Effects of tunicamycin on growth hormone binding in rat adipocytes

Digestion of covalently linked [125I]human (h) GH-receptor complexes with neuraminidase or endoglycosidase F reduced the mass of the principal hormone receptor complex from about 130 kilodaltons (kDa) to 120 and 110 kDa, respectively, suggesting that about 20% of the mass of the GH receptor of rat adipocytes consists of N-linked sialocarbohydrates. Incubation of adipocytes with tunicamycin, an inhibitor of N-linked glycosylation, decreased the incorporation of [35S]methionine into membrane glycoproteins by more than 50% in 4 h and decreased specific binding of [125I]hGH by about 70% after 8 h. Decreased binding and incorporation of [35S]methionine were seen only after a lag time of about 2 h. Cross-linking of [125I] hGH to cells that had been treated with tunicamycin resulted in the appearance of a new labeled species of hormone-receptor complex with an apparent mass of about 110 kDa. This band appeared after a delay of about 3 h and reached approximately equal prominence with the 130 kDa band at 5 h. By 8 h, the 110 kDa complex was the predominant band in radioautograms, but some of the 130 kDa species remained. Scatchard analysis of binding data in tunicamycin-treated adipocytes indicated that decreased binding of [125I]hGH resulted from a 3- to 4-fold decrease in affinity accompanied by only a small (30%) decline in receptor number. Tunicamycin did not affect the rate of receptor turnover in cells that were also treated with cycloheximide to block protein synthesis, but receptor turnover decelerated with increasing time of incubation. Treatment with tunicamycin for 8 h markedly slowed the rate at which specifically bound [125I]hGH disappeared from adipocytes, suggesting that N-linked carbohydrates may play some role in internalization and processing of labeled hormone. We conclude that 1) N-linked carbohydrates contribute about 20 kDa to the apparent mass of the GH receptor of rat adipocytes; 2) N-linked glycosylation is not required for GH receptors to be inserted into the adipocyte membrane in the proper orientation and to retain their ability to recognize and bind GH; 3) N-linked sugar chains are required for maintenance of a normal high affinity of receptors for GH; 4) N-linked carbohydrates are necessary for normal rates of internalization and processing of bound hGH.     

Growth hormone receptors in isolated rat adipocytes

Specific GH binding sites in isolated rat adipocytes have been partially characterized. Binding of [125I]iodohuman(h)GH was rapid, reversible, and was time and temperature dependent. Maximum specific binding occurred at 37 C in approximately 40 min at pH 7.4. Bound labeled hGH was rapidly dissociable, with the addition of excess unlabeled hormone. Specific binding is inhibited by as little as 1.0-1.5 ng/ml hGH, and 50% inhibition was obtained with 15-20 ng/ml. No inhibition was observed with insulin, glucagon, hPRL, or hTSH at concentrations up to 1 micrograms/ml. This receptor does not discriminate between monkey GH, rat GH, bovine GH, and porcine GH. Specific binding varied linearly with cell concentration. Scatchard analysis revealed linear plots with a Ka of approximately 10(9) M-1 and 15,000 sites per cell. There was less than 15% degradation of [125I]iodo-hGH over 90 min at 37 C. There was a striking increase in [125I]iodo-hGH binding to adipocytes at pH 4.85. Scatchard analysis of binding at pH 4.85 revealed a curvilinear plot with an apparent increase of sites per cell from 15,000 to 60,000, and a modest increase in the apparent affinity constant of the high affinity, low capacity sites using the two-compartment model for curvilinear plots. The GH receptors in rat fat cells displayed no ability to bind labeled hPRL or human placental lactogen, consistent with minimal recognition of lactogenic peptides by these receptors. Thus, the rat adipocyte contains specific binding sites for GH that fulfill the major criteria for receptor binding. The presence of such receptors in these cells may facilitate the study of GH receptors in relation to the biological effects of the hormone on adipose tissue in various metabolic settings.     

Relationship between binding and biological effects of human growth hormone in rat adipocytes

Insulin-like responses to human GH (hGH) were produced in adipocytes isolated from the epididymal fat of normal rats 3 h after excision of the tissues. Insulin-like responses consisted of increased oxidation of glucose and incorporation of its carbons into total lipid, increased oxidation of L-[1-14C]leucine, and antagonism of the lipolytic actions of epinephrine. Refractoriness to these effects of hGH in the fourth hour of incubation was produced by the addition of as little as 3 ng/ml hGH as soon as possible after excision of the tissues. These cells also responded to the delayed lipolytic effect of hGH in the presence of theophylline. The cells were found to have high affinity, low capacity, specific binding sites for 125I-labeled hGH. Monoiodination of hGH did not interfere with its capacity to produce biological responses. Specific binding equilibrated rapidly and appeared to saturate at about 100 ng/ml. In cells that were capable of exhibiting an insulin-like response to hGH, rat and ovine GH successfully competed with [125I]hGH for binding sites, but porcine insulin, at a concentration of 100 mU/ml, failed to reduce the binding of [125I]hGH, indicating that GH does not produce its insulin-like effects by interacting with the insulin receptor. Binding of [125I]hGH in cells that are refractory to the insulin-like effects of GH is indistinguishable from binding in responsive cells. Scatchard analysis of the data for both responsive and refractory cells gave linear plots consistent with a single class of about 20,000 receptors/cell, which become half-saturated at a concentration of approximately 20 ng/ml. This corresponds well with 30-50 ng/ml needed for half-maximal insulin-like responses and the 3-10 ng/ml ED50 for induction of refractoriness or lipolysis. It thus appears unlikely that there are appreciable spare receptors for insulin-like responses. These findings make it likely that refractoriness to the insulin-like effects of GH occurs at a postreceptor site.     

Regulation of insulin-like growth factor II receptors by growth hormone and insulin in rat adipocytes.

The acute and long-term effects of growth hormone (GH) on the binding of insulin-like growth factor II (IGF-II) were evaluated in adipose cells from hypophysectomized rats given replacement therapy with thyroxine and hydrocortisone and in cells from their sham-operated littermates. After the cells were incubated with insulin and/or GH, the recycling of IGF-II receptors was metabolically inhibited by treating the cells with KCN. IGF-II binding was 100 +/- 20% higher in cells from GH-deficient animals when compared with sham-operated controls. These GH-deficient cells also showed an increased sensitivity for insulin as compared with control cells (the EC50 for insulin was 0.06 ng/ml in GH-deficient cells and 0.3 ng/ml in control cells). However, the maximal incremental effect of insulin on IGF-II binding was reduced approximately 27% by hypophysectomy. GH added to the incubation medium increased the number of IGF-II binding sites by 100 +/- 18% in cells from hypophysectomized animals. This increase was rapidly induced (t1/2, approximately 10 min), but the time course was slower than that for the stimulatory effect of insulin. Half-maximal effect of GH on IGF-II binding was obtained at approximately equal to 10 ng/ml. Thus, GH added in vitro exerted a rapid insulin-like effect on the number of IGF-II receptors. GH also appears to play a regulating role for maintaining the cellular number of IGF-II receptors and, in addition, modulates the stimulatory effect of insulin on IGF-II binding.     

Growth hormone maintains its own receptors in rat adipocytes

Hypophysectomy decreased the capacity of adipocytes isolated from epididymal fat to bind [125I]human GH [( 125I]hGH) specifically without changing the apparent affinity for hGH. Specific binding of hGH by adipocytes of both normal and hypophysectomized rats appeared saturated when incubated with 75-80 ng/ml or higher concentrations of GH regardless of whether binding was studied for 2 h at 37 C or for 16 h at 0 C. Maximum binding of hGH by normal adipocytes was approximately 0.45 ng/10(6) cells, and that by adipocytes of hypophysectomized rats ranged from 0.15-0.25 ng/10(6) cells. In cells of both normal and hypophysectomized rats, only 25-30% of the hormone specifically bound at 37 was removed by digestion with trypsin, and about 75% was displaced by incubation with 5 M magnesium chloride, suggesting that these adipocytes internalized a significant fraction of bound hormone and that hypophysectomy did not alter the extent of internalization. Previously bound hormone was lost from normal adipocytes with a half-time of about 32 min and from adipocytes of hypophysectomized rats with a half-time of about 45 min, suggesting that hypophysectomy slowed the rate of processing bound hormone. To determine which pituitary hormone(s) might be required to maintain GH binding, we measured the binding of [125I]hGH at 3 or 30 ng/ml by fat cells prepared from hypophysectomized rats after various treatment regimens. Administration of bovine GH ip at a dose of 10 micrograms/rat every 4 h for 24 h doubled the binding of [125I]hGH by adipocytes prepared 4 h after the last injection. Similar results were obtained in fat cells examined 4 h after only one injection of 60 micrograms bovine GH to rats hypophysectomized 2-4 weeks previously. When binding was measured 16-24 h after GH administration, there was no apparent effect on restoration of binding even after treatment with 100 micrograms GH/day for up to 6 days, suggesting that the effects of GH in maintaining receptor number are transient. In accord with the apparently short-lived ability of GH to maintain its receptors on fat cells, GH binding was significantly reduced in adipocytes obtained form both hypophysectomized and sham-operated rats as early as 4 h after surgery, and by 8 h after surgery, declined to a level as low as that in adipocytes of chronically hypophysectomized rats. Twenty-four hours after surgery, GH binding by cells of sham-operated animals returned to normal. Fasting for 24 h also reduced GH binding by adipocytes of normal rats to a level comparable to that in adipocytes of fed hypophysectomized animals.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adenosine 3',5'-monophosphate-dependent loss of growth hormone binding in rat adipocytes

GH exerts a number of metabolic effects on adipose tissue. Depending on the circumstances, it may increase or decrease glucose metabolism and lipolysis. These effects appear to be mediated by a single class of receptors, which bind GH with high affinity. Incubation of isolated rat adipocytes with a variety of lipolytic agents, including catecholamines, forskolin, or (Bu)2cAMP, decreased the specific binding of [125I]human (h) GH within 10 min. In the presence of 10 microM forskolin, GH binding declined to less than 20% of the control value within 50 min. Cholera and pertussis toxins, which increase cAMP secondary to ADP ribosylation of guanine nucleotide-binding proteins associated with hormone receptors, also decreased the binding of GH. None of these agents affected the rate of loss of cell-associated 125I when added to cells that had previously equilibrated with [125I]hGH. The inhibitory effects of forskolin and (Bu)2cAMP were at least as great when binding was measured in the presence of the protease inhibitor leupeptin, suggesting that increased rates of internalization and processing of bound hormone could not account for the decline in binding. Scatchard plots of data obtained in the presence of forskolin or (Bu)2cAMP were linear and parallel to control plots, indicating that the decline in binding could be accounted for by a decrease in the number of binding sites, with no change in affinity. To determine whether phosphorylation affected binding to receptors already present in the membrane or modified the turnover of receptors, we studied adipocyte ghosts, whose cellular apparatus for receptor turnover is disrupted. Incubation of adipocyte ghosts with cAMP-dependent protein kinase decreased the binding of [125I]hGH by 25%. The data suggest that cAMP-dependent phosphorylation of the GH receptor or a closely associated membrane protein renders the receptor incapable of binding GH.     

Acute growth hormone deficiency rapidly alters glucose metabolism in rat adipocytes. Relation to insulin responses and binding

To investigate cellular aspects of the antagonism between endogenous GH and insulin in adipose tissue, we examined glucose metabolism, insulin responses, and insulin binding in adipocytes isolated from rats made GH deficient by treatment with antibodies to rat GH (ArGH), which neutralize the biological actions of GH. Adipocytes were incubated in the presence or absence of insulin, and their ability to convert [14C] glucose to CO2 and lipid was measured. Basal glucose utilization was significantly elevated in adipocytes from ArGH-treated rats after 6, 3, or even 1 h of serum treatment compared to that in controls treated with nonimmune serum. This suggests that endogenous GH suppresses glucose metabolism in adipocytes from normal rats. In the presence of insulin, the absolute values of glucose utilization were also higher in cells from ArGH-treated rats than in controls. However, because basal glucose oxidation was higher with GH deficiency, the percent stimulation by insulin was not different in adipocytes from ArGH-treated rats and controls. In keeping with this, the binding of [125I]iodoinsulin was not altered by GH deficiency. These findings indicate that endogenous GH suppresses basal glucose metabolism in adipocytes, and that this effect is rapidly reversed by treatment of normal rats with ArGH. Furthermore, this effect of GH on adipocyte metabolism is independent of changes in insulin binding and appears to account for the enhanced responses to insulin observed with GH deficiency.     

Covalent cross-linking of growth hormone-releasing factor to pituitary receptors

We have used a bifunctional cross-linker, disuccinimidyl suberate, to covalently attach [125I]human pancreatic GH-releasing factor (GHRF) (-1-40)OH to bovine pituitary membranes and rat anterior pituitary cells. Covalently radiolabeled membrane and cell preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. In the former case, we observed the specific labeling of a polypeptide with an apparent mol wt of 75,000 +/- 3,000. The labeling of this species was specific for GHRF, as evidenced by the fact that it was inhibited in a dose-dependent fashion with increasing concentration of unlabeled GHRF. Furthermore, the radiolabeling was inhibited in the presence of excess unlabeled GHRF analogs but not unrelated peptides such as insulin and rat GH. The size of the radiolabeled band was the same in both bovine pituitary membranes and rat anterior pituitary cells. The extent of radiolabeling was dependent on the amount of membrane or the number of cells present during the binding reaction. These observations indicate that the mol wt 75,000 species is a ligand-binding subunit of the GHRF receptor in the pituitary. Under nonreducing conditions, a species much larger than mol wt 200,000 was specifically radiolabeled, again in both bovine pituitary membranes and rat cells. This result suggests the possibility that the ligand-binding subunit might be disulfide-linked to other subunit(s) forming homo- and heterooligomers.     

Refractoriness to growth hormone is associated with increased intracellular calcium in rat adipocytes.

In adipocytes that have been deprived of growth hormone (GH) for at least 3 hr, GH elicits a transient insulin-like response that is followed by a period of refractoriness to further insulin-like stimulation. Exposure of adipocytes to GH in the first hour of a 3-hr incubation prevents the appearance of insulin-like sensitivity. Intracellular Ca2+ concentration [( Ca2+]i) was measured in individual adipocytes that were loaded with fura-2 hexakis(acetoxymethyl) ester after preincubation in the presence (refractory) or absence (sensitive) of recombinant human GH at 100 ng/ml. Using a dual nitrogen laser imaging microscope with computer-assisted image processing to measure fluorescence changes, we observed that resting [Ca2+]i was 220 +/- 10 nM in refractory adipocytes and 110 +/- 6 nM in sensitive adipocytes (P less than 0.001). GH had no acute effect on [Ca2+]i in sensitive adipocytes but caused a sustained 3-fold increase in [Ca2+]i in refractory cells within 3 min (P less than 0.001). Insulin did not change [Ca2+]i in either sensitive or refractory adipocytes. In refractory cells treated with insulin and GH simultaneously, insulin completely blocked the rise in [Ca2+]i due to GH. Oxytocin elicited a prompt increase in [Ca2+]i followed by a quick return to resting levels in both sensitive and refractory cells. These findings indicate that basal [Ca2+]i is increased in refractory cells and that GH produces a sustained rise in [Ca2+]i only in refractory adipocytes. We suggest that the sustained increase in [Ca2+]i produced by GH in refractory cells prevents the expression of the insulin-like response.     

Nucleotide regulation of growth hormone-releasing factor binding to rat pituitary receptors

The specific binding of a GRF radioligand, [His1,125I-Tyr10,Nle27]hGRF-1-32NH2, to rat pituitary homogenates is reduced by the addition of GTP and its nonhydrolyzable analogs 5'-guanylylimidodiphosphate (GppNHp) and guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). GDP and cAMP had no effect while the nonhydrolyzable ATP analogs 5'-adenylylimidodiphosphate and adenosine 5'-O-(3-thiotriphosphate) did elicit a significant reduction in GRF binding. The effect of GppNHp was half-maximal at 0.2 microM, and the maximum inhibition achieved was 85%. The effect of 0.1 microM GppNHp on GRF competitive displacement experiments indicated a significant reduction in affinity for the ligand (Kd = 0.51 +/- 0.11 nM in the absence of GppNHp and 2.1 +/- 1.1 nM in its presence) without an effect on receptor number. The GRF radioligand dissociates slowly from its receptor (t1/2 = 250 +/- 50 min), but the addition of 0.1 microM GppNHp converts approximately half of the receptors present to a more rapidly dissociating form (t1/2 = 9 +/- 10 min). These results are consistent with existing models for receptor-G-protein interactions, and thus, we conclude that transduction of the GRF response across the cell membrane involves a guanine nucleotide-binding protein, presumably Gs.     

Comparison of growth hormone binding and metabolic response in rat adipocytes of epididymal, subcutaneous, and retroperitoneal origin

We undertook a comparison of human growth hormone (hGH) binding and metabolic responses in rat adipocytes of epididymal, subcutaneous, and retroperitoneal origin to determine whether the site of fat depot biopsy might affect the response to hGH stimulation. The results showed highest specific binding in epididymal (3.6%), followed by subcutaneous (2.3%) and retroperitoneal adipocytes (1.5%); half-maximal binding was achieved at 14-18 ng/ml hGH for the three sites. Scatchard analysis of the binding data from each site was linear; there was no significant difference in binding affinities (2.1 to 3.3 X 10(9), M-1), but the number of binding sites was statistically higher in epididymal (9.8 X 10(3) as compared to subcutaneous (7.5 X 10(3), P less than 0.05) and retroperitoneal cells (3.3 X 10(3), P less than 0.01). Stimulation with 5 to 2500 ng pituitary hGH produced a dose-related increase in glucose incorporation, with the largest increase in epididymal fat cells (31%, P less than 0.05) followed by subcutaneous cells (18%, P less than 0.05); no significant increase was seen with retroperitoneal cells. Biosynthetic hGH produced a similar pattern of glucose incorporation in the three sites. Addition of hGH antibodies blocked the glucose incorporation in epididymal adipocytes using both pituitary-derived and biosynthetic hGH. It seems clear that this insulin-like effect is caused by hGH, not an insulin-like impurity. We conclude that the number of binding sites, perhaps related to adipose cell size, differs in adipose tissue from different locations and this influences the metabolic response to hGH stimulation.     

Growth hormone binding to specific receptors stimulates growth and function of cloned insulin-producing rat insulinoma RIN-5AH cells

Binding of 125I-labeled human GH (hGH) to a cloned rat insulin-producing cell line RIN-5AH in monolayer culture was studied along with some physiological effects of the hormone on these cells. Binding was time and temperature dependent, and steady state binding was observed in 60 min at 37 C with [125I]hGH at 4.2 pM, whereas at 24 C, binding had not reached a steady state after 120 min. The binding was largely reversible, since 80% of initially bound [125I]hGH dissociated from the cells upon incubation in hGH-free buffer for 120 min. Half-maximal binding was obtained when cells were incubated in the presence of 3.0 X 10(-10) M unlabeled hGH. Rat GH as well as human placental lactogen were able to compete for binding sites, but with less affinity. Other non-GH peptides at 6.7 micrograms/ml did not affect [125I]hGH binding. Scatchard analysis revealed curvilinear plots, and approximately 2700 high affinity binding sites were calculated. Culture of RIN-5AH in the presence of 1 microgram/ml hGH for 4 days resulted in an 80% increase in insulin content as well as an 18% increase in cell number and DNA and protein content compared to those in cells cultured in the absence of hGH. The dose dependence of the insulinotropic effect showed that half-maximal and maximal stimulation were observed in cells cultured in the presence of 10 and 100 ng/ml, respectively. Insulin release to the medium during the 4-day culture period was not affected by hGH. These data suggest that GH, through binding to specific receptors in the cell membrane, directly stimulates proliferation and function of pancreatic beta-cells.     

Specific binding of iodinated growth hormone to rat liver in vivo.

The specific uptake by rat liver of human (hGH) and bovine (bGH) GHs labeled with 125I was studied by an in vivo procedure. A significant reduction of the uptake was observed when labeled hormones were injected together with different amounts of the corresponding native GH. This reduction was dose dependent, and the concentration of native hormone that prevents 50% of the liver uptake of the labeled hormone was close to 12 microgram/100 g BW. In normal rats, only native hGH or bGH significantly decreased the liver uptake of [125I]iodo-bGH, while bovine PRL (oPRL) or heat-denatured bGH were inactive. The highest inhibition of the uptake of [125I]iodo-hGH by rat liver was obtained when this labeled hormone was injected either together with hGH or with bGH plus oPRL while partial displacement was observed with bGH or oPRL. These data suggest that hGH binds to both somatotropic and lactogenic sites in the liver of normal rats. In hypophysectomized animals, only the somatogenic binding sites could be detected.     

Identification of growth hormone binding protein in rat serum

The present report describes the initial characterization of a specific, high-affinity growth hormone binding protein (GH-BP) in adult male rat serum. GH-BP activity was measured by incubation of rat serum with [125I]hGH and [125I]rGH and separation of bound from free GH by dextran-coated charcoal. [125I]hGH binding to rat serum was dependent on serum concentration and incubation time, equilibrium being reached within 10 min both at 4 and 37 degrees C. Binding was rapidly and completely reversible and specific for somatogenic (but not lactogenic) hormones. Scatchard analysis yielded a linear plot with an affinity (Ka) of 1.51 +/- 0.63 x 10(8) M-1. Preliminary data obtained in various physiological conditions showed that GH-BP activity in adult male rats was 5.95 +/- 0.20%/0.1 ml serum. Significantly higher values were obtained in sera of female (21.66 + 0.79%/0.1 ml serum) and pregnant rats (23.02 +/- 1.15%/0.1 ml serum). A closer analysis of these binding values by Scatchard analysis revealed that the binding capacity in pregnant rats (50.5 +/- 5.8 pmol/0.1 ml serum) was significantly higher than in adult female estrous rats (19.2 +/- 6.5 pmol/0.1 ml serum), both being much higher than in adult male rats (2.5 +/- 0.6 pmol/0.1 ml serum). The GH-BP activity of 10-day-old rats was only approximately 63% of the adult male rat value. The presence of high-affinity GH-specific binding protein in rat serum suggests a probable action in regulation of GH activity. The detailed physiological role of rat serum GH-BP is currently being investigated.     

Thyroid hormone regulates rat pituitary insulin-like growth factor-I receptors

As we previously obtained evidence that insulin-like growth factor-I (IGF-I) inhibits T3-induced GH secretion and GH mRNA expression without affecting basal GH secretion in thyroidectomized rat pituitary cells grown in hypothyroid medium, we examined changes in IGF-I receptors in the pituitary gland, as induced by thyroid hormone. Thyroidectomized rats and a quantitative receptor autoradiographic method were used. The density of [125I]IGF-I-binding sites in the anterior pituitary gland decreased 4 weeks after thyroidectomy; that is a significant decrease in the number of the receptors compared to findings in control rats (P less than 0.01). The affinity (Kd) remained unchanged. There were no changes in binding parameters in the ventroposterior thalamic nucleus in the brain, renal cortex, and liver parenchyma. The ip administration of T4 once a day (48 micrograms/kg) for 1-2 weeks compensated for the decrease in the binding capacity of [125I]IGF-I-binding sites to that of the control values (P less than 0.01). We propose that IGF-I receptors in the anterior pituitary gland may be regulated by thyroid hormone.     

Growth hormone receptors are up-regulated in diabetic adipocytes

Using an isolated fat cells (IFC) perifusion system and bovine growth hormone (bGH), we demonstrate that the lipolytic response in normal rat IFC is markedly enhanced after preincubation with bGH. In contrast, when IFC are prepared from diabetic animals or in the spontaneous diabetic BB rat (SDR-BB), no such preincubation is necessary. These IFC respond immediately to bGH with maximal release of glycerol. Using a binding assay established for rat growth hormone (rGH) receptors, we measured the number of GH receptors in IFC from these rats. We demonstrate a 75% increase in GH receptors after preincubation with GH in normal rat IFC, and a 125% increase in GH receptors in diabetic IFC, without preincubation. These data support the concept that enhanced sensitivity to GH is an important feature of diabetes in rats and that this sensitivity is at least in part controlled by up-regulation of GH receptors.