Insulin-like Effects Of Growth Hormone Research Articles

Signaling Mechanism for the Insulin-like Effects of Growth Hormone - Another Example of a Classical Hormonal Negative Feedback Loop

Growth hormone (GH) exerts many effects in addition to its ability to stimulate growth. The metabolic effects are either chronic diabetogenic or acute insulin-like. The latter effects are only seen in cells that have been deprived of the hormone for a few hours. After exposure to GH the ability of the cells to respond with insulin-like effects disappears within a couple of hours, a negative feedback loop, which is a part of the chronic effects of the hormone. The insulin-like effects are mediated by the cytosolic tyrosine kinase Janus kinase 2 (JAK2) upon GH-GH receptor interaction, resulting in tyrosine phosphorylation of downstream targets including the GH receptor itself and insulin receptor substrate-1 (IRS-1) and IRS-2. Analogous to the post-receptor events for insulin this results in recruitment of phosphatidylinositol-3 kinase (PI3-kinase) to the IRS-proteins. Downstream PI3-kinase protein kinase B/Akt participates in the activation of glucose transporters (GLUT4) and increased glucose uptake as well as activation of phosphodiesterase 3B and hydrolysis of cAMP leading to a net dephosphorylation of the hormone sensitive lipase and inhibition of lipolysis. Simultaneously, JAK2 phosphorylates STAT-family transcription factors that move into the nucleus and activate the transcription of, among others, genes coding for negatively regulatory proteins called Suppressors of cytokine signalling (SOCS). The turnover of SOCS is rapid and in their presence JAK2 will still activate STAT-proteins (and the diabetogenic effects), but no longer phosphorylate the IRS-proteins (and induce insulin-like effects), closing the loop of yet another classical hormonal negative feedback loop.

SOCS-3 is involved in the downregulation of the acute insulin-like effects of growth hormone in rat adipocytes by inhibition of Jak2/IRS-1 signaling.

One of the long-term effects of growth hormone (GH) in adipocytes is to maintain a state of refractoriness to insulin-like effects, a refractoriness which otherwise declines within a few hours of GH starvation. Here, we examined differences in GH signaling and the possible role for the recently identified family of suppressors of cytokine signaling (SOCS) proteins in the transition between the refractory and the responsive states in rat adipocytes. The ability of GH to stimulate lipogenesis and tyrosine phosphorylation of the GH receptor (GHR), Janus kinase 2 (Jak2), insulin receptor substrate-1 (IRS-1) and -2 (IRS-2) was greatly reduced in refractory as compared to responsive primary rat adipocytes. However, phosphorylation of Signal Transducer and Activator of Transcription 5 (Stat5) was not affected. SOCS-3 and CIS mRNA levels were significantly higher in refractory compared to responsive cells and could be induced by GH, whereas the level of SOCS-2 mRNA was unchanged. With overexpression of GHR, Jak2 and IRS-1 along with each of these SOCS proteins in human A293 cells, we could demonstrate that both SOCS-1 and SOCS-3 completely inhibited the GH-stimulated tyrosine phosphorylation of IRS-1, whereas SOCS-2 and CIS did not. Our data suggest that GH induces refractoriness to the insulin-like effects in a negative-feedback manner by inhibiting GH-induced GHR/Jak2/IRS-1/IRS-2 phosphorylation through upregulation of SOCS-3, which almost completely blocks Jak2 activation.

Stimulation of Lipolysis but Not of Leptin Release by Growth Hormone Is Abolished in Adipose Tissue from Stat5a and b Knockout Mice

The present studies examined the effects of growth hormone (GH) on lipolysis and leptin release by adipose tissue from mice incubated for 24 h in primary culture. In adipose tissue from control mice GH enhanced lipolysis without affecting leptin release. The lipolytic action of GH was unaffected in adipose tissue from Stat5b−/− male mice but leptin release was enhanced by GH in fat from Stat5b−/− mice. In adipose tissue from Stat5ab−/− female mice no significant lipolytic action of GH was seen but leptin release was enhanced by GH. An insulin-like effect of GH on glucose conversion to lactate was also seen in mice deficient in Stat5ab−/−. These results suggest that the lipolytic action of GH involves the Stat5 proteins while the insulin-like effects of GH on glucose metabolism and leptin release involve different mechanisms.

Tyrosine phosphorylation of the growth hormone (GH) receptor and Janus tyrosine kinase-2 is involved in the insulin-like actions of GH in primary rat adipocytes.

The nature of tyrosine phosphorylations induced by GH in relation to the insulin-like metabolic effects in primary rat adipocytes was investigated. Unlike other cells, e.g. 3T3-F442A fibroblast, in which GH is believed to initiate cell differentiation through activation of the Janus tyrosine kinase-2 (JAK2), the adipocytes are metabolically active and fully differentiated cells that do not proliferate. Thus, it cannot be assumed that the same molecular mechanisms relay the acute insulin-like effects of GH. In adipocytes responsive to these effects, we found that GH induced tyrosine phosphorylation of a 114-kilodalton membrane protein, identified as the GH receptor, and a 130-kilodalton cytosolic protein, identified as JAK2. In contrast, these phosphorylations were not seen in adipocytes refractory to these effects of GH. The GH concentration dependency (ED50,1-2 nM) of these phosphorylations coincided with the increase in lipogenesis and the decrease in noradrenaline-induced lipolysis caused by the hormone. In analogy with the effects of insulin, the onset of phosphorylation was rapid (t1/2, < 1 min) and preceded the metabolic responses. The observations that a small fraction of the receptor pool became tyrosine phosphorylated and that the level of phosphorylation induced by GH decreased at higher GH concentrations agree with the concept that GH-induced dimerization of the receptor is necessary for signal transduction. We conclude that tyrosine phosphorylation of JAK2 and the GH receptor seems to be involved in the signal transduction mechanism leading to the insulin-like effects of GH in adipocytes. Importantly, the signal pathways for GH and insulin clearly differ at the receptor level, but seem to converge at or before the level of insulin receptor substrate 1 or 2 phosphorylation that has been shown to occur in response to both of these hormones.

The acute insulin-like effects of growth hormone in primary adipocyte-signaling mechanisms.

The acute insulin-like effects of growth hormone in primary adipocyte-signaling mechanisms.

Growth Hormone Stimulates the Tyrosine Phosphorylation of the Insulin Receptor Substrate-1 and Its Association with Phosphatidylinositol 3-Kinase in Primary Adipocytes

Insulin receptor substrate-1 (IRS-1) is tyrosine-phosphorylated in response to insulin resulting in association with and activation of phosphatidylinositol 3-kinase (PI 3-kinase), thereby initiating some of the effects of insulin. We have recently shown that the insulin-like effects of growth hormone (GH) in adipocytes can be inhibited by the selective PI 3-kinase inhibitor wortmannin (Ridderstråle, M., and Tornqvist, H. (1994) Biochem. Biophys. Res. Commun. 203, 306-310), suggesting a similar role for PI 3-kinase in GH action. Here we show that IRS-1 is tyrosine-phosphorylated in a time- and dose-dependent manner in response to GH in primary rat adipocytes. This phosphorylation coincided with the extent of interaction between IRS-1 and the 85-kDa subunit of PI 3-kinase as evidenced by coimmunoprecipitation. Stimulation with 23 nM GH increased the PI 3-kinase activity associated with IRS1 4-fold. Our data suggest that GH-induced tyrosine phosphorylation of IRS-1 and the subsequent docking of PI 3-kinase are important postreceptor events in GH action. The mechanism for the phosphorylation of IRS-1 induced by GH is unknown, but involvement of JAK2, the only known GH receptor-associated tyrosine kinase, seems possible.

PI-3-Kinase Inhibitor Wortmannin Blocks the Insulin-Like Effects of Growth Hormone in Isolated Rat Adipocytes

The effect of wortmannin, a selective phosphatidylinositol 3-kinase inhibitor, on the insulin-like effects of growth hormone in isolated adipocytes from rat was investigated. Wortmannin inhibited both the lipogenic and the antilipolytic effects (IC50 ≍ 20 nM) with no effect on [125I]-growth hormone binding to the adipocytes. These data suggest that phosphatidylinositol 3-kinase might play an important role in the insulin-like actions of growth hormone.

Cellular mechanism of the insulin-like effect of growth hormone in adipocytes. Rapid translocation of the HepG2-type and adipocyte/muscle glucose transporters.

The cellular mechanism whereby growth hormone (GH) acutely stimulates adipocyte glucose uptake was studied in cultures of primary rat adipocytes differentiated in vitro. Preadipocytes were isolated by collagenase digestion of inguinal fat-pads from young rats and were differentiated in the presence of 3-isobutyl-1-methylxanthine, insulin and dexamethasone. The development of an adipocyte morphology (i.e. lipid inclusions) was observed over 6 days after initiation of differentiation. Coincident with this phenotypic change was an increase in glyceraldehyde-3-phosphate dehydrogenase (GPDH) activity and in cellular content of the HepG2-type (Glut1) and adipocyte/muscle (Glut4) glucose transporter isoforms as determined by Western immunoblotting of total cellular protein. Age-matched undifferentiated cells expressed the Glut1 transporter and low levels of GPDH, but neither accumulated lipid nor exhibited measurable expression of the Glut4 protein. On day 6 after the initiation of differentiation, GH and insulin stimulated 2-deoxy[14C]glucose uptake in a dose- and time-dependent fashion in adipocytes cultured under serum-free conditions for at least 15 h. Western-blot analysis of subcellular fractions revealed that both GH and insulin rapidly (within 20 min) stimulated translocation of the Glut1 and Glut4 proteins from a low-density microsomal fraction to the plasma membrane. Confirmatory evidence was provided in immunocytochemical experiments utilizing antisera directed against the C-terminal region of the Glut4 protein and a fluorescein isothiocyanate-labelled second antibody. Observation of the cells via confocal laser microscopic imaging was consistent with glucose transporter redistribution from an intracellular region to the plasma membrane after treatment with GH or insulin. On the basis of these data, we suggest that the insulin-like effect of GH on adipocyte glucose transport involves translocation of the Glut1 and Glut4 proteins to the plasma membrane. Furthermore, stimulation of glucose-transporter translocation by both GH and insulin may indicate a common cell signalling element between the adipocyte GH and insulin receptors or, alternatively, the existence of multiple cellular mechanisms for stimulating glucose-transporter translocation.

The insulin-like effect of growth hormone on insulin-like growth factor II receptors is opposed by cyclic AMP. Evidence for a common post-receptor pathway for growth hormone and insulin action

The counter-regulatory effects of beta-adrenergic stimulation and cyclic AMP on the insulin-like action of growth hormone (GH) on the subcellular distribution of insulin-like growth factor II (IGF-II) receptors were studied in fat cells from hypophysectomized (Hx) and sham-operated rats. For comparison, the effect of insulin on this process was also studied. Basal IGF-II binding was increased by approx. 2-fold in cells from Hx as compared with sham-operated animals. The stimulatory effect of insulin was decreased in Hx cells, mainly due to a basal redistribution but also to a reduced total number of receptors. GH exerted an acute insulin-like effect in cells from Hx rats and stimulated the translocation of IGF-II receptors from an intracellular pool to the plasma membrane. beta-Adrenergic stimulation with isoprenaline or addition of the non-metabolizable cyclic AMP-analogue N6-monobutyryl cyclic AMP induced a cellular resistance to both GH and insulin and also reduced the responsiveness to these hormones. Adenosine exerted a modulatory effect on both hormones. Binding of 125I-labelled GH to its receptors was not significantly changed by any of these factors. It is concluded that: (1) beta-adrenergic stimulation and cyclic AMP induce a cellular GH resistance at a level distal to the GH-binding site, and (2) the insulin-like effect of GH shares a common pathway with insulin which occurs at the post-binding level.

Induction of Responsiveness of Rat Diaphragm to Ovine Growth Hormone after Administration of Reserpine

The diaphragm of the pituitary intact rat is insensitive to the insulin-like effects of growth hormone unless weanling animals are used, and even then these effects are not achieved reliably. We report here that an intraperitoneal injection of reserpine is able to induce consistent responsiveness to ovine growth horomone (oGH) in hemidiaphragms from 20-27 day old rats as assessed by stimulation of 3H-AIB transport and 14C-phenylalanine incorporation into protein. Maximal stimulation of 3H-AIB transport (approximately 40%) can be elicited by addition of oGH (5 micrograms/ml) to hemidiaphragms after a 2 mg/kg injection of reserpine given 5 h prior to sacrifice. The degree of stimulation does not alter significantly if the rats are sacrificed 3, 5 or 12 h after administration of reserpine, although it decreases by 24 h. Administration of reserpine 3 h before sacrifice also leads to a 50% increase in 14C-phenylalanine incorporation into protein in rat diaphragms in response to the addition of oGH (5 micrograms/ml). The induced sensitivity to oGH is not due to inhibition of GH secretion by reserpine as demonstrated by RIA of plasma GH. Addition of a monoclonal antibody to the GH receptor (MAb263) did not result in a stimulation or inhibition of 3H-AIB uptake or stimulation of protein synthesis in reserpinized rat hemidiaphragms. These results suggest that reserpine can induce tissue responsiveness in rats 20-27 d.o. independent of plasma GH levels. Our results also imply that the type 1 GH receptor of Barnard, Bundesen, Rylatt and Waters (1985) does not mediate the insulin like actions of GH on rat diaphragm.

Sphingosine, an inhibitor of protein kinase C, suppresses the insulin-like effects of growth hormone in rat adipocytes.

Insulin, human growth hormone (hGH), and phorbol 12-myristate 13-acetate all stimulate lipogenesis in rat adipocytes preincubated without hGH for 4 hr. As previous data suggested that protein kinase C plays an important role in the action of insulin and in the insulin-like effects of hGH in rat adipocytes, we tested the effects of sphingosine, a potent inhibitor of protein kinase C, on the lipogenic activity of both hormones. At 50 microM, sphingosine had no effect on basal lipogenesis but completely abolished the action of phorbol 12-myristate 13-acetate and decreased by 65% and 89%, respectively, the effects of hGH and insulin. At higher concentrations (100 microM), sphingosine abolished both basal and hormone-stimulated lipogenesis; this effect was partially reversible after washing the cells. Similar effects of sphingosine on basal and stimulated glucose uptake were seen in parallel, suggesting that sphingosine inhibits lipogenesis at the glucose-uptake step in rat adipocytes. N-Acetylsphingosine and sphingomyelin, two analogs of sphingosine that are inactive on protein kinase C, did not inhibit lipogenesis induced by hGH, insulin, or phorbol 12-myristate 13-acetate. Sphingosine did not inhibit insulin binding to rat adipocytes at concentrations up to 200 microM but decreased hGH binding to its receptors by 44% at 50 microM. These data suggest a direct link between the inhibition of protein kinase C and that of lipogenesis and provide new evidence for the involvement of protein kinase C in the mechanism of action of growth hormone and insulin in rat adipocytes.

Receptor binding properties and insulin-like effects of human growth hormone and its 20 kDa-variant in rat adipocytes.

The natural 20 kDa-variant of human growth hormone (hGH) binds with high affinity to IM-9 human lymphocyte receptors, in agreement with its potency in biological assays for growth promoting and lactogenic activities. In contrast, 20 kDa-hGH has only 3% of the potency of 22 kDa-hGH in binding to the receptors of normal and hypophysectomized rat adipocytes. In agreement with the binding potency, 20 kDa-hGH is only 3% as potent as 22 kDa-hGH in stimulating lipogenesis in normal rat adipocytes preincubated for a few hours in hGH-free medium. The 20 kDa-hGH is also much weaker than 22 kDa-hGH in stimulating lipogenesis in adipocytes from hypophysectomized rats. These data strongly support the concept that the rat adipocyte receptor, which mediates the insulin-like effects of growth hormone, is different from the receptor found on human IM-9 lymphocytes. Preincubation of rat adipocytes with hGH induces a refractoriness to subsequent activation of lipogenesis by hGH but does not abolish the response to insulin, while preincubation with insulin slightly potentiates the hGH response and does not change the insulin response. Additivity studies and a detailed comparison of the lipogenic effects of insulin and hGH suggest that hGH shares only a subset of the metabolic pathways activated by insulin.

Pituitary secretions related to adrenocorticotropic hormone induce sensitivity of adipose tissue to the insulin-like actions of growth hormone.

In its initial encounter with growth hormone (GH) in vitro, epididymal fat excised from GH-deficient rats responds with an insulin-like increase in glucose metabolism. Tissues freshly excised from normal rats are refractory to the insulin-like effects of GH, but become sensitive immediately after surgical stress. Reversal of refractoriness is prevented by administration of the opioid antagonist, naloxone, just prior to stress, suggesting a possible role of beta-endorphin or related peptides. These experiments were undertaken to determine the source of these peptides which might equally well be released from the pituitary, adrenal medullae, or nerve endings in response to stress. Since adrenalectomy, like stress, also results in increased secretion of adrenocorticotropic hormone (ACTH) and related peptides, we studied the effects of GH on glucose oxidation in adipose tissue obtained from adrenalectomized rats and found a significant insulin-like response to GH in tissues studied 4 days after adrenalectomy. This effect was not due to GH deficiency, since plasma concentrations were only slightly reduced by adrenalectomy. Administration of naloxone (250 micrograms/rat), 30 or 60 min before sacrifice, or dexamethasone (100 micrograms/injection), 60 and 120 min before sacrifice, prevented a response to GH without affecting circulating levels of GH. The effects of adrenalectomy could not be reproduced by preincubation of adipose tissue from normal nonstressed rats with ACTH and beta-endorphin, but were duplicated by preincubation of adipose tissue for 15 min in medium in which pituitary glands had previously incubated in the presence of corticotropin-releasing hormone (0.1 microM) and arginine vasopressin (0.2 microM). Addition of naloxone (250 micrograms/ml) blocked this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

The antilipolytic, insulin-like effect of growth hormone is caused by a net decrease of hormone-sensitive lipase phosphorylation.

The mechanism of the antilipolytic effect of GH and the cause of refractoriness to its own action was studied in isolated rat adipocytes. Human GH rapidly inhibited catecholamine-stimulated lipolysis rate, with a time course similar to that of insulin, but only in cells which had been preincubated in the absence of GH for 2-3 h. Half-maximal inhibition was obtained with a GH concentration of 100 ng/ml. Parallel determinations of the lipolysis rate (with a pH-stat titration technique) and the extent of phosphorylation of hormone-sensitive lipase, the rate-controlling enzyme in adipose tissue lipolysis, were made. The extent of lipase phosphorylation, 1.7-fold enhanced by previous noradrenaline stimulation, was rapidly reversed by addition of GH, and the decrease was followed by a parallel decrease in the lipolysis rate. The time course and magnitude of these effects was similar to those obtained with exposure of the cells to insulin, indicating that the antilipolytic effect of both hormones was exerted through the same mechanism: a net dephosphorylation of the hormone-sensitive lipase. To study the refractoriness of the fat cells to the action of GH (which was not found with insulin) adipocytes were prepared from hypophysectomized rats 24 h after surgery. With such cells no preincubation was required to obtain the effects of GH on the lipolysis rate and extent of hormone-sensitive lipase phosphorylation. The effects of GH on both of the parameters studied were similar to those obtained in nonhypophysectomized rats. These results suggest that the refractoriness of the fat cells to GH may be explained by a functional inhibition at a site(s) in the series of metabolic events initiated by GH action, which precedes the activation of the hormone-sensitive lipase.

The effect of synthetic fragment 31–44 of human growth hormone on glucose uptake by isolated adipose tissue

Synthetic tetradecapeptide corresponding to amino acid sequence 31–44 of human growth hormone molecule and possessing a lipotropic activity was tested for the ability to stimulate glucose uptake by isolated epididymal fat pads of fed rats. Tetradecapeptide 31–44 (1 μg/ml), growth hormone (1 μg/ml) and insulin (50 μU/ml) stimulated in about equal degree the uptake of [U- 14C]glucose by adipose tissue. Tissue samples were preliminary incubated for 3–4 hours in the absence of hormones to eliminated the refractoriness to the insulin-like effects of growth hormone. Without preincubation the tissue was refractory to the action of growth hormone and tetradecapeptide 31–44, but was sensitive to insulin. The data obtained together with the findings of Lewis et al., which showed that 20K structural variant of human growth hormone having the deletion of residues 32–46 cannot stimulate glucose uptake and lipolysis in rats, make it possible to suggest that both activities are associated with fragment 31–44.

Comparison of Several Insulin-Like Effects of Growth Hormone

Three different insulin-like effects of growth hormone were studied in segments of adipose tissue obtained from hypophysectomized rats. The onset of each response was preceded by a characteristic lag period: antilipolysis was seen only after a 10--15 minute exposure to growth hormone; stimulation of glucose oxidation was significant 20 minutes after exposure to growth hormone and increased leucine oxidation was seen only after 30 minutes. Each of the responses was measurable without a detectable delay when the tissues were exposed to hormone during a prior incubation period. Accelerated leucine oxidation was detected when 0.01 microgram/ml growth hormone was present in the incubation medium; the other responses required a minimum of 0.1 microgram/ml. Inhibitors of protein synthesis of concentrations which decreased the incorporation of 14C leucine into protein by 99% had no effect on either the antilipolytic action of growth hormone or the stimulatory action on glucose oxidation, but abolished the acceleration of leucine oxidation. In contrast to findings in diaphragm muscle, theophylline was without effect on any of the insulin-like actions of growth hormone in adipose tissue, even though it decreased the basal rate of glucose and leucine oxidation.

Acute suppressive effect of human growth hormone on basal insulin secretion in man.

The acute effects of human growth hormone (GH) on the basal levels of glucose and insulin in blood were investigated in 11 healthy men. GH doses of 5, 10, 20, and 40 mug/kg body weight were given iv as a constant-rate infusion over 30 min, and resulted in peak hormone levels (30 min) of 20.5 plus or minus 1.0, 48.5 plus or minus 2.2, 108.2 plus or minus 4.5, and 229.2 plus or minus 14.6 ng/ml, respectively. There was a small (max 9.8 plus or minus 2.6%) but significant decrease in the blood glucose level, observed already at 15 min after the beginning of the GH infusion and persisting up to 90 min. The highest dose of GH induced the most marked changes, but there was otherwise no clear correlation between dose and effect. The basal plasma insulin levels showed a more marked (max 16.0 plus or minus 4.7%) decrease which was not correlated, in time or in magnitude, with the changes in blood glucose. In some subjects, in whom no significant decrease in blood glucose was observed, plasma insulin still demonstrated a similar fall (max 20.2 plus or minus 7.6%). Neither were these changes in plasma insulin correlated to the dose of GH within the range used in this study. The findings suggested that the early, insulin-like effect of GH on blood glucose is distinct from its effect on the pancreas. The latter is a suppressive one, consistent with earlier findings on glucose-induced insulin release.

The insulin-like effect of growth hormone

The insulin-like effect of growth hormone