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Endocrinology Vol. 143, No. 10 3766-3772
Copyright © 2002 by The Endocrine Society

ARTICLE

Discrimination between Signaling Pathways in Regulation of Specific Gene Expression by Insulin and Growth Hormone in Hepatocytes

Pascale Ribaux, Asllan Gjinovci, Henry B. Sadowski and Patrick B. Iynedjian

Division of Clinical Biochemistry and Experimental Diabetes Research (P.R., A.G., P.B.I.), University of Geneva School of Medicine, CH-1211 Geneva 4, Switzerland; and Department of Pharmacology and Biological Chemistry (H.B.S.), Mount Sinai School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Patrick B. Iynedjian, M.D., Division of Clinical Biochemistry, University of Geneva School of Medicine, 1, Rue Michel-Servet, CH-1211 Geneva 4, Switzerland. E-mail: iynedjian{at}medecine.unige.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin and GH can activate common signaling elements in many tissues and cell lines. We investigated the possibility of overlap in signaling pathways activated by insulin and GH in a key target cell, the hepatocyte. In primary cultures of rat hepatocytes, GH caused a dose- and time-dependent increase in tyrosine phosphorylation of signal transducer and activator of transcription 5. This was accompanied by the induction of the mRNA encoding suppressor of cytokine signaling 2. Neither of these effects took place in companion hepatocytes challenged with insulin. By contrast, insulin caused a rapid and sustained phosphorylation of protein kinase B, accompanied by a massive induction of the mRNA encoding glucokinase. GH had no detectable effect on phosphorylation of protein kinase B or level of glucokinase mRNA. Insulin also elicited brief hyperphosphorylation of ERK1 and 2, an effect not seen in GH-stimulated hepatocytes. Thus, there was a clear demarcation of signaling events triggered in hepatocytes by insulin and GH, and this was accompanied by hormone-specific responses with respect to the induction of gene expression. Additionally, the current results show that signal transducer and activator of transcription 5 activation is neither necessary nor sufficient for the insulin-dependent induction of hepatic glucokinase.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN AND GH REGULATE the function of target cells by binding to cell surface receptors of distinct classes. The insulin receptor belongs to the family of receptors with intrinsic tyrosine kinase activity, which phosphorylate intracellular scaffolding molecules such as the insulin-receptor-substrates 1–4 (IRS) (1). The phosphorylation of these substrates triggers the activation of two signaling modules, the ERK1 and 2 module and the phosphoinositide 3-kinase (PI3K) module. The PI3K pathway, via effector protein kinases including phosphoinositide-dependent protein kinase 1 and protein kinase B (PKB)/cAKT, is thought to be the preponderant pathway for the metabolic effects of insulin, including the transcriptional regulation of genes for key enzymes of metabolism (2). By contrast, the GH receptor is a representative of the cytokine receptors, which transduce signals via extrinsic tyrosine kinase partners known as Janus kinases (JAKs), specifically JAK2 for the GH receptor. Major substrates for the GH receptor-associated JAK2 are signal transducer and activator of transcription (STAT)5a and STAT5b, two closely related members of the STAT family (3). Phosphorylation of the STAT proteins results in their dimerization, nuclear translocation, and binding to cognate cis-acting DNA elements, thereby contributing to the regulation of target gene transcription (4).

Although the insulin and GH receptors are prototypes of receptors classically associated with distinct signaling cascades, the ability of the two hormones to elicit common physiological responses has long been recognized. For instance, short-term insulinlike effects of GH in adipose cells including an inhibition of lipolysis and stimulation of lipogenesis have been well documented (5, 6). At the signaling level, both insulin and GH can activate the ERK1 and 2 kinase cascade in some target cells (7, 8, 9). More importantly, phosphorylation of IRS proteins can occur in GH-stimulated cells (10, 11), and a modest activation of PI3K was detected in rat liver after administration of GH (12). Also, the phosphorylation of PKB was stimulated by GH in some cell lines (13, 14). Conversely, direct protein-protein interaction between the insulin receptor and STAT5b was demonstrated in the yeast two-hybrid system (15, 16). In cultured rhabdomyosarcoma cells and C2C12 myotubes, the addition of insulin resulted in tyrosine phosphorylation of STAT5 (17, 18). The level of insulin-induced tyrosine phosphorylation of STAT5a and STAT5b was markedly enhanced in C2C12 myotubes overexpressing the insulin receptor and typical cytokine-responsive genes such as the suppressor of cytokine signaling (SOCS) genes were induced (18).

Given the potential for overlap between insulin and GH signaling pathways, how are specific responses to each hormone ensured? This issue is examined here in a key target cell for both insulin and GH, the parenchymal liver cell. Using isolated rat hepatocytes in primary culture, we investigated the effects of both hormones on the phosphorylation of PKB, STAT5, and ERK1 and 2. As a physiologic read-out of insulin action, we were particularly interested in the induction of glucokinase gene expression because of a recent report suggesting a role for STAT5 in glucokinase induction (19). The possibility to mimic the insulin effect on glucokinase with GH was examined, and the regulation of the SOCS2 gene by the two hormones was also investigated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Hepatocytes culture
Hepatocytes were isolated from male Wistar rats weighing approximately 230 g and fasted for 2 d before the experiments. Experiments were approved by the State Commissioner on Animal Care. Hepatocytes were obtained after collagenase perfusion of the liver as described (20) and were allowed to attach to the culture dishes in seeding medium consisting of RPMI-1640 medium supplemented with glutamine, penicillin plus streptomycin, and 10% fetal bovine serum as described previously (21). After 1.5 h, floating cells and seeding medium were aspirated, and the attached hepatocytes were fed fresh RPMI 1640 culture medium supplemented as above, except that the concentration of fetal bovine serum was reduced to 4% (maintenance medium). When specified, dexamethasone (10-8 M), triiodothyronine (10-7 M), and glucagon (10-9 M) were added to the maintenance culture medium after the period of cell attachment. The hepatocytes were again supplied with fresh maintenance medium 20 h after seeding. This medium contained dexamethasone and triiodothyronine when indicated but was devoid of glucagon in all cases. Hormonal stimulation of the cells with human GH (Humatrope, Lilly France SA, Fegersheim, France) or insulin (Actrapid HM, Novo Nordisk A/S, Bagsvaerd, Denmark ) was started 1–2 h later.

Protein extraction and immunoblotting
Cell monolayers in six-well dishes were washed with 2 ml ice-cold PBS and incubated on ice for 10 min with 0.2 ml lysis buffer (Cell Signaling Technology, Beverly, MA) containing 20 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Na pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin supplemented with 1 mM phenylmethylsulfonyl fluoride, and 2 mM dithiothreitol. The cell lysates were collected by scraping with a plastic spatula and transferred to microfuge tubes, mixed vigorously for 1 min, and centrifuged at 4 C and 14,000 x g for 10 min. The supernatant was assayed for protein concentration by the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA). Specified amounts of protein were resolved by SDS-PAGE. Western transfer to nitrocellulose membrane (Bio-Rad Laboratories, Inc.) and immunoblotting were performed as described (22). Primary antibodies were a mouse monoclonal antibody recognizing STAT5 phosphorylated at residue Tyr 694 (23); a rabbit polyclonal antibody recognizing PKB phosphorylated at residue Ser 473 (Cell Signaling Technology); a rabbit antibody recognizing total PKB {alpha}, ß, and {gamma} (Cell Signaling Technology); and a rabbit antibody recognizing the peptide sequence around phosphorylated Thr 202-Tyr 204 of Erk1 (Cell Signaling Technology). Secondary antibodies were affinity-purified goat antibodies against rabbit or mouse immunoglobulins conjugated with peroxidase (Bio-Rad Laboratories, Inc.). The signal was revealed by enhanced chemiluminescence (SuperSignal West Pico, Pierce Chemical Co., Rockford, IL). Quantification was accomplished by densitometer analysis of the x-ray films, using calibration curves obtained with graded inputs of cell protein.

RNA extraction and Northern blotting
Total RNA extraction from cell monolayer in 10-cm dishes, RNA electrophoresis, blotting, and hybridization with 32P-labeled cDNA probes were performed as described previously (21, 24). The probes used were a rat liver glucokinase cDNA (25) and a mouse SOCS2 cDNA (26) subcloned into the pBluescript SK vector. To verify equal loading of all gel lanes, blots were stripped and probed with a glyceraldehyde-3-phosphate dehydrogenase cDNA (27). Quantification of specific mRNAs was accomplished by phosphor imaging of the membranes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dose-response relationship for GH effects in hepatocytes
Initial experiments were done to test the sensitivity of our hepatocyte culture system to the action of GH. For this purpose, the phosphorylation of STAT5 on tyrosine was monitored in cells exposed to various concentrations of GH. Protein was harvested from hepatocytes at 10 min after the addition of GH on the basis of kinetics experiments presented below, and tyrosine phosphorylated STAT5 (both STAT5a and STAT5b) was estimated by immunoblotting with a phospho-specific STAT5 antibody. As shown in Fig. 1AGo, the addition of increasing doses of GH between 0.8 and 500 ng/ml produced a dose-dependent increase of tyrosine phosphorylated STAT5. Quantification of the level of STAT5 phosphorylation as a function of GH concentration is presented in Fig. 1BGo.



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  		Figure 1. Effect of GH on tyrosine phosphorylation of STAT5 and amount of SOCS2 mRNA in hepatocytes. Hepatocytes were isolated, maintained in primary culture for 22 h in the absence of dexamethasone, and exposed to the indicated concentrations of GH. For the extraction of total protein and assay of tyrosine phosphorylated STAT5, cells were lysed 10 min after GH addition. Samples containing 40 µg protein were subjected to SDS-PAGE in a 7.5% polyacrylamide gel, transferred to nitrocellulose and immunoblotted with an antiphospho-STAT5 antibody. For the isolation of total cell RNA and assay of SOCS2 mRNA, cells were harvested 6 h after GH addition. Samples containing 20 µg RNA were electrophoresed in agarose gels, transferred to nylon membranes and hybridized with a 32P-radiolabeled SOCS2 cDNA probe. A, A representative immunoblot showing the effect of GH on STAT5 phosphorylation is illustrated. The position of standard molecular weight markers is indicated on the left. The asterisk designates a nonspecific protein band that verifies equal loading of all lanes. B, Dose-response plot showing the effects of GH on STAT5 phosphorylation (empty circle) and SOCS2 mRNA level (filled square). The quantification of phosphorylated STAT5 in immunoblots was done by densitometer analysis as described in Materials and Methods. The quantification of SOCS2 mRNA in Northern blots was performed by phosphor imaging. Results are the means for two different culture experiments; vertical lines indicate the range between the two values.

 
A putative genomic target for STAT5 is the SOCS2 gene (18, 28). The relative level of mRNA coding for SOCS2 was assayed by Northern blot of total cellular RNA extracted from hepatocytes that had been cultured for 6 h with various doses of GH. As shown in Fig. 1BGo, GH induced an increase in the amount of SOCS2 mRNA over a range of concentrations that coincided with those effecting the phosphorylation of STAT5. These data are compatible with the notion that STAT5 activation might play an essential role in the transcriptional regulation of SOCS2. The maximal increase of SOCS2 mRNA over the baseline amounted to a 2.5-fold increase (see also below). The GH-dependent induction of SOCS2 mRNA in primary cultures of hepatocytes has been reported previously (29).

Comparison of the effects of GH and insulin on STAT5 phosphorylation and SOCS2 mRNA regulation
Having established that hepatocytes maintained in the primary culture system are responsive to GH, we performed a side-by-side comparison of the effects of GH and insulin on the extent of tyrosine phosphorylation of STAT5. Hepatocytes cultured with GH or insulin for time periods between 10 min and 4 h were harvested for the isolation of total cell proteins and immunoblot assay of the phosphorylation status of STAT5 (Fig. 2Go, A and B). As expected, GH elicited a robust phosphorylation of STAT5, with a peak at 10 min after the addition of GH to the cells. The level of STAT5 phosphorylation subsequently declined but remained significantly above the baseline for the entire 4-h duration of the experiment. By contrast, no tyrosine phosphorylation of STAT5 could be detected at any time following the addition of insulin to companion cells (Fig. 2Go, A and B). It should be pointed out that in the current immunoblotting assay, an increase in STAT5 phosphorylation of less than 10% of the GH-induced level could have been accurately quantified.



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  		Figure 2. Effects of GH and insulin on phosphorylation of STAT5 and ERK1 and 2 in hepatocytes. Primary hepatocytes were cultured for 22 h in the absence or presence of 10 nM dexamethasone (Dex), 100 nM triiodothyronine (T3), and/or 1 nM glucagon (Gluc) as specified. Thereafter, cells were exposed to 500 ng/ml GH or 30 nM insulin (Ins) for the indicated times and harvested for extraction of total cell protein. Samples of 31 µg protein were subjected to immunoblotting with an antiphospho-STAT5 antibody. Samples of 70 µg protein were used for immunoblotting with antiphospho-ERK1 and 2. A, Representative antiphospho-STAT5 immunoblot from cells cultured in the absence of hormones before stimulation with GH or insulin. B, Plot showing the time course of effect of GH and insulin on STAT5 phosphorylation in the absence (empty square, GH; empty triangle, insulin) or presence of dexamethasone (filled circle, GH; cross, insulin). Quantification was done by densitometer analysis. Data are the means from two independent culture experiments; vertical lines indicate the range between the two values. C, Representative antiphospho-STAT5 immunoblot from cells maintained with dexamethasone plus triiodothyronine, glucagon, or both before stimulation with GH or insulin for 10 min. D, Representative antiphospho-ERK1 and 2 immunoblot from cells cultured in the absence of hormones before stimulation with GH or insulin for the indicated times.

 
We tested the hypothesis that the hormonal milieu provided to the hepatocytes during the cell culture period preceding the stimulation with GH or insulin might modulate the ensuing STAT5 phosphorylation response. To this effect, the hepatocytes were first cultured in the presence of dexamethasone, which was added to the culture medium after the attachment period and maintained throughout the experiment. As shown in Fig. 2BGo, strong phosphorylation of STAT5 in response to GH occurred with as well as without dexamethasone. On the other hand, insulin remained unable to elicit the phosphorylation of STAT5 on tyrosine, regardless of the presence or absence of dexamethasone during the prestimulation phase of cell culture. Similar results were obtained when the culture medium was supplemented with triiodothyronine and glucagon, separately or together, in addition to dexamethasone. Under all conditions, GH triggered a potent stimulation of tyrosine phosphorylation of STAT5, whereas no measurable phosphorylation was induced by insulin (Fig. 2CGo).

The apparent inability of insulin to activate the JAK2-STAT5 signaling pathway prompted us to monitor another signaling pathway in the hepatocytes, namely the ERK1 and 2 kinase cascade. To this end, the threonine/tyrosine phosphorylation status of ERK1 and 2 was assessed by immunoblotting with phospho-specific antibodies in hepatocytes challenged with GH or insulin. There was no measurable increase in ERK1 or 2 phosphorylation at any time after GH addition to the cells. On the other hand, insulin triggered a readily discernible burst of hyperphosphorylation of ERK1 and 2. The effect of insulin was maximal at 5 min and amounted to an approximately 3-fold increase in the level of phosphorylation of ERK1 and 2 (Fig. 2DGo).

The effects of GH and insulin on SOCS2 mRNA were investigated next. In hepatocytes that were cultured in the absence or presence of dexamethasone, GH induced a steady accumulation of SOCS2 mRNA within 2 h of hormone addition. A further slight increase to a final level 2- to 3-fold above basal was noted at 6 h (Fig. 3Go). In marked contrast, insulin was essentially devoid of effect in both the absence and presence of dexamethasone, although a very modest rise (not reaching statistical significance) was noted at 1 h post treatment. At the 6-h time point, SOCS2 mRNA was slightly reduced below the basal level in hepatocytes cultured with insulin. Interestingly, the basal level of SOCS2 mRNA was decreased approximately 50% in hepatocytes cultured with dexamethasone, compared with the level in the absence of dexamethasone. This difference is not apparent in Fig. 3CGo because levels of SOCS2 mRNA were expressed relative to levels at the time of GH or insulin additions, without or with dexamethasone, respectively. As may be seen, the extent of SOCS2 mRNA induction by GH was marginally larger in the presence of dexamethasone than in its absence (Fig. 3CGo).



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  		Figure 3. Effects of GH and insulin on level of SOCS2 mRNA in hepatocytes. Primary hepatocytes were cultured for 22 h in the absence or presence of 10 nM dexamethasone (Dex). Then cells were exposed to 500 ng/ml GH or 30 nM insulin for the indicated times and harvested for extraction of total RNA. Samples of 20 µg RNA were subjected to electrophoresis, Northern transfer and hybridization with a radioactive SOCS2 cDNA probe. A, Representative autoradiograph of a blot from cells cultured in the absence of dexamethasone. B, Representative autoradiograph of a blot from cells cultured in the presence of dexamethasone. The positions of ribosomal 28S and 18S RNA are indicated on the left. C, Plot showing the time course of effect of GH and insulin on SOCS2 mRNA in the absence (empty square, GH; empty triangle, insulin) or presence of dexamethasone (filled circle, GH; cross, insulin). Quantification was done by phosphor imaging; mRNA amounts were expressed relative to the level at 0 time, which was assigned a value of 1. Data are the means ± SD from three independent culture experiments.

 
Comparison of the effects of insulin and GH on PKB phosphorylation and glucokinase gene expression
The PI3K/phosphoinositide-dependent protein kinase 1/PKB signaling pathway appears to be crucial to mediate at least some of the metabolic effects of insulin, including the induction of hepatic glucokinase (22). We monitored the activity of this pathway by measuring the extent of phosphorylation of PKB on Ser 473 in hepatocytes stimulated with insulin or GH. Total cellular proteins were extracted at various times after hormone addition and subjected to Western blotting, first with an antibody recognizing phospho-Ser 473 specifically, followed by stripping and incubation with antibodies to total PKB. As can be seen in Fig. 4AGo, insulin caused a rapid, massive, and sustained increase in specific phosphorylation of Ser 473 in PKB. Insulin-dependent PKB phosphorylation seemed somewhat increased in dexamethasone-treated cells, although the range of values with or without dexamethasone overlapped (Fig. 4BGo). GH had no detectable effect on Ser 473 phosphorylation of PKB in presence as well as absence of dexamethasone. Note that the threshold of detection of an increase in PKB phosphorylation in the current immunoblotting assay was lower than 10% of the level of phosphorylation induced by insulin.



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  		Figure 4. Effects of insulin and GH on phosphorylation of PKB in hepatocytes. Experimental details were as described in the legend of Fig. 2Go. Western transfer was performed with samples of 50 µg protein. Nitrocellulose membranes were blotted with an antibody specific to PKB phosphorylated on Ser 473, stripped, and reblotted with antibodies reactive to both nonphosphorylated and phosphorylated PKB. A, Representative immunoblots from cells cultured in the absence of dexamethasone. Upper panel, PKB phosphorylated on Ser 473; lower panel, total PKB. B, Plot showing the time course of effect of insulin and GH on PKB phosphorylation in the absence (empty square, GH; empty triangle, insulin) or presence of dexamethasone (filled circle, GH; cross, insulin). Quantification was done by densitometer analysis. Data are the means from two independent culture experiments; vertical lines indicate the range between the two values.

 
A major effect of insulin in the liver is the induction of glucokinase at the transcriptional level (30). A side-by-side comparison of the effects of insulin and GH on glucokinase mRNA in cultured hepatocytes is shown in Fig. 5Go. As expected, a time-dependent build-up of glucokinase mRNA took place in hepatocytes challenged with insulin. In contrast, hepatocytes cultured with GH did not induce glucokinase mRNA. Note that the data illustrated in Fig. 5AGo were obtained with cells that had been cultured in the presence of dexamethasone because dexamethasone is permissive for insulin-dependent induction of glucokinase. Quantitative data obtained by phosphor imaging of Northern blots from three independent culture experiments are depicted in Fig. 5BGo.



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  		Figure 5. Effects of insulin and GH on level of glucokinase mRNA in hepatocytes. Details for hepatocyte culture and Northern blotting of RNA were as described in Fig. 3Go. Membranes were hybridized with a radiolabeled glucokinase cDNA probe. A, Representative autoradiograph of a blot from cells cultured in the presence of dexamethasone. B, Plot showing the time course of effect of insulin and GH on glucokinase mRNA in the absence (empty square, GH; empty triangle, insulin) or presence of dexamethasone (filled circle, GH; cross, insulin). Quantification was done by phosphor imaging; mRNA amounts were expressed relative to the level at 6 h in the presence of dexamethasone and insulin, which was assigned a value of 100. Data are the means ± SD from three independent culture experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several recent reports have pointed to substantial cross-talk between signaling pathways activated by insulin and GH in a variety of cell systems. Here we have addressed the question of overlap between insulin and GH signaling in a major physiologic target cell for both hormones, the hepatocyte. In response to insulin, primary cultures of hepatocytes displayed a sustained increase in phosphorylation of PKB at Ser 473, a requisite for full activation of this protein kinase, and a strong induction of glucokinase mRNA. In addition, the classical MAPK signaling cascade was activated in insulin-stimulated hepatocytes, as evidenced by a burst of hyperphosphorylation of ERK1 and 2. None of these effects took place after stimulation of hepatocytes with GH. In contrast, GH elicited robust tyrosine phosphorylation of STAT5 and accumulation of mRNAs for SOCS2, whereas none of these effects was seen in insulin-stimulated hepatocytes. Thus, nonoverlapping intracellular signals were triggered by insulin and GH in liver cells, and this led to the induction of separate mRNAs.

A DNA element capable of binding STAT5 was identified using EMSA in the upstream region of the liver promoter of the human glucokinase (19). When included in a glucokinase promoter construct driving the chloramphenicol acetyl transferase gene, this STAT-binding element appeared to sustain modest insulin-stimulated reporter activity in transiently transfected Cos-7 cells overexpressing the insulin receptor and STAT5. On this basis, Sawka-Verhelle et al. (19) hypothesized that STAT5 activation might play a role in the regulation of hepatic glucokinase gene expression by insulin. In contrast to the work of Sawka-Verhelle et al. (19), who used engineered cell lines and assayed the activity of glucokinase promoter-luciferase gene constructs, the present experiments were performed with primary hepatocytes, and were concerned with the response of the endogenous glucokinase gene. They show clearly that insulin-induced accumulation of glucokinase mRNA, which reflects the transcriptional stimulation of the gene (21), occurred without detectable tyrosine phosphorylation of STAT5. Furthermore, tyrosine phosphorylation of STAT5 and induction of the SOCS2 mRNA by GH were not accompanied by a rise of glucokinase mRNA. We therefore concluded that tyrosine phosphorylation of STAT5 is neither necessary nor sufficient to elicit an increase in glucokinase mRNA in cultured rat hepatocytes.

Tyrosine phosphorylation of STAT5 was detected in mouse liver during the fasting-refeeding transition (15). Because insulin hypersecretion occurs under this nutritional condition, the phosphorylation of STAT5 was tentatively ascribed to the action of insulin. Phosphorylation of STAT5 was also noted after perfusion of the isolated mouse liver with insulin (15). The results of the present study do not confirm a direct effect of insulin on tyrosine phosphorylation of STAT5 in hepatocytes. Rather, they suggest the hypotheses that hormones distinct from insulin might have been responsible for the phosphorylation of STAT5 in the liver of the intact animal upon refeeding, and that nonparenchymal cells might have contributed to STAT5 phosphorylation in the liver perfusion system. The alternative possibility, that cultured hepatocytes could lose this particular aspect of insulin responsiveness during the tissue culture process, cannot be formally ruled out. It is noteworthy that glucocorticoids, thyroid hormones, or glucagon, added to the medium during the phase of adaptation of the hepatocytes to culture, could not unmask a potential for insulin to effect significant tyrosine phosphorylation of STAT5. Further studies will be needed to resolve the differences between results in the perfused liver and primary cultures of hepatocytes.

The ability of insulin to activate the STAT5 pathway might well vary among different cell types. Thus, Sadowski et al. (18) have produced strong evidence for insulin-dependent increase in tyrosine phosphorylation of STAT5a and STAT5b and steady-state levels of mRNAs encoding SOCS proteins in the C2C12 myoblasts and myotubes. The insulin effect appeared to be mediated by direct contact between the liganded insulin receptor and STAT5, leading to the phosphorylation of this factor by the intrinsic tyrosine kinase of the receptor or receptor-associated JAKs. Whether similar mechanisms operate in nonimmortalized muscle cells or in other extrahepatic cells of the body is unknown.

Previous work in one of our laboratories has shown that activation of PKB is sufficient to induce glucokinase gene expression in an insulin-like manner (22). Evidence for GH-dependent activation of the PI3K and PKB pathways in cultured cells (13, 14, 31) prompted us to ask whether GH might induce glucokinase gene expression in hepatocytes. Our experiments failed to detect any phosphorylation of PKB on Ser 473 and any induction of glucokinase mRNA in hepatocytes challenged with GH. The lack of GH effect on PKB phosphorylation differs from earlier findings by Shoba et al. (31), who reported transient GH-dependent stimulation of PKB phosphorylation in cultured hepatocytes. These authors also reported that ERK1 and 2 were transiently hyperphosphorylated in hepatocytes after the addition of GH, whereas our experiments did not reveal any significant effect of GH on the ERK1 and 2 pathway. These discrepancies are not understood but might reflect two critical differences in cell culture protocols. First, in the work by Shoba et al. (31), GH was supplied to the hepatocytes immediately after a period of 4 h of attachment of the cells to the dishes. The attachment phase of the culture was conducted in the presence of a high concentration of insulin, which would by itself be expected to induce a strong and protracted phosphorylation of PKB. Under our conditions, acute stimulation with GH took place after a period of 22 h of adaptation of the hepatocytes to culture in medium containing 4% fetal bovine serum but without insulin supplement. Secondly, in the protocol of Shoba et al. (31), extracellular matrix proteins (Matrigel, BD Biosciences, Bedford, MA) were added to the culture medium concomitantly with GH, but no control condition with addition of extracellular matrix proteins in the absence of GH was presented. It is therefore impossible to discern the real contribution of GH to the reported effects, because the engagement of integrins by extracellular matrix proteins is known by itself to trigger hormone-independent activation of the ERK1 and 2 and PI3K/PKB signaling pathways (32).

Another factor that might influence the hormonal responses of cultured hepatocytes is the nutritional status of the rats at the time of cell isolation. In our laboratory, rats are routinely fasted for 2 d prior the isolation of the hepatocytes because this maneuver ensures strong induction of glucokinase in response to insulin. Recently, Beauloye et al. (33) reported that the GH-dependent activation of the JAK-STAT pathway is reduced in liver during starvation, and one could argue that some effects of GH might be blunted in hepatocytes isolated from fasted rather than from fed rats. However, it is important to point out that the hepatocytes in the present experiments were highly sensitive to GH as well as to insulin. This was evidenced by the potent stimulation of STAT5 phosphorylation and SOCS2 mRNA accumulation by GH. In spite of these clear-cut effects, threonine/tyrosine hyperphosphorylation of ERK1 and 2 or Ser 473 phosphorylation of PKB were not detected in GH-stimulated hepatocytes. Therefore, we conclude that the activation of the ERK1 and 2 cascade and the stimulation of the PI3K/PKB pathway are not consistent features of the response of primary hepatocytes to GH. This conclusion is in line with the observation of Chow et al. (34), who failed to detect any increase in tyrosine phosphorylation of IRS1 in the livers of intact rats after an acute administration of GH, whereas the same animals displayed strong tyrosine phosphorylation of hepatic JAK2 and STAT5. In the studies of Yamauchi et al. (12), PI3K was activated in the liver after a single injection of GH, but the extent of activation was extremely small, compared with the effect of insulin, and no data were produced for the activation of PKB downstream of PI3K. Evidence for the activation of PKB in GH-treated cells or cells treated with other cytokines is mostly restricted to engineered cells overexpressing the receptors for these cytokines. The physiological relevance of such findings remains to be proven.


   Footnotes
 
This work was supported by Grant 3200-52445-97 from the Swiss National Science Foundation.

Abbreviations: IRS, Insulin-receptor-substrate; JAK, Janus kinase; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription.

Received March 15, 2002.

Accepted for publication June 3, 2002.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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