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Original Studies |
Pituitary Research Unit, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010; and Department of Physiology and Pharmacology, Center for Molecular and Cellular Biology, University of Queensland (M.J.W.), St. Lucia, Queensland 4072, Australia
Address all correspondence and requests for reprints to: Dr. Kin-Chuen Leung, Pituitary Research Unit, Garvan Institute of Medical Research, 384 Victoria Street, Sydney, New South Wales 2010, Australia. E-mail: k.leung{at}garvan.unsw.edu.au
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There is strong evidence that insulin is essential for GH stimulation of hepatic IGF-I production. Insulin-dependent diabetes mellitus is characterized by a state of GH resistance. Circulating IGF-I levels are low despite high serum GH levels (6, 7), and growth is poor (8, 9). Insulin treatment normalizes serum GH concentrations (10), elevates serum IGF-I levels (11), and stimulates growth (12). These observations suggest that insulin is essential for GH stimulation of IGF-I production and growth. Animal studies also show that GH binding to the liver is reduced in diabetic rats and mice (12, 13) and is increased with insulin treatment (12), providing strong evidence that insulin positively regulates hepatic GHRs.
The mechanism by which insulin regulates hepatic GHRs is not well understood, and there may be multiple sites for receptor regulation. Most previous studies have focused on GHR biosynthesis and internalization as the mechanism for regulation (14, 15). Recently, we identified a novel mechanism of GHR regulation by demonstrating that insulin down-regulates surface GHRs in osteoblasts (16) by impairing receptor translocation from the intracellular pool to the cell surface (17). It is not known whether surface translocation is also a mechanism for regulation of surface GHR status in other tissues, such as the liver. More importantly, there are virtually no data on the regulation of hepatic GHRs in humans, mainly because of the difficulty in obtaining primary hepatocytes for studies.
HuH7 is a human hepatoma cell line with differentiated phenotype (18). It has been shown to express GHR messenger ribonucleic acid (mRNA) (19, 20), although GH binding has not been examined. It responds to GH in stimulation of GHR expression (19) and to insulin in proliferative and functional studies (21, 22). In the present study we have characterized the GH-binding properties of HuH7, validated its suitability for the study of GHRs, and investigated the regulation of GHR expression by insulin.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HuH7 cells were routinely grown in monolayer culture at 37 C in 5% CO2/95% air in MEM (Trace Biosciences, Sydney, Australia) supplemented with 10% FBS, 25 mM HEPES, 2 mM L-glutamine, 50 IU/mL penicillin, and 50 µg/mL streptomycin (Life Technologies, Inc., Melbourne, Australia). To examine the effects of insulin and IGF-I on GH binding to cell monolayers, confluent cultures were set up in 6-cm culture dishes (Falcon, Franklin Lakes, NJ) and treated in triplicate with human insulin (Novo Nordisk, Bagsvaerd, Denmark) or human IGF-I (GroPep Pty. Ltd., Adelaide, Australia) at predetermined concentrations in MEM with 0.2% BSA (MEM/BSA) at 37 C in 5% CO2/95% air for 18 h.
Assay of GH binding in monolayers was performed in 2 mL MEM/BSA with 125I-labeled human GH (2 x 105 cpm) in the presence or absence of 10 µg unlabeled GH at 23 C for 3 h. [125I]GH was prepared by radiolabeling recombinant human GH produced in the Garvan Institute of Medical Research (23) with Na125I (ARI, Sydney, Australia) by the Iodogen method (Pierce Chemical Co., Rockford, IL) to a specific radioactivity of 25–40 µCi/µg. In the specificity study a predetermined amount of unlabeled human GH or human PRL (NIDDK, Bethesda, MD) was also added. At the end of the incubation, the cultures were washed five times with ice-cold PBS containing 0.2% BSA (PBS/BSA), detached with trypsin/ethylenediamine tetraacetate for cell counting, and solubilized in 0.5 mol/L NaOH and 0.1% Triton X-100 for radioactivity measurement. Specific binding of [125I]GH was calculated as the difference between total and nonspecific binding and was corrected for cell number.
Total GH binding
GH binding to surface and intracellular membranes derived from cell sonicates was used as an estimate of total cellular content of functional GHRs. Accordingly, monolayer cultures were set up in 15-cm culture dishes. After 18-h treatment with insulin, the cells were detached by scraping in 4 mL PBS containing a mixture of protease inhibitors (1 trypsin inhibitory unit/mL aprotinin, 10 mmol/L benzamidine, and 0.2 mmol/L phenylmethylsulfonylfluoride; Sigma, St. Louis, MO). After centrifugation, the packed cells were resuspended in 2.5 mL ice-cold inhibitor mixture and disrupted by sonication. Total cellular membranes were obtained by centrifugation of the cell sonicates at 150,000 x g for 15 min at 4 C. The protein content of the membrane preparations was determined by the bicinchoninic acid assay (Pierce Chemical Co.).
To measure total GH binding, the membrane pellets were resuspended at
protein concentrations of 0.5–1.0 mg/mL in 300 µL binding assay
buffer (25 mmol/L Tris-Cl (pH 7.4), 0.1% BSA, and 10 mmol/L
MgCl2) and incubated in triplicate with
105 cpm [125I]GH in the
presence or absence of 10 µg unlabeled GH. After incubation at 4 C
for 18 h, the membrane suspensions were centrifuged, washed twice
with 1 mL ice-cold PBS/BSA, and counted on a 
-counter.
Surface GH binding
As the monolayer GH binding assay measures the sum of surface-bound and internalized [125I]GH, insulin effects on GH binding confined to the cell surface were determined by performing the assay at low temperature to prevent GHR internalization (17, 24). Accordingly, monolayer cultures set up in 6-cm dishes were treated in triplicate with insulin and assayed for GH binding as described above, except that the binding assay was carried out at 4 C for 3 h.
Intracellular GH binding
The content of intracellular GHRs was determined by measuring GH binding to membrane preparations after removal of surface receptors with trypsin treatment. This was done by incubating the cells with 0.25 g/L trypsin at 23 C for 15 min with continuous agitation, followed by addition of equal volume of 1 trypsin inhibitory unit/mL aprotinin. The cells were washed once with PBS, resuspended in 2.5 mL ice-cold inhibitor mixture, and disrupted by sonication. GH binding assay was set up with membrane preparations as described above for total GH binding.
RT-PCR for GHRs
The abundance of GHR mRNA was estimated by RT-PCR assay using the forward (5'-GGATAAGGAATATGAAGTGC-3') and reverse (5'-GATTTCTCATGGTCACTGC-3') primers in exons 7 and 10 of the human GHR gene, respectively (25). Total RNA of insulin-treated cultures was extracted using the TRIzol reagent kit (Life Technologies, Inc., Melbourne, Australia) with procedures as recommended by the manufacturers. The RNA integrity was assessed by visual inspection under UV for the 18S and 28S ribosomal RNA bands after separation by agarose gel electrophoresis and staining with ethidium bromide. RT of sample RNA was performed using the Superscript preamplification system (Life Technologies, Inc.), in which 5 µg sample RNA and random hexamers were used. At the end of the RT reaction, RNA in the mixture was removed by digestion with ribonuclease H and ribonuclease T1 (Life Technologies, Inc.).
PCR was performed with 2 µL of the RT reaction mixture in 50 µL 10 mmol/L Tris-Cl, pH 8.3, containing 50 mmol/L KCl, 2 mmol/L MgCl2, 1.5 U AmpliTaq Gold (Perkin-Elmer Corp., Branchburg, NJ), 2 µmol/L deoxy-NTP, and 50 pmol of the forward and reverse primers. After heating to 94 C for 12 min, 35 cycles were run at 94 C for 45 s, 58 C for 30 s, and 72 C for 30 s, with a final extension at 72 C for 5 min. Then, 20 µL of the PCR product were analyzed by electrophoretic separation on 3% Super Fine Resolution agarose (Amresco, Solon, OH) and staining with ethidium bromide. To control for loading, RT-PCR assay for ß-actin was performed in parallel using 2 µL of the RT reaction mixture and primers specific for human ß-actin (forward, 5'-TGAAGGTGACAGCAGTCGGTT-3'; reverse, 5'-TACACGAAAGCAATGCTATCACCT-3'), with the same procedures for the GHR PCR except that 24 cycles were carried out.
Western analysis of GHRs
The content of immunoreactive GHR protein was assessed by
immunoprecipitation and Western analysis using a monoclonal antibody
against the receptor extracellular domain (MAb263) (26)
and an antiserum against the cytoplasmic domain
(
GHRcyto) (27). HuH7 monolayers
were solubilized in 2 mL lysis buffer [50 mmol/L Tris-Cl (pH 7.2),
0.14 mol/L NaCl, 10 mmol/L NaF, 1 mmol/L
Na3VO4, and 0.4% Triton
X-100] with Complete protease inhibitor cocktail (Roche Molecular Biochemicals, Sydney, Australia). The lysates were
then incubated with MAb263 (1:400) and GHRcyto
(1:200) at 4 C for 18 h, followed by precipitation with ImmunoPure
Protein A/G agarose gel (Pierce Chemical Co.) and washing
with lysis buffer. After boiling in Laemmli sample buffer containing
100 mmol/L dithiothreitol, the samples were separated by SDS-PAGE on
7.5% gel according to the method of Laemmli (28) and
blotted onto PolyScreen polyvinylidene difluoride membrane (NEN Life Science Products, Boston, MA). The membrane was treated
sequentially with blocking buffer (20 mmol/L Tris-HCl (pH 7.4), 150
mmol/L NaCl, 5% skim milk powder, and 0.1% Tween-20),
GHRcyto (1:400 in blocking buffer), and donkey
antirabbit Ig-horseradish peroxidase. The bands were visualized using
the Renaissance chemiluminescence reagent (NEN Life Science Products) and quantified by densitometry.
Internalization
The rate of GHR internalization was measured as the level of
cell-associated [125I]GH after removal of the
surface-bound radioligand by trypsin treatment at the end of the
monolayer GH binding assay. The binding assay was set up at 23 C for
3 h, followed by treatment with 0.25 g/L trypsin at 23 C for 15
min. After three washes with PBS/BSA, the cells were lysed with 0.5
mol/L NaOH and 0.1% Triton X-100, and radioactivity in the lysates was
measured by -counting. Parallel studies without trypsin treatment
were set up to determine the levels of total radioactive incorporation
in the same experiments.
Surface translocation
The translocation of GHRs to the cell surface was measured as the recovery of GH binding to whole cells after removal of receptors on the cell surface by trypsin treatment as described previously (17). The insulin-treated cultures were incubated with 0.25 g/L trypsin at 23 C for 15 min, followed by addition of equal volume of 1 trypsin inhibitory unit/mL aprotinin. After centrifugation, the cells were resuspended in MEM/BSA, dispensed at a concentration of 2 x 106 cells/mL to 6-well plates and allowed to recover at 23 C for 4 h with continuous gentle shaking. GH binding assay (1 h at 23 C) was set up before and after the recovery with the addition of 105 cpm of [125I]GH with or without 10 µg unlabeled GH to the cell suspension. At the end of the assay, the cells were washed twice with ice-cold PBS/BSA before radioactivity measurement.
Effects of PD98059 and wortmannin
To determine the postreceptor signaling pathways involved in the insulin regulation of GHRs, the cultures were treated with 10 µmol/L PD98059 (Calbiochem, Alexandria, Australia), 100 nmol/L wortmannin (Sigma), or 0.01% dimethylsulfoxide vehicle at 37 C for 1 h before the addition of insulin at 10 or 1000 nmol/L. PD98059 and wortmannin are inhibitors of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways, respectively. After incubation for 18 h, the cultures were studied for monolayer and total GH binding and receptor surface translocation as described.
GH-induced Janus kinase-2 (JAK2) phosphorylation
The effects of insulin on GH responsiveness of the cells were examined by measuring JAK2 phosphorylation. The insulin-treated cultures were incubated with 1 µg/mL GH at 37 C for 2 min, washed with 1 mmol/L Na3VO4 in PBS, and solubilized in lysis buffer with protease inhibitor cocktail as in the GHR Western study. The lysates were then incubated with 4 µg rabbit anti-JAK2 polyclonal antibody (HR-758, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 4 C for 18 h and precipitated with ImmunoPure Protein A/G agarose gel, followed by SDS-PAGE separation and blotting onto a polyvinylidene difluoride membrane. The membrane was blocked with blocking buffer containing 1% BSA in place of skim milk powder and incubated with 10 µg antiphosphotyrosine monoclonal antibody (4G10, Upstate Biotechnology, Inc., Lake Placid, NY) in the same buffer overnight at 4 C. The signal was developed with sheep antimouse Ig-horseradish peroxidase and the Renaissance chemiluminescence assay, and quantified by densitometry.
The protein content of JAK2 in the samples was determined after stripping the membrane with 62.5 mmol/L Tris-Cl, pH 6.8, containing 100 mmol/L 2-mercaptoethanol and 2% SDS at 50 C for 30 min. The membrane was then blocked with blocking buffer containing skim milk powder and reprobed with 10 µg HR-758 and donkey antirabbit Ig-horseradish peroxidase, and signal was developed with the chemiluminescence method.
Statistical analyses
All points in the GH binding assays were measured in triplicate. All experiments were repeated at least three times, and the mean ± SEM of results from multiple experiments of the same study are presented. The degree of significance of differences between groups was calculated using Student’s t test or ANOVA (StatView 4.02, Abacus Concepts, Inc., Berkeley, CA) where appropriate and was set at P < 0.05. Simple regression analysis was performed for Scatchard analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To establish the suitability of HuH7 cells for the study of GHRs,
expression of GHR mRNA and [125I]GH binding to
these cells were determined. RT-PCR assay revealed the presence of GHR
mRNA in the cells at a level lower than that in normal human liver
(Fig. 1a
). In contrast, no GHR
mRNA was detected in another human hepatoma cell line, HepG2. The
GH-binding properties of HuH7 were determined by measuring
[125I]GH binding to total cell membranes in the
presence of increasing amounts of unlabeled human GH and human PRL. As
shown in Fig. 1b, GH, but not PRL, displaced
[125I]GH binding, with Scatchard analysis
revealing a Ka of 1.43 ± 0.27 x
109
mol/L-1 (n = 4;
mean ± SEM; Fig. 1c) and a binding capacity of
10.32 ± 1.55 fmol/mg protein.
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Insulin exerted a biphasic effect on monolayer GH binding, with
binding increasing maximally by 2-fold at a concentration of 10 nmol/L
(P < 0.0001) and declining progressively to that of
untreated control at 1000 nmol/L (Fig. 2a). Scatchard analysis revealed that the
elevation of GH binding at 10 nmol/L insulin resulted from an increase
in GHR number (171 ± 12% of the control value; n = 3;
P = 0.004; Fig. 2b), with no effect on binding affinity
(0.40 ± 0.14 and 0.40 ± 0.12 x
109
mol/L-1 for control and
treated cultures, respectively). Unlike insulin, IGF-I at
concentrations of up to 100 nmol/L did not affect monolayer GH binding
(Fig. 2a).
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IR3, against the IGF-I receptor (29). GH
binding was 169 ± 12% and 83 ± 13% of the control value
at 10 and 1000 nmol/L insulin, respectively. Addition of IR3 did not
affect GH binding at either insulin concentration (163 ± 10% and
101 ± 20%, respectively). Surface GH binding and internalization
Because GH is rapidly internalized after binding to its receptor,
the effects of insulin on monolayer binding of
[125I]GH might reflect alteration in the number
of surface GHRs and/or the rate of receptor internalization. To examine
insulin action on GH binding confined to the cell surface, the
monolayer binding assay was performed at 4 C to prevent GHR
internalization. Under these conditions, surface GH binding varied in a
biphasic mode in response to insulin (Fig. 3a), with the levels increasing maximally
to 215 ± 19% of the control value at 10 nmol/L insulin
(P = 0.004) and falling to 100 ± 21% at 1000
nmol/L, a pattern very similar to that seen for monolayer GH
binding.
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).
Therefore, the data on monolayer GH binding reflect the effects of
insulin on the population of surface GHRs and not on the rate of
receptor internalization. Receptor biosynthesis
Insulin action on total GHRs was examined by measuring
[125I]GH binding to membranes of cells
disrupted by sonication. In contrast to its effects on surface GH
binding, insulin increased total GH binding in a
concentration-dependent manner, with the levels increasing by 4.5-fold
to a plateau at a concentration of 100 nmol/L (P <
0.0001; Fig. 4a). The maximal level of GH
binding induced by insulin was not different from that induced by the
addition of 10% serum to the culture medium (431 ± 58%).
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, insulin markedly increased receptor mRNA levels. It also increased
receptor protein content in a concentration-dependent manner (Fig. 4c),
with the level increasing by 5-fold at a concentration of 1000 nmol/L
(P < 0.005; Fig. 4d). Thus, insulin increased total
GHRs by up-regulating receptor biosynthesis. Intracellular GH binding
We next determined whether reciprocal changes in intracellular
GHRs might account for the discrepancy between surface and total GH
binding induced by insulin. Intracellular GHRs were estimated by
performing GH binding studies on total cellular membranes after
removing surface receptors by trypsin treatment. As shown in Fig 5, intracellular GH binding increased
with increasing insulin concentrations by 5 times that of untreated
control at 1000 nmol/L (P < 0.0001). When expressed as
a fraction of total GH binding, intracellular GH binding increased
progressively from 29.3 ± 4.1% in untreated control to 42.2
± 5.6%, 50.8 ± 9.5% (P < 0.05), and 87.0
± 13.6% (P = 0.001) at 10, 100, and 1000 nmol/L
insulin, respectively, suggesting an accumulation of intracellular
GHRs.
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To elicit the mechanism by which insulin caused a dissociation
between surface and total GH binding, its effect on surface
translocation of receptors was investigated. Receptor translocation was
measured as the reappearance of GH binding to whole cells 4 h
after trypsin treatment to remove surface receptors. In untreated
control, GH binding increased by 98.9 ± 19.5
cpm/106 cells (n = 4) during the 4-h period
of recovery. Insulin caused a concentration-dependent reduction in the
reappearance of surface GH binding, with the level significantly
reduced to 32.2 ± 0.7% of the control at 10 nmol/L
(P = 0.005) and completely inhibited at 1000 nmol/L
(P < 0.0005; Fig 6).
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To determine the insulin receptor signaling pathways involved in
regulating total GH binding, PD98059, an inhibitor of the MAPK pathway,
and wortmannin, an inhibitor of PI3K, were used. In the absence of
inhibitors, insulin increased total GH binding by 228 ± 16% and
319 ± 28% of the control value at 10 and 1000 nmol/L,
respectively (P = 0.0002; Fig. 7a). PD98059 alone had no effect on total
GH binding (138 ± 43%), but completely inhibited the increase in
GH binding induced by insulin (127 ± 37% and 136 ± 55%,
respectively; P < 0.05). In contrast, wortmannin did
not influence the action of insulin on total GH binding.
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). PD98059 had no effect on
insulin-induced inhibition of surface translocation. In contrast,
wortmannin not only blocked this effect at 10 nmol/L insulin, but
resulted in an increased receptor translocation by 2-fold at 1000
nmol/L insulin. Therefore, PD98059 and wortmannin had distinct effects
on the insulin-induced changes in GHR biosynthesis and surface
translocation.
The effects of these inhibitors on insulin regulation of monolayer GH
binding are shown in Fig. 7c. Without inhibitors, insulin induced a
biphasic effect, as previously observed, increasing monolayer GH
binding to 223 ± 23% of the control value at 10 nmol/L insulin
(P < 0.01) but having no significant effect at 1000
nmol/L. The addition of PD98059 blocked the increase in GH binding at
10 nmol/L insulin (124 ± 7%; P < 0.025). In
contrast, wortmannin did not significantly affect the increase in GH
binding induced by insulin at 10 nmol/L (223 ± 33%), and
significantly enhanced GH binding at 1000 nmol/L (324 ± 50%;
P < 0.05).
GH-induced JAK2 phosphorylation
To investigate the impact of insulin treatment on GHR action, JAK2
phosphorylation in response to GH was examined. As shown in Fig. 8a, GH specifically stimulated JAK2
phosphorylation. However, the effect of insulin on GH-induced
phosphorylation was dose dependent and biphasic. GH-induced
phosphorylation was increased by 6-fold at 10 nmol/L insulin
(P < 0.005) and fell progressively to a level not
significantly different from the untreated control value at 1000 nmol/L
(Fig. 8b). The total protein content of JAK2 was not affected by
insulin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As insulin can bind to the IGF-I receptor at an affinity about 100-fold lower than that of IGF-I (31), it is possible that the biphasic mode of regulation of monolayer GH binding may have arisen from activation of the IGF-I receptor. However, we found no effect on GH binding of IGF-I at concentrations up to 100 nmol/L, nor was the effect of insulin affected by an antagonistic antibody for the IGF-I receptor. Thus, it is likely that insulin acts specifically through its own receptor to regulate hepatic GHRs.
Animal studies have shown that insulin increases total GH binding to rat liver (12). However, it is not clear from previous studies whether GHR transcription is stimulated by insulin. Although the GHR mRNA level was reduced in the liver of diabetic animals (13, 32), it was not restored with insulin treatment (32). The present study provides strong evidence that insulin increases the total cellular content of hepatic GHRs by up-regulating receptor mRNA expression in humans. However, these findings are opposite that reported recently by Ji et al. (33), who showed that insulin decreased the GHR content of a rat hepatoma cell line. The reason for the disagreement is not obvious, but this may be the result of differential exon 1 promoter usage between cell lines or species (1). The present study shows that insulin increased GHR mRNA abundance and the total cellular content of immunoreactive and functional receptors, revealing internal consistency of its cellular effects.
Insulin caused a dissociation between surface and total GHRs, suggesting that it regulates the subcellular distribution of the receptors. Studies on components of receptor turnover reveal that the loss of surface receptors resulted from inhibition of GHR translocation from the intracellular pool to the cell surface, rather than any effect on receptor internalization. These observations in hepatic cells are similar to our previous findings in osteoblasts (17). However, unlike HuH7, receptor biosynthesis in osteoblasts is not affected by insulin (17), suggesting that a tissue-specific difference may exist at different levels of GHR regulation.
Insulin induced a concentration-dependent increase in GHR biosynthesis, but simultaneously inhibited surface translocation. However, the net effect of reducing receptor surface availability only occurred at concentrations greater than 10 nmol/L, a concentration causing 70% inhibition of surface translocation. These data suggest that up-regulation of surface GHRs can occur with as little as 30% of intracellular receptors available for translocation to the cell surface. At concentrations above 10 nmol/L, the inhibitory effect of insulin on surface translocation overrides the compensatory effect of a 4- to 5-fold increase in receptor biosynthesis. This may be a result of differing ratios of signaling effectors to insulin receptor for the two pathways.
It is well established that insulin triggers a range of intracellular events imparted by the activation of a number of postreceptor signaling pathways (34). Our data show that insulin regulation of GHR biosynthesis and surface translocation are mediated by distinct pathways. Studies with PD98059 reveal that insulin up-regulates receptor biosynthesis via the MAPK pathway, which is known to be responsible for the gene transcriptional action of insulin (35). On the other hand, the wortmannin studies demonstrate that the effect of insulin on receptor translocation is mediated by the PI3K pathway, confirming our previous finding in osteoblasts (17). The PI3K pathway is associated with insulin-induced transport of intracellular proteins such as GLUT4 and receptors for IGF-II and transferrin to the cell surface (36, 37), and the two receptors recycle as component proteins of vesicles containing GLUT4. The insulin effect on GHRs is unique because inhibition rather than stimulation of surface translocation occurs. This suggests that the mechanism for insulin regulation of GHR translocation does not involve an association with GLUT4-containing vesicles.
The studies with inhibitors give insight into how surface GHR availability is governed by insulin through a balance between regulatory effects on receptor biosynthesis and surface translocation. We show that PD98059 specifically inhibited the stimulatory action of insulin on GHR biosynthesis, and wortmannin attenuated the inhibition of receptor translocation. Therefore, it is anticipated that PD98059 would suppress insulin-induced increase in surface GHRs, whereas wortmannin would increase surface receptors by restoring translocation. However, our data show that the net effect on surface GHRs was dependent on the insulin concentration. At a low concentration of insulin, PD98059 inhibited the increase in surface GHRs induced by insulin, whereas wortmannin had no effect. In marked contrast, surface receptors at a high insulin concentration were not affected by PD98059, but were increased by 3-fold with wortmannin. These data thus suggest that the numbers of surface GHRs are largely dependent on total receptor content when surface translocation is partially impaired. However, at high insulin concentrations, surface translocation is a critical mechanism in determining GHR surface availability regardless of the intracellular abundance of receptors.
The significance of insulin regulation of surface GHRs was examined by studying its effects on GH-induced JAK2 phosphorylation, which is the first step involved in the cellular activation of GH action (38). GH-induced JAK2 phosphorylation was significantly modulated by the prevailing concentration of insulin in parallel with the effect of insulin on surface GHRs. Thus, JAK2 phosphorylation was potentiated by a low insulin concentration and attenuated by a high insulin concentration. These observations support the view that insulin modulates GH responsiveness by regulating surface GHR availability. Recently, Wojcik et al. (39) identified a mutation (I153T) in the extracellular domain of the GHR that causes Laron dwarfism. This mutation results in defective translocation of intracellular receptors to the cell surface and, hence, GH insensitivity. This finding highlights the functional importance of GHR surface translocation as a mechanism for controlling GH action.
The present data give insight into a critical role for insulin in regulating hepatic responsiveness to GH as well as the mechanisms involved. Surface translocation of GLUT4 is a well recognized intracellular response to insulin and provides a rapid mechanism that subserves the metabolic needs of the cells. That GHRs are also regulated at the translocational level is consistent with the view that GH is a metabolic hormone (40), whose action can be acutely controlled in response to metabolic cues. The significance of a dominant negative effect of translocation over GHR biosynthesis is unclear, but may represent a rapid mechanism for regulating the metabolic action of GH. It is well documented that GH and insulin interact closely to regulate substrate metabolism with the actions of one opposing, but complementing, those of the other (40). The dominant restraining effect on GHR translocation may be a mechanism of limiting hepatic GH action in the presence of hyperinsulinemia.
Using a human hepatic cell line, we demonstrate that insulin regulates hepatic responsiveness to GH by regulating the surface availability of GHRs. The mechanisms involve distinct and concentration-dependent effects on receptor biosynthesis and surface translocation using different signal transduction pathways. The dual divergent mechanisms of peptide hormone receptor regulation have not been previously described and may have significance beyond insulin and the GHR.
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Acknowledgments |
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IR3. |
Footnotes |
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Received May 24, 2000.
Revised August 16, 2000.
Accepted August 25, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-fetoprotein secretion in human hepatoma
cell lines proliferating in chemically defined medium. Cancer Res. 45:6379–6383.[Medline]
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C. A. Hedman, J. Frystyk, T. Lindstrom, J.-W. Chen, A. Flyvbjerg, H. Orskov, and H. J Arnqvist Residual {beta}-Cell Function More than Glycemic Control Determines Abnormalities of the Insulin-Like Growth Factor System in Type 1 Diabetes J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6305 - 6309. [Abstract] [Full Text] [PDF]
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 P. Kotokorpi, C. Gardmo, C. S. Nystrom, and A. Mode Activation of the Glucocorticoid Receptor or Liver X Receptors Interferes with Growth Hormone-Induced akr1b7 Gene Expression in Rat Hepatocytes Endocrinology, December 1, 2004; 145(12): 5704 - 5713. [Abstract] [Full Text] [PDF]
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 M. M. Buijs, J. A. Romijn, J. Burggraaf, M. L. de Kam, M. Frolich, M. T. Ackermans, H. P. Sauerwein, A. F. Cohen, A. E. Meinders, and H. Pijl Glucose homeostasis in abdominal obesity: hepatic hyperresponsiveness to growth hormone action Am J Physiol Endocrinol Metab, July 1, 2004; 287(1): E63 - E68. [Abstract] [Full Text] [PDF]
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 R. P. Rhoads, J. W. Kim, B. J. Leury, L. H. Baumgard, N. Segoale, S. J. Frank, D. E. Bauman, and Y. R. Boisclair Insulin Increases the Abundance of the Growth Hormone Receptor in Liver and Adipose Tissue of Periparturient Dairy Cows J. Nutr., May 1, 2004; 134(5): 1020 - 1027. [Abstract] [Full Text] [PDF]
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T. K. Hansen, C. H. Gravholt, H. Orskov, M. H. Rasmussen, J. S. Christiansen, and J. O. L. Jorgensen Dose Dependency of the Pharmacokinetics and Acute Lipolytic Actions of Growth Hormone J. Clin. Endocrinol. Metab., October 1, 2002; 87(10): 4691 - 4698. [Abstract] [Full Text] [PDF]
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T. K. Hansen, J. O. L. Jorgensen, and J. S. Christiansen Body Composition and Circulating Levels of Insulin, Insulin-Like Growth Factor-Binding Protein-1 and Growth Hormone (GH)-Binding Protein Affect the Pharmacokinetics of GH in Adults Independently of Age J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2185 - 2193. [Abstract] [Full Text] |
). Total specific GH
binding (Bo) was 0.63 ± 0.06% of the amount of
[125I]GH added. c, Scatchard plot of GH binding derived
from the GH displacement curve in b. The r2 and
P values for the plot are 0.69 and less than 0.0001,
respectively.
, P < 0.0001;
¶, P < 0.005; 
, P <
0.001. b, Scatchard plots of GH binding to untreated control (
) or with insulin at 10 nmol/L (
) and 1000 nmol/L (
). The
cultures were then assayed for total and monolayer GH binding and
receptor translocation. a, Total GH binding. The untreated control
level was 2658 ± 834 cpm/mg protein. Significance
vs. untreated control: *, P =
0.0002; vs. 10 nmol/L insulin alone: #,
P < 0.05; vs. 1000 nmol/L insulin
alone: 
,
P = 0.0005; vs. wortmannin alone:
