to the reduced liver IGF-I production observed in this situation.

	Acknowledgments

 
We thank Prof. J. Kolanowski and A. Saliez for helpful assistance in performing the adrenalectomy; Prof. P. De Nayer and P. Granger for serum corticosterone; E. Saunier, F. Dif, and Profs. M. Maes, P. Malvaux, J. M. Ketelslegers, and D. Maiter for continuous support; and Dr. Parlow for kindly providing us rGH.

	Footnotes

 
This work was supported by a visiting scholarship grant from the European Society of Pediatric Endocrinology (to V.B.), NIH Grants DK-46395 and DK-58259 and a V.A. Merit Review grant (to S.J.F.), and grants from the Cliniques Universitaires Saint-Luc (to V.B.); Danone Belgium (to J.P.T); the Fund for Scientific Medical Research, Belgium (to J.P.T.; no. 3.4548.99); and Université Catholique de Louvain, Belgium (to J.P.T.).

Abbreviations: ADU, Arbitrary densitometric units; adx, adrenalectomized; GHR, GH receptor; IP, immunoprecipitation; JAK2, Janus kinase 2; SOCS, suppressors of cytokine signaling; STAT5, signal transducer and activator of transcription 5.

Received July 9, 2001.

Accepted for publication November 15, 2001.

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