The anabolic actions of GH are mediated by the production of insulin-like growth factor I (IGF-I) from the liver and by local production of IGF-I in extrahepatic tissues. Insulin facilitates the hepatic production of IGF-I by up-regulating GH receptors (GHRs) in the liver and augmenting the IGF-I response to GH. Although GHRs have also been identified in extrahepatic tissues that produce IGF-I, the possibility that IGF-I and insulin might partake in GHR regulation, thereby modulating the effects of GH locally has not received detailed study. The aim of this study was to investigate whether IGF-I and insulin are involved in the local regulation of GHRs, using osteoblasts as a model of GH-responsive extrahepatic tissues. We have used UMR106.06, a well differentiated rat osteoblast-like cell line that expresses GHRs and exhibits a mitogenic response to GH. IGF-I and insulin (0-10 nM) increased cell number and reduced [125I]GH binding in a concentration-dependent manner, with ED50 values of 0.8 and 0.3 nM, respectively. Although IGF-I increased cell number maximally by 36.9 +/- 1.2% (mean +/- SE) above the control value and insulin by 104.8 +/- 5.7% (P < 0.001), they decreased GH binding to 47.0 +/- 9.3% (P < 0.01) and 29.8 +/- 8.7% of the control value (P < 0.001), respectively. Scatchard analysis revealed that the down-regulation of GH binding was attributed to reduced receptor numbers and not binding affinity. The effects of IGF-I and insulin at submaximal concentrations were additive, although the combined effects did not exceed the maximal effect of either growth factor alone. Addition of an anti-IGF-I receptor antibody (alpha IR3) reversed the inhibition of GH binding induced by IGF-I, but not that caused by insulin; similarly, an antiinsulin receptor antibody (29B4) attenuated the inhibitory effect of insulin only. Addition of alpha IR3 alone or an ant-IGF-I antibody (Sm1.2) decreased cell number and increased GH binding in a concentration-dependent mode. GH at 1.5 nM significant increased cell number by 19.3 +/- 2.4% above the control level (P < 0.01), an increase that was reversed by alpha IR3. GH increased GH binding by 32.4 +/- 7.2% (P < 0.05) in cells treated with alpha IR3 to remove the secondary effect of IGF-I. In summary, IGF-I and insulin acted via specific receptors to stimulate cell proliferation and down-regulate GHRs in osteoblasts. GH stimulated cell proliferation, an action mediated by local production of IGF-I, and GH enhanced its own binding. The collective data suggest the presence of a peripheral negative feedback loop that allows IGF-I to limit locally the response of extrahepatic tissues to circulating GH.
Oral estrogen administration attenuates the metabolic action of growth hormone (GH) in humans. To investigate the mechanism involved, we studied the effects of estrogen on GH signaling through Janus kinase (JAK)2 and the signal transducers and activators of transcription (STATs) in HEK293 cells stably expressing the GH receptor (293GHR), HuH7 (hepatoma) and T-47D (breast cancer) cells. 293GHR cells were transiently transfected with an estrogen receptor-α expression plasmid and luciferase reporters with binding elements for STAT3 and STAT5 or the β-casein promoter. GH stimulated the reporter activities by four- to sixfold. Cotreatment with 17β-estradiol (E 2 ) resulted in a dose-dependent reduction in the response of all three reporters to GH to a maximum of 49–66% of control at 100 nM ( P < 0.05). No reduction was seen when E 2 was added 1–2 h after GH treatment. Similar inhibitory effects were observed in HuH7 and T-47D cells. E 2 suppressed GH-induced JAK2 phosphorylation, an effect attenuated by actinomycin D, suggesting a requirement for gene expression. Next, we investigated the role of the suppressors of cytokine signaling (SOCS) in E 2 inhibition. E 2 increased the mRNA abundance of SOCS-2 but not SOCS-1 and SOCS-3 in HEK293 cells. The inhibitory effect of E 2 was absent in cells lacking SOCS-2 but not in those lacking SOCS-1 and SOCS-3. In conclusion, estrogen inhibits GH signaling, an action mediated by SOCS-2. This paper provides evidence for regulatory interaction between a sex steroid and the GH/JAK/STAT pathway, in which SOCS-2 plays a central mechanistic role.
Human HT-1080 fibrosarcoma cells produce urokinase-type plasminogen activator (u-PA) and type 1 plasminogen activator inhibitor (PAI-1). We found that after incubation of monolayer cultures with purified native human plasminogen in serum-containing medium, bound plasmin activity could be eluted from the cells with tranexamic acid, an analogue of lysine. The bound plasmin was the result of plasminogen activation on the cell surface; plasmin activity was not taken up onto cells after deliberate addition of plasmin to the serum-containing medium. The cell surface plasmin formation was inhibited by an anticatalytic monoclonal antibody to u-PA, indicating that this enzyme was responsible for the activation. Preincubation of the cells with diisopropyl fluorophosphate-inhibited u-PA led to a decrease in surface-bound plasmin, indicating that a large part, if not all, of the cell surface plasminogen activation was catalyzed by surface-bound u-PA. In the absence of plasminogen, most of the cell surface u-PA was present in its single-chain proenzyme form, while addition of plasminogen led to formation of cell-bound two-chain u-PA. The latter reaction was catalyzed by cell-bound plasmin. Cell-bound u-PA was accessible to inhibition by endogenous PAI-1 and by added PAI-2, while the cell-bound plasmin was inaccessible to serum inhibitors, but accessible to added aprotinin and an anticatalytic monoclonal antibody. A model for cell surface plasminogen activation is proposed in which plasminogen binding to cells from serum medium is followed by plasminogen activation by trace amounts of bound active u-PA, to form bound plasmin, which in turn serves to produce more active u-PA from bound pro-u-PA. This exponential process is subject to regulation by endogenous PAI-1 and limited to the pericellular space.
Abstract : This report summarizes the experimental results obtained in the evaluation of the effects of alcohol and drugs on rats under ambient and low temperatures as measured by avoidance response. Based on LD50 determinations, the toxicity of the tranquilizers, chlorpromazine and chlordiazepoxide, and the sedative hypnotics, morphine and promethazine, increased with acute exposure to cold. The toxicity of amphetamine, a stimulant, was lowered at reduced temperatures. (Author)
We previously described significant changes in GH-binding protein (GHBP) in pathological human pregnancy. There was a substantial elevation of GHBP in cases of noninsulin-dependent diabetes mellitus and a reduction in insulin-dependent diabetes mellitus. GHBP has the potential to modulate the proportion of free placental GH (PGH) and hence the impact on the maternal GH/insulin-like growth factor I (IGF-I) axis, fetal growth, and maternal glycemic status. The present study was undertaken to investigate the relationship among glycemia, GHBP, and PGH during pregnancy and to assess the impact of GHBP on the concentration of free PGH. We have extended the analysis of specimens to include measurements of GHBP, PGH, IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), IGFBP-2, and IGFBP-3 and have related these to maternal characteristics, fetal growth, and glycemia. The simultaneous measurement of GHBP and PGH has for the first time allowed calculation of the free component of PGH and correlation of the free component to indexes of fetal growth and other endocrine markers. PGH, free PGH, IGF-I, and IGF-II were substantially decreased in IUGR at 28–30 weeks gestation (K28) and 36–38 weeks gestation (K36). The mean concentration (±sem) of total PGH increased significantly from K28 to K36 (30.0 ± 2.2 to 50.7 ± 6.2 ng/mL; n = 40), as did the concentration of free PGH (23.4 ± 2.3 to 43.7 ± 6.0 ng/mL; n = 38). The mean percentage of free PGH was significantly less in IUGR than in normal subjects (67% vs. 79%; P < 0.01). Macrosomia was associated with an increase in these parameters that did not reach statistical significance. Multiple regression analysis revealed that PGH/IGF-I and IGFBP-3 account for 40% of the variance in birth weight. IGFBP-3 showed a significant correlation with IGF-I, IGF-II, and free and total PGH at K28 and K36. Noninsulin-dependent diabetes mellitus patients had a lower mean percentage of free PGH (65%; P < 0.01), and insulin-dependent diabetics had a higher mean percentage of free PGH (87%; P < 0.01) than normal subjects. Mean postprandial glucose at K28 correlated positively with PGH and free PGH (consistent with the hyperglycemic action of GH). GHBP correlated negatively with both postprandial and fasting glucose. Although GHBP correlated negatively with PGH (r =− 0.52; P < .001), free PGH and total PGH correlated very closely (r = 0.98). The results are consistent with an inhibitory function for GHBP in vivo and support a critical role for placental GH and IGF-I in driving normal fetal growth.
We have reported that insulin and IGF-1 act through their own receptors to down-regulate GH binding in rat osteoblast-like cells (UMR106.06). In this study, we investigated the mechanisms of GH receptor (GHR) down-regulation by the two growth factors at 5 nM after 24 h incubation in serum-free condition. Insulin and IGF-1 significantly decreased GH binding of cell monolayers to 33±5% (mean±SEM) and 40±3% of control respectively (p<0.05). No corresponding decreases in the levels of functional GHR, immunoreactive protein and GHR mRNA of treated cultures were found, showing that the down-regulation was not at the transcriptional or translational level. As the GH binding assay with cell monolayers measures the sum of surface-bound and internalized I-hGH, effects of insulin and IGF-1 on the compartmentalizaton of cell-associated radioactivity were investigated. Acid washing of cultures at the end of binding assay to remove surface-bound tracer revealed no significant difference in GHR internalization rates between control and treated cultures. The effects of insulin and IGF-1 on GHR translocation was assessed by measuring recovery of GH binding activity of whole cells after stripping of surface GHR by trypsin. GH binding of control culture increased 3 fold 2 h after trypsin treatment, whereas no recovery of binding activity was seen in treated cultures. In conclusion, insulin and IGF-1 down-regulate GH binding in osteoblasts by preventing translocation of the receptor to the cell surface. This is a novel mechanism of GHR regulation. (Supported by the NHMRC of Australia).
GH-responsive markers of the IGF system and of collagen turnover hold promise as the basis of a GH doping test.The purpose of this study was to determine the influence of age, gender, body mass index (BMI), ethnicity, and sporting type on GH-responsive serum markers in a large cohort of elite athletes from different ethnic backgrounds.The study was designed as a cross-sectional study.A total of 1103 elite athletes (699 males, 404 females), aged 22.2 +/- 5.2 yr, from 12 countries and 10 major sporting categories participated in this study.Serum IGF-I, IGF binding protein-3 (IGFBP-3), acid labile subunit (ALS), and collagen markers [N-terminal propeptide of type I procollagen (PINP), C-terminal telopeptide of type I collagen (ICTP), N-terminal propeptide of type III procollagen (PIIINP)] were measured.There was a significant negative correlation (r = -0.14 to -0.58, P < 0.0005) between age and each of the GH-responsive markers. Serum IGF-I, IGFBP-3, and ALS were all lower (P < 0.05), whereas the collagen markers PINP, ICTP, and PIIINP were higher (P < 0.05) in men than in women. Multiple regression analysis indicated that age, gender, BMI, and ethnicity accounted for 23-54% of total between-subject variability of the markers. Age and gender cumulatively accounted for 91% of the attributable variation of IGF-I and more than 80% for PINP, ICTP, and PIIINP. Gender exerted the greatest effect on ALS (48%), and BMI accounted for less than 12% attributable variation for all markers. The influence of ethnicity was greatest for IGFBP-3 and ALS; however, for the other markers, it accounted for less than 6% attributable variation. Analysis of 995 athletes indicated that sporting type contributed 5-19% of attributable variation.Age and gender were major determinants of variability of GH-responsive markers except for IGFBP-3 and ALS. Ethnicity is unlikely to confound the validity of a GH doping test based on IGF-I and these collagen markers.
The anabolic actions of GH are mediated by the production of insulin-like growth factor I (IGF-I) from the liver and by local production of IGF-I in extrahepatic tissues. Insulin facilitates the hepatic production of IGF-I by up-regulating GH receptors (GHRs) in the liver and augmenting the IGF-I response to GH. Although GHRs have also been identified in extrahepatic tissues that produce IGF-I, the possibility that IGF-I and insulin might partake in GHR regulation, thereby modulating the effects of GH locally has not received detailed study. The aim of this study was to investigate whether IGF-I and insulin are involved in the local regulation of GHRs, using osteoblasts as a model of GH-responsive extrahepatic tissues. We have used UMR106.06, a well differentiated rat osteoblast-like cell line that expresses GHRs and exhibits a mitogenic response to GH. IGF-I and insulin (0-10 nM) increased cell number and reduced [125I]GH binding in a concentration-dependent manner, with ED50 values of 0.8 and 0.3 nM, respectively. Although IGF-I increased cell number maximally by 36.9 +/- 1.2% (mean +/- SE) above the control value and insulin by 104.8 +/- 5.7% (P < 0.001), they decreased GH binding to 47.0 +/- 9.3% (P < 0.01) and 29.8 +/- 8.7% of the control value (P < 0.001), respectively. Scatchard analysis revealed that the down-regulation of GH binding was attributed to reduced receptor numbers and not binding affinity. The effects of IGF-I and insulin at submaximal concentrations were additive, although the combined effects did not exceed the maximal effect of either growth factor alone. Addition of an anti-IGF-I receptor antibody (alpha IR3) reversed the inhibition of GH binding induced by IGF-I, but not that caused by insulin; similarly, an antiinsulin receptor antibody (29B4) attenuated the inhibitory effect of insulin only. Addition of alpha IR3 alone or an ant-IGF-I antibody (Sm1.2) decreased cell number and increased GH binding in a concentration-dependent mode. GH at 1.5 nM significant increased cell number by 19.3 +/- 2.4% above the control level (P < 0.01), an increase that was reversed by alpha IR3. GH increased GH binding by 32.4 +/- 7.2% (P < 0.05) in cells treated with alpha IR3 to remove the secondary effect of IGF-I. In summary, IGF-I and insulin acted via specific receptors to stimulate cell proliferation and down-regulate GHRs in osteoblasts. GH stimulated cell proliferation, an action mediated by local production of IGF-I, and GH enhanced its own binding. The collective data suggest the presence of a peripheral negative feedback loop that allows IGF-I to limit locally the response of extrahepatic tissues to circulating GH.
The Endocrine Society of Australia, Proceedings 2006, 49th Annual Scientific Meeting, Queensland, Australia, The New Zealand Society of Endocrinology Proceedings 2004 and 2005 Annual Meetings
'/%3e%3c/defs%3e%3cpath fill='%23012169' d='M0 0h10080v5040H0z'/%3e%3cpath stroke='%23fff' stroke-width='.6' d='m0 0 6 3m0-3L0 3' clip-path='url(%23b)' transform='scale(840)'/%3e%3cpath stroke='%23e4002b' stroke-width='.4' d='m0 0 6 3m0-3L0 3' clip-path='url(%23c)' transform='scale(840)'/%3e%3cpath stroke='%23fff' stroke-width='840' d='M2520 0v2520M0 1260h5040'/%3e%3cpath stroke='%23e4002b' stroke-width='504' d='M2520 0v2520M0 1260h5040'/%3e%3cg fill='%23fff'%3e%3cuse xlink:href='%23d' x='2520' y='3780'/%3e%3cuse xlink:href='%23a' x='7560' y='4200'/%3e%3cuse xlink:href='%23a' x='6300' y='2205'/%3e%3cuse xlink:href='%23a' x='7560' y='840'/%3e%3cuse xlink:href='%23a' x='8680' y='1869'/%3e%3cuse xlink:href='%23e' x='8064' y='2730'/%3e%3c/g%3e%3c/svg%3e)
'/%3e%3cuse xlink:href='%23a' transform='rotate(72 0 0)'/%3e%3cuse xlink:href='%23a' transform='rotate(-72 0 0)'/%3e%3cuse xlink:href='%23a' transform='scale(-1 1) rotate(72)'/%3e%3c/g%3e%3c/defs%3e%3cpath fill='%23012169' d='M0 0h1200v600H0z'/%3e%3cpath stroke='%23FFF' stroke-width='60' d='m0 0 600 300M0 300 600 0' clip-path='url(%23b)'/%3e%3cpath stroke='%23C8102E' stroke-width='40' d='m0 0 600 300M0 300 600 0' clip-path='url(%23c)'/%3e%3cpath stroke='%23FFF' stroke-width='100' d='M300 0v300M0 150h600' clip-path='url(%23b)'/%3e%3cpath stroke='%23C8102E' stroke-width='60' d='M300 0v300M0 150h600' clip-path='url(%23b)'/%3e%3cuse xlink:href='%23d' fill='%23FFF' transform='matrix(45.4 0 0 45.4 900 120)'/%3e%3cuse xlink:href='%23d' fill='%23C8102E' transform='matrix(30 0 0 30 900 120)'/%3e%3cg transform='rotate(82 900 240)'%3e%3cuse xlink:href='%23d' fill='%23FFF' transform='rotate(-82 519.022 -457.666) scale(40.4)'/%3e%3cuse xlink:href='%23d' fill='%23C8102E' transform='rotate(-82 519.022 -457.666) scale(25)'/%3e%3c/g%3e%3cg transform='rotate(82 900 240)'%3e%3cuse xlink:href='%23d' fill='%23FFF' transform='rotate(-82 668.57 -327.666) scale(45.4)'/%3e%3cuse xlink:href='%23d' fill='%23C8102E' transform='rotate(-82 668.57 -327.666) scale(30)'/%3e%3c/g%3e%3cuse xlink:href='%23d' fill='%23FFF' transform='matrix(50.4 0 0 50.4 900 480)'/%3e%3cuse xlink:href='%23d' fill='%23C8102E' transform='matrix(35 0 0 35 900 480)'/%3e%3c/svg%3e)
'%3e%3cpath fill='%23012169' d='M0 0v30h60V0z'/%3e%3cpath stroke='%23fff' stroke-width='6' d='m0 0 60 30m0-30L0 30'/%3e%3cpath stroke='%23C8102E' stroke-width='4' d='m0 0 60 30m0-30L0 30' clip-path='url(%23b)'/%3e%3cpath stroke='%23fff' stroke-width='10' d='M30 0v30M0 15h60'/%3e%3cpath stroke='%23C8102E' stroke-width='6' d='M30 0v30M0 15h60'/%3e%3c/g%3e%3c/svg%3e)