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Insulin Inhibits Growth Hormone Signaling via the Growth Hormone Receptor/JAK2/STAT5B Pathway*

Open AccessPublished:May 07, 1999DOI:https://doi.org/10.1074/jbc.274.19.13434
      Insulin is important for maintaining the responsiveness of the liver to growth hormone (GH). Insulin deficiency results in a decrease in liver GH receptor (GHR) expression, which can be reversed by insulin administration. In osteoblasts, continuous insulin treatment decreases the fraction of cellular GHR localized to the plasma membrane. Thus, it is not clear whether hyperinsulinemia results in an enhancement or inhibition of GH action. We asked whether continuous insulin stimulation, similar to what occurs in hyperinsulinemic states, results in GH resistance. Our present studies suggest that insulin treatment of hepatoma cells results in a time-dependent inhibition of acute GH-induced phosphorylation of STAT5B. Whereas total protein levels of JAK2 were not reduced after insulin pretreatment for 16 h, GH-induced JAK2 phosphorylation was inhibited. There was a concomitant decrease in GH binding and a reduction in immunoreactive GHR levels following pretreatment with insulin for 8–24 h. In summary, continuous insulin treatment in rat H4 hepatoma cells reduces GH binding, immunoreactive GHR, GH-induced phosphorylation of JAK2, and GH-induced tyrosine phosphorylation of STAT5B. These findings suggest that hepatic GH resistance may develop when a patient exhibits chronic hyperinsulinemia, a condition often observed in patients with obesity and in the early stage of Type 2 diabetes.
      Growth hormone (GH)
      The abbreviation used is: GH, growth hormone; GHR, growth hormone receptor; JAK, Janus activating kinase; STAT, signal transducers and activators of transcription; PRL, prolactin; IGF, insulin growth factor; GHBP, growth hormone binding protein; h, human; o, ovine; b, bovine.
      1The abbreviation used is: GH, growth hormone; GHR, growth hormone receptor; JAK, Janus activating kinase; STAT, signal transducers and activators of transcription; PRL, prolactin; IGF, insulin growth factor; GHBP, growth hormone binding protein; h, human; o, ovine; b, bovine.
      is one of the prime regulators of body composition (
      • Ho K.K.
      • O'Sullivan A.J.
      • Hoffman D.M.
      ). Along with other hormones and growth factors it increases muscle mass and decreases subcutaneous and visceral fat (
      • Bjorntorp P.
      ,
      • Cuneo R.C.
      • Judd S.
      • Wallace J.D.
      • Perry-Keene D.
      • Burger H.
      • Lim-Tio S.
      • Strauss B.
      • Stockigt J.
      • Topliss D.
      • Alford F.
      • Hew L.
      • Bode H.
      • Conway A.
      • Handelsman D.
      • Dunn S.
      • Boyages S.
      • Cheung N.W.
      • Hurley D.
      ). Abdominal adiposity is prevalent in human diseases of impaired GH function, including Laron syndrome, a GH-resistant syndrome due to mutation of the GH receptor (GHR), and Prader-Willi syndrome in which there is diminished circulating GH (
      • Laron Z.
      ,
      • Angulo M.
      • Castro-Magana M.
      • Mazur B.
      • Canas J.A.
      • Vitollo P.M.
      • Sarrantonio M.
      ). Abdominal obesity is also associated with human peripheral insulin resistance, hyperinsulinemia, and Type 2 diabetes (
      • Bjorntorp P.
      ,
      • Bjorntorp P.
      ). Common to all of these conditions is an increase in the ratio of insulin to GH (
      • Ho K.K.
      • O'Sullivan A.J.
      • Hoffman D.M.
      ,
      • Bjorntorp P.
      ,
      • Bjorntorp P.
      ).
      The GHR belongs to the superfamily of cytokine receptors and in humans and rabbits the full-length GHR is translated from a single mRNA (
      • Baumann G.
      ). Circulating GHBP results from proteolytic cleavage of the plasma membrane-associated GHR (
      • Baumann G.
      ). However, a recent study suggests that primate GHBP may also arise from an alternatively spliced mRNA, a mechanism first indicated in rodents (
      • Baumann G.
      ,
      • Martini J.F.
      • Pezet A.
      • Guezennec C.Y.
      • Edery M.
      • Postel-Vinay M.C.
      • Kelly P.A.
      ). Binding of GH to its receptor results in dimerization of the receptor followed by tyrosine phosphorylation of GHR itself and tyrosine phosphorylation and activation of Janus activating kinase 2 (JAK2) (
      • Argetsinger L.S.
      • Campbell G.S.
      • Yang X.
      • Witthuhn B.A.
      • Silvennoinen O.
      • Ihle J.N.
      • Carter-Su C.
      ,
      • Carter-Su C.
      • Schwartz J.
      • Smit L.S.
      ). Activation of JAK2 by GH, and by other cytokines and growth factors, leads to phosphorylation and activation of one or more signaltransducers and activators oftranscription (STAT) (
      • Carter-Su C.
      • Schwartz J.
      • Smit L.S.
      ,
      • Witthuhn B.A.
      • Quelle F.W.
      • Silvennoinen O.
      • Yi T.
      • Tang B.
      • Miura O.
      • Ihle J.N.
      ,
      • Dusanter-Fourt I.
      • Muller O.
      • Ziemiecki A.
      • Mayeux P.
      • Drucker B.
      • Djiane J.
      • Wilks A.
      • Harpur A.G.
      • Fischer S.
      • Gisselbrecht S.
      ). The JAK-STAT pathway, is a major pathway for GH regulation of gene transcription. Although GH promotes activation of STAT1, STAT3, STAT5A, and STAT5B, gene disruption experiments indicate that STAT5B is necessary for GH regulation of sexually dimorphic hepatic genes (
      • Carter-Su C.
      • Schwartz J.
      • Smit L.S.
      ,
      • Udy G.B.
      • Towers R.P.
      • Snell R.G.
      • Wilkins R.J.
      • Park S.H.
      • Ram P.A.
      • Waxman D.J.
      • Davey H.W.
      ).
      In vivo insulin appears to be necessary for normal liver GH responsiveness, probably by maintaining liver GHR levels (
      • Baxter R.C.
      • Bryson J.M.
      • Turtle J.R.
      ,
      • Bereket A.
      • Lang C.H.
      • Blethen S.L.
      • Gelato M.C.
      • Fan J.
      • Frost R.A.
      • Wilson T.A.
      ,
      • Menon R.K.
      • Arslanian S.
      • May B.
      • Cutfield W.S.
      • Sperling M.A.
      ,
      • Menon R.K.
      • Stephan D.A.
      • Rao R.H.
      • Shen-Orr Z.
      • Downs Jr., L.S.
      • Roberts Jr., C.T.
      • Leroith D.
      • Sperling M.A.
      ). In Type 1 diabetic patients and streptozotocin-treated rodents, insulin deficiency is correlated with hepatic GH resistance which, in most studies, is associated with reduced levels of circulating GHBP in patients or decreased liver GHR in rodents (
      • Baxter R.C.
      • Bryson J.M.
      • Turtle J.R.
      ,
      • Bereket A.
      • Lang C.H.
      • Blethen S.L.
      • Gelato M.C.
      • Fan J.
      • Frost R.A.
      • Wilson T.A.
      ,
      • Menon R.K.
      • Arslanian S.
      • May B.
      • Cutfield W.S.
      • Sperling M.A.
      ,
      • Menon R.K.
      • Stephan D.A.
      • Rao R.H.
      • Shen-Orr Z.
      • Downs Jr., L.S.
      • Roberts Jr., C.T.
      • Leroith D.
      • Sperling M.A.
      ,
      • Kratzsch J.
      • Kellner K.
      • Zilkens T.
      • Schmidt-Gayk H.
      • Selisko T.
      • Scholz G.H.
      ,
      • Hanaire-Broutin H.
      • Sallerin-Caute B.
      • Poncet M.F.
      • Tauber M.
      • Bastide R.
      • Chale J.J.
      • Rosenfeld R.
      • Tauber J.P.
      ,
      • Hanaire-Broutin H.
      • Sallerin-Caute B.
      • Poncet M.F.
      • Tauber M.
      • Bastide R.
      • Rosenfeld R.
      • Tauber J.P.
      ,
      • Mercado M.
      • Baumann G.
      ,
      • Clayton K.L.
      • Holly J.M.
      • Carlsson L.M.
      • Jones J.
      • Cheetham T.D.
      • Taylor A.M.
      • Dunger D.B.
      ,
      • Mercado M.
      • Molitch M.E.
      • Baumann G.
      ,
      • Baxter R.C.
      • Turtle J.R.
      ). In streptozotocin-treated rats, circulating insulin-like growth factor 1 (IGF-1), whose mRNA expression is regulated by GH, is reduced as is GH binding capacity. Insulin treatment restores IGF-1 levels and in some, but not all experiments, restores GH binding (
      • Baxter R.C.
      • Bryson J.M.
      • Turtle J.R.
      ,
      • Menon R.K.
      • Stephan D.A.
      • Rao R.H.
      • Shen-Orr Z.
      • Downs Jr., L.S.
      • Roberts Jr., C.T.
      • Leroith D.
      • Sperling M.A.
      ,
      • Maes M.
      • Ketelslegers J.M.
      • Underwood L.E.
      ,
      • Bornfeldt K.E.
      • Arnqvist H.J.
      • Enberg B.
      • Mathews L.S.
      • Norstedt G.
      ,
      • Chen N.Y.
      • Chen W.Y.
      • Kopchick J.J.
      ). In Type 1 diabetic patients intraperitoneal insulin administration restores GHBP levels better than subcutaneous insulin treatment (
      • Hanaire-Broutin H.
      • Sallerin-Caute B.
      • Poncet M.F.
      • Tauber M.
      • Bastide R.
      • Chale J.J.
      • Rosenfeld R.
      • Tauber J.P.
      ,
      • Hanaire-Broutin H.
      • Sallerin-Caute B.
      • Poncet M.F.
      • Tauber M.
      • Bastide R.
      • Rosenfeld R.
      • Tauber J.P.
      ,
      • Mercado M.
      • Molitch M.E.
      • Baumann G.
      ). This suggests that peripheral (subcutaneous) insulin administration may result in portal insulin concentrations insufficient, compared with intraperitoneal insulin infusion, to properly regulate hepatic GHR expression and therefore circulating GHBP. Patients with Type 2 diabetes and peripheral insulin resistance also exhibit reduced circulating IGF-1 levels, possibly due to a decrease in GH responsiveness, but it has not yet been studied whether liver GHR or circulating GHBP levels are reduced accordingly (
      • Kratzsch J.
      • Kellner K.
      • Zilkens T.
      • Schmidt-Gayk H.
      • Selisko T.
      • Scholz G.H.
      ,
      • Mercado M.
      • Molitch M.E.
      • Baumann G.
      ).
      In vitro studies are also inconsistent concerning whether insulin can increase GH binding and GHR mRNA. For example, in a study with primary cultures of rat hepatocytes, insulin treatment increases GH binding 4-fold with no significant effect on GHR mRNA expression (
      • Tollet P.
      • Enberg B.
      • Mode A.
      ). In a study measuring the subcellular localization of GHR in osteoblasts, continuous insulin treatment decreases the fraction of cellular GHR presented at the plasma membrane via inhibition of surface translocation of GHR with no effect on the total cellular content of GHR (
      • Leung K.C.
      • Waters M.J.
      • Markus I.
      • Baumbach W.R.
      • Ho K.K.
      ). However, changes in GH-induced signaling pathways have not been investigated in these studies measuring insulin-induced changes in GHR and GH binding.
      There is impaired GH action after 60 years of age in humans, possibly resulting from decreases in circulating GHBP levels, and therefore most likely hepatic GHR levels (
      • Maheshwari H.
      • Sharma L.
      • Baumann G.
      ). Also, there may be defects in GH stimulation of the JAK-STAT signaling pathway in aging humans, as there are in aging mice (
      • Xu X.
      • Bennett S.A.
      • Ingram R.L.
      • Sonntag W.E.
      ). Therefore, investigation of factors that affect GH responsiveness may help in the understanding of aging-related changes in GH action.
      In the present study, insulin pretreatment for 8–24 h was found to reduce the acute effect of GH on STAT5B phosphorylation in rat H4 hepatoma cells. The GH-induced tyrosine phosphorylation of JAK2, immunoreactive GHR, and binding of 125I-hGH were also reduced following insulin pretreatment. Inhibition of GH-induced STAT5B phosphorylation, immunoreactive GHR, and GH binding were all reduced by insulin pretreatment with similar kinetics. Our study indicates an extensive reduction in hepatoma cell GH responsiveness following conditions that may mimic the chronic hyperinsulinemia observed in some obese patients and patients in the early stages of Type 2 diabetes.

      DISCUSSION

      Although usually thought of as a “counter-regulatory” hormone, with many actions opposing those of insulin, GH can have acute insulin-like effects, especially in the setting of GH deficiency (
      • Ho K.K.
      • O'Sullivan A.J.
      • Hoffman D.M.
      ). In contrast, GH excess induces insulin resistance (
      • Luger A.
      • Prager R.
      • Gaube S.
      • Graf H.
      • Klauser R.
      • Schernthaner G.
      ) but little is known about the ability of insulin to promote GH resistance. A significant fraction of the adult population exhibit peripheral insulin resistance and hyperinsulinemia. This is true both of Type 2 diabetic patients and a large number of individuals, many of which are obese, but do not exhibit overt diabetes (
      • Bjorntorp P.
      ). In the present study, prolonged treatment of hepatoma cells with high insulin concentrations, similar to those in the hepatic portal circulation in patients with hyperinsulinemia (see below), resulted in reduced GH binding and a severe diminution of GH-induced STAT5B phosphorylation.
      There are two closely related STAT5 isoforms, STAT5A and STAT5B (
      • Liu X.
      • Robinson G.W.
      • Gouilleux F.
      • Groner B.
      • Hennighausen L.
      ). They clearly mediate different functions and the loss of either cannot be compensated by the other isoform in vivo, as indicated by studies with knockout mouse models (
      • Udy G.B.
      • Towers R.P.
      • Snell R.G.
      • Wilkins R.J.
      • Park S.H.
      • Ram P.A.
      • Waxman D.J.
      • Davey H.W.
      ,
      • Feldman G.M.
      • Rosenthal L.A.
      • Liu X.
      • Hayes M.P.
      • Wynshaw-Boris A.
      • Leonard W.J.
      • Hennighausen L.
      • Finbloom D.S.
      ). Acute insulin treatment stimulates STAT5B tyrosine phosphorylation in Chinese hamster ovary cells overexpressing either the insulin receptor or STAT5B (
      • Chen J.
      • Sadowski H.B.
      • Kohanski R.A.
      • Wang L.H.
      ,
      • Sawka-Verhelle D.
      • Filloux C.
      • Tartare-Deckert S.
      • Mothe I.
      • Van O.E.
      ). This change in phosphorylation is not observed in native Chinese hamster ovary cells or hepatoma cells expressing the normal complement of insulin receptors (
      • Chen J.
      • Sadowski H.B.
      • Kohanski R.A.
      • Wang L.H.
      ). In the present work, it is shown that continuous insulin treatment does not significantly alter STAT5B protein concentrations, STAT5B phosphorylation, or STAT5 tyrosine phosphorylation in rat H4 hepatoma cells.
      Patients with peripheral insulin resistance and hyperinsulinemia often exhibit abdominal adiposity, a morphology similar to that found in patients or animal models with GH deficiency or a disruption of GH signaling, such as STAT5B knockout mice (
      • Laron Z.
      ,
      • Angulo M.
      • Castro-Magana M.
      • Mazur B.
      • Canas J.A.
      • Vitollo P.M.
      • Sarrantonio M.
      ,
      • Bjorntorp P.
      ,
      • Udy G.B.
      • Towers R.P.
      • Snell R.G.
      • Wilkins R.J.
      • Park S.H.
      • Ram P.A.
      • Waxman D.J.
      • Davey H.W.
      ,
      • Zhou Y.
      • Xu B.C.
      • Maheshwari H.G.
      • He L.
      • Reed M.
      • Lozykowski M.
      • Okada S.
      • Cataldo L.
      • Coschigamo K.
      • Wagner T.E.
      • Baumann G.
      • Kopchick J.J.
      ). This lead us to hypothesize that hyperinsulinemia may result in GH resistance. This hypothesis is consistent with our presented results indicating that continuous treatment with high concentrations of insulin reduced the ability of GH to stimulate phosphorylation of STAT5B in hepatoma cells. As determined with two different antibodies measuring a GH-induced mobility shift in STAT5B due to phosphorylation and directly measuring tyrosine phosphorylation of STAT5, GH-induced STAT5B phosphorylation was severely reduced by insulin pretreatment for 8–24 h. In addition, the GH-induced tyrosine phosphorylation of immunoprecipitated JAK2 was almost completely inhibited by insulin pretreatment with little change in JAK2 protein levels.
      Since a reduction in GH binding could contribute to the reductions in STAT5B and JAK2 phosphorylation, GH binding was measured using125I-hGH. Ovine PRL was ineffective in competing for125I-hGH, so we conclude that there were few, if any, lactogenic binding sites on rat H4 hepatoma cells, a cell line derived from an hepatocarcinoma of a male rat (
      • Pitot H.C.
      • Peraino C.
      • Morse Jr., P.A.
      • Potter V.R.
      ), and the specific125I-hGH binding was to somatogenic sites. Due to the low levels of GH-specific binding in H4 cells, we choose to measure binding for 2 h at room temperature. The measured specific binding is likely due to both cell surface-binding sites and a small amount of internalized 125I-hGH·GHR complexes. The percentage that is degraded or released into the media is thought to be minimal due to the room temperature incubation and relatively short time of incubation (
      • Lobie P.E.
      • Mertani H.
      • Morel G.
      • Morales-Bustos O.
      • Norstedt G.
      • Waters M.J.
      ,
      • Gavin J.R.
      • Saltman R.J.
      • Tollefsen S.E.
      ). The low amount of total and specific GH binding in this cell line is not surprising since GHR mRNA levels in H4 cells are 40% or less of those in rat liver (
      • Ooi G.T.
      • Cohen F.J.
      • Tseng L.Y.
      • Rechler M.M.
      • Boisclair Y.R.
      ). To our knowledge these represent the first experiments to demonstrate GH binding in H4 cells.
      Insulin pretreatment for 16 h reduced specific125I-hGH specific binding to somatogenic binding sites to 26% of the specific binding prior to pretreatment, a decrease in GH binding comparable to the reduction of GH-induced STAT5B phosphorylation. Previous studies are inconsistent on whether insulin increases GH binding and GHR mRNA or not (
      • Baxter R.C.
      • Bryson J.M.
      • Turtle J.R.
      ,
      • Bereket A.
      • Lang C.H.
      • Blethen S.L.
      • Gelato M.C.
      • Fan J.
      • Frost R.A.
      • Wilson T.A.
      ,
      • Menon R.K.
      • Arslanian S.
      • May B.
      • Cutfield W.S.
      • Sperling M.A.
      ,
      • Menon R.K.
      • Stephan D.A.
      • Rao R.H.
      • Shen-Orr Z.
      • Downs Jr., L.S.
      • Roberts Jr., C.T.
      • Leroith D.
      • Sperling M.A.
      ,
      • Kratzsch J.
      • Kellner K.
      • Zilkens T.
      • Schmidt-Gayk H.
      • Selisko T.
      • Scholz G.H.
      ,
      • Hanaire-Broutin H.
      • Sallerin-Caute B.
      • Poncet M.F.
      • Tauber M.
      • Bastide R.
      • Chale J.J.
      • Rosenfeld R.
      • Tauber J.P.
      ,
      • Hanaire-Broutin H.
      • Sallerin-Caute B.
      • Poncet M.F.
      • Tauber M.
      • Bastide R.
      • Rosenfeld R.
      • Tauber J.P.
      ,
      • Mercado M.
      • Baumann G.
      ,
      • Clayton K.L.
      • Holly J.M.
      • Carlsson L.M.
      • Jones J.
      • Cheetham T.D.
      • Taylor A.M.
      • Dunger D.B.
      ,
      • Mercado M.
      • Molitch M.E.
      • Baumann G.
      ,
      • Baxter R.C.
      • Turtle J.R.
      ,
      • Maes M.
      • Ketelslegers J.M.
      • Underwood L.E.
      ,
      • Bornfeldt K.E.
      • Arnqvist H.J.
      • Enberg B.
      • Mathews L.S.
      • Norstedt G.
      ,
      • Chen N.Y.
      • Chen W.Y.
      • Kopchick J.J.
      ,
      • Tollet P.
      • Enberg B.
      • Mode A.
      ). This may be due to multiple, and sometimes separate, controlling factors for GHR expression and for GH binding. For example, while in vivodata suggests that insulin treatment of insulin-deficient individuals might increase GHBP (and therefore most likely GHR expression), high physiological levels of GHBP may feedback to decrease GH-receptor mRNA levels and low levels of GHBP may increase GH-receptor mRNA expression (
      • Kratzsch J.
      • Kellner K.
      • Zilkens T.
      • Schmidt-Gayk H.
      • Selisko T.
      • Scholz G.H.
      ,
      • Clayton K.L.
      • Holly J.M.
      • Carlsson L.M.
      • Jones J.
      • Cheetham T.D.
      • Taylor A.M.
      • Dunger D.B.
      ,
      • Mullis P.E.
      • Wagner J.K.
      • Eble A.
      • Nuoffer J.M.
      • Postel-Vinay M.C.
      ). Additionally, continuous insulin treatment of osteoblasts decreases the fraction of cellular GHR presented at the plasma membrane via inhibition of surface translocation of GHR with no effect on the total cellular content of GHR (
      • Leung K.C.
      • Waters M.J.
      • Markus I.
      • Baumbach W.R.
      • Ho K.K.
      ). Thus, there may multiple feedback mechanisms controlling GHR expression and the subcellular localization of GHR. Most in vivo studies do not maintain high insulin concentrations for extended periods and therefore do not study the effects of insulin under conditions similar to patients with insulin resistance and hyperinsulinemia. The effect of insulin on GHR expression may be dependent on the concentration of insulin, the duration of insulin treatment and may be tissue and cell-type type specific (
      • Chen N.Y.
      • Chen W.Y.
      • Kopchick J.J.
      ).
      Decreases in GH binding could be due to insulin-dependent changes in GHR synthesis or degradation, or insulin-dependent reduction of cell surface GHR without changes in total GHR expression. GHR normally migrates as a broad band with M r 110–125 due to its complex and variable glycosylation (
      • Asakawa K.
      • Hedo J.A.
      • McElduff A.
      • Rouiller D.G.
      • Waters M.J.
      • Gorden P.
      ,
      • Frick G.P.
      • Tai L.R.
      • Baumbach W.R.
      • Goodman H.M.
      ). Using 2 different antibodies that recognize the cytoplasmic domain of the GHR, there was no significant change in immunoreactive GHR during the first 4 h of insulin treatment. However, immunoreactive GHR was reduced by insulin pretreatment for 8–24 h, with a maximum reduction to less than one-fourth of the untreated cells with insulin for 16 h. Densitometric analysis of the broad GH bands indicated a similar reduction of GHR using either antiserum. Insulin treatment (16 h) at 10 and 100 nmreduced GHR to a similar extent, while 1 nm insulin resulted in an intermediate reduction and 0.1 nm insulin did not alter the amount of immunoreactive GHR, respectively. Thus, chronic hyperinsulinemia resulted in a similar reduction in GH binding, whole cell immunoreactive GHR, and the ability of GH to induce JAK2 and STAT5B phosphorylation.
      The finding of reduced GHR by both binding studies and Western analysis suggests that insulin might reduce expression of GHR, thereby decreasing the number of available somatogenic binding sites and decreasing the levels of immunoreactive GHR. A second, more remote possibility, is that insulin pretreatment results in a post-translational alteration of the GHR resulting in decreased GH binding. This modification would then also have to result in a reduced immuno-detectability by Western blot analysis, possibly due to a conformational change in the cytoplasmic region of the GHR, to which the GHR antibodies were raised. Since two separate polyclonal antibodies were used, it is unlikely that a covalent modification would result in such similar decreases in immunoreactivity of the GHR and in GH binding. However, we cannot exclude this possibility.
      In a number of species, IGF-1 may have some actions similar to those of insulin. Since GH is one of the main regulators of IGF-1 expression, elevated IGF-1 may also act to reduce GHR and GH responsiveness as part of a negative feedback system. However, in the present studies the effects of insulin are through the insulin receptor since H4 hepatoma cells, like the H35 cells from which H4 cells were derived and rat liver, contain few if any IGF-1 receptors (
      • Massague J.
      • Blinderman L.A.
      • Czech M.P.
      ,
      • Krett N.L.
      • Heaton J.H.
      • Gelehrter T.D.
      ).
      The similarity between the timing and percentage decrease in GHR levels and STAT5B phosphorylation following insulin pretreatment suggests that the decrease in GHR is the cause of the reduction of GH-induced STAT5B phosphorylation. The remaining GHR are still functional since a small amount of GH-induced STAT5B phosphorylation is observed even following extended insulin pretreatment. The reduction of GHR levels and GH-induced STAT5B phosphorylation required more than 4 h of elevated insulin and the insulin concentration needed to be 1 nm or greater. In normal human subjects basal insulin concentrations in the hepatic portal circulation are approximately 0.2 nm, severalfold higher than peripheral insulin concentrations (
      • Blackard W.G.
      • Nelson N.C.
      ). Peak post-prandial insulin concentrations reach 0.5–1.0 nm in the peripheral circulation and approximately 3 nm in the portal circulation (
      • Blackard W.G.
      • Nelson N.C.
      ,
      • Cerasi E.
      • Hallberg D.
      • Luft R.
      ). However, in obese individuals with peripheral insulin resistance, peak post-prandial systemic insulin concentrations reach 3–7 nm and this increase in insulin concentration is more prolonged than in normal subjects (
      • Malherbe C.
      • Heller F.
      • Gasparo M.
      • Hertogh R.
      • Hoet J.J.
      ). Portal circulation insulin concentrations were not measured in these obese individuals, but if the ratio of peripheral to portal insulin remains consistent, portal circulation concentrations of insulin would be expected in the range of 20–50 nm. Therefore, normal postprandial concentrations of insulin may be insufficient to result in substantial or prolonged GH resistance. However, in patients with peripheral insulin resistance, where there are elevated basal insulin levels and extended postprandial peripheral insulin concentrations above 1 nm, peripheral GH resistance may develop. In these same individuals, where insulin concentrations in the hepatic portal circulation may rise to well above 10 nmfor extended periods, there is an even greater possibility for the development of hepatic GH resistance. Thus, prior to commencement of exogenous GH administration, knowledge of a patient's insulin sensitivity and circulating insulin concentrations may be warranted.

      Acknowledgments

      We thank A. Keeton, M. Amsler, S.-O. Kim, and J. Jiang for helpful and insightful discussions and suggestions. We are grateful to A. F. Parlow, Pituitary Hormones and Antisera Center, Harbor-UCLA Medical Center (Torrance, CA), and the NIDDK, National Institutes of Health, National Hormone & Pituitary Program for the gift of bGH, to Dr. Ron Chance (Eli Lilly, Co., Indianapolis, IN) for the porcine sodium insulin and hGH, which was also kindly provided by Eli Lilly Co.

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