Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cultured rat granulosa cells have provided a useful model to examine the hormonal regulation of inhibin secretion. In the present study we have used the cloned rat inhibin alpha- and beta A-subunit cDNAs to characterize the influences of gonadotropins, growth factors, and GnRH on inhibin subunit mRNA levels in granulosa cells obtained from immature estrogen-treated rats. Cells were cultured in medium with or without added hormones. Total RNA from cultured cells was extracted and hybridized with 32P-labeled inhibin alpha- and beta A-subunit cRNA or beta-actin cDNA probes, and inhibin subunit mRNA levels were normalized with beta-actin mRNA levels. Treatment of granulosa cells with FSH increased inhibin alpha- and beta A-subunit mRNA levels in a dose-dependent manner. Similarly, LH, but not PRL, increased alpha- and beta A-subunit mRNA levels in granulosa cells pretreated with FSH to induce functional LH and PRL receptors. The effects of FSH and LH on inhibin subunit mRNA levels were mimicked by forskolin, which increased alpha- and beta A-subunit transcripts in a dose- and time-dependent manner, suggesting involvement of the cAMP-dependent protein kinase-A pathway. Since several growth factors have been shown to influence inhibin secretion, their effects on inhibin subunit mRNA levels were also studied. Treatment of cells with transforming growth factor-beta 1 increased both basal and FSH-stimulated inhibin alpha- and beta A-subunit mRNA content, whereas insulin-like growth factor-I had no significant effect. In contrast, both epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) markedly suppressed both basal and FSH-stimulated inhibin subunit transcript levels. The inhibitory effects of EGF and basic FGF were dose dependent and persisted from 12-72 h of incubation. The regulatory peptide GnRH, which decreases inhibin secretion, was also found to suppress FSH-stimulated inhibin alpha- and beta A-subunit mRNA levels in a dose-dependent manner. Furthermore, the effects of GnRH could be counteracted by coincubation with a GnRH antagonist, suggesting the involvement of specific GnRH-binding sites in GnRH action. These studies indicate that, except for insulin-like growth factor-I, the effects of gonadotropins, growth factors (EGF, basic FGF, and transforming growth factor-beta 1), and GnRH on inhibin secretion are related to their regulation of inhibin alpha- and beta A-subunit mRNA levels.
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PMID:Regulation of inhibin subunit messenger ribonucleic acid levels by gonadotropins, growth factors, and gonadotropin-releasing hormone in cultured rat granulosa cells. 211 34

Relaxin is a member of the insulin family of polypeptide hormones and is known to exert its biological effects on various parts of the mammalian reproductive system. Biologically active human relaxin has been chemically synthesized based on the nucleotide sequence obtained from an ovarian cDNA clone. In the present study synthetic human relaxin was radiolabled by phosphorylation with cAMP-dependent protein kinase and [gamma-32P]ATP to a specific activity of 5000 Ci/mmol. The phosphorylated relaxin was purified on cation exchange high performance liquid chromatography and was shown to co-migrate with relaxin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mass spectrometry revealed a single phosphorylated site on the B chain of relaxin. The 32P-relaxin was able to bind to a goat anti-relaxin antibody, and this binding could be displaced by unlabeled relaxin in a concentration-dependent manner. A comparison of the concentration responses of cellular cAMP production stimulated by relaxin and phosphorylated relaxin in a primary human uterine cell line showed that phosphorylation did not affect the in vitro biological efficacy of relaxin. This made it suitable for in situ autoradiographic localization of relaxin binding sites in rat uterine, cervical, and brain tissue sections. Displacement of the binding of 100 pM 32P-relaxin by 100, 10, and 3 nM unlabeled relaxin, but not by 100 nM insulin, insulin-like growth factor-I, and an insulin-like growth factor-I analog, demonstrated the high affinity and specificity of such binding. We conclude that 32P-labeled human relaxin is biologically and immunologically active and that this novel probe binds reversibly and with high affinity to classical (e.g. uterus) and unpredicted (e.g. brain) tissues.
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PMID:Preparation of biologically active 32P-labeled human relaxin. Displaceable binding to rat uterus, cervix, and brain. 216 Sep 76

Rat astrocytes synthesize and secrete two types of plasminogen activators (PAs), tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), whose functions are related to cell proliferation, migration, and differentiation during development. The regulation of PAs produced by brain astrocytes is poorly understood. In a previous report we demonstrated that t-PA and u-PA are each independently regulated by cAMP-dependent protein kinase and protein kinase-C. In the present study we examined the effects of three well characterized astrocyte mitogens, insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF), on the PA activities produced and secreted by rat astrocytes in vitro. We found that IGF-I and EGF increase cell-associated total PA activity in astrocyte-conditioned medium (CM). The effects of both growth factors were dose and time dependent, and maximal stimulation was achieved after 72 h of treatment with the highest dose tested (100 nM). IGF-I stimulated the cell-associated PA activity more than the CM activity, whereas EGF showed an opposite pattern, suggesting that the secretion of PA is differentially modulated by IGF-I and EGF. PDGF had no effect on astrocyte PA activities at any dose or time point included in the study. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis/zymography showed type-specific changes in CM and cell-associated PA activity after growth factor treatment. IGF-I stimulated only t-PA, whereas EGF induced a marked increase in u-PA activity and a more limited increase in t-PA. PDGF did not modify either t-PA or u-PA activity. In summary, our results show that IGF-I and EGF each had different effects on PA activities, whereas PDGF had no effect. This diversity in the patterns of growth factor regulation of PAs suggests that the production of astrocyte PAs is not simply related to mitogenesis. More likely, astrocyte PAs are involved in a wide range of growth factor-mediated actions in the developing brain.
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PMID:Differential regulation of astrocyte plasminogen activators by insulin-like growth factor-I and epidermal growth factor. 819 86

Insulin-like growth factor-I (IGF-I) is essential for somatic growth and promotes bone cell replication and differentiation. IGF-I production by rat osteoblasts is stimulated by activation of cAMP-dependent protein kinase (PKA). In this report, we define two interacting PKA-regulated pathways that control IGF-I gene transcription in cultured human osteoblasts. Stimulation of cAMP led to a 12-fold increase in IGF-I mRNA and enhanced IGF-I promoter activity through a DNA response element termed HS3D and the transcription factor CCAAT/enhancer-binding protein delta (C/EBPdelta). Under basal conditions, C/EBPdelta was found in osteoblast nuclei but was transcriptionally silent. Treatment with the PKA inhibitor H-89 caused redistribution of C/EBPdelta to the cytoplasm. After hormone treatment, the catalytic subunit of PKA accumulated in osteoblast nuclei. Inhibition of active PKA with targeted nuclear expression of PKA inhibitor had no effect on the subcellular location of C/EBPdelta but prevented hormone-induced IGF-I gene activation, while cytoplasmic PKA inhibitor additionally caused the removal of C/EBPdelta from the nucleus. These results show that IGF-I gene expression is controlled in human osteoblasts by two PKA-dependent pathways. Cytoplasmic PKA mediates nuclear localization of C/EBPdelta under basal conditions, and nuclear PKA stimulates its transcriptional activity upon hormone treatment. Both mechanisms are indirect, since PKA failed to phosphorylate human C/EBPdelta in vitro.
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PMID:Hormonal control of insulin-like growth factor I gene transcription in human osteoblasts: dual actions of cAMP-dependent protein kinase on CCAAT/enhancer-binding protein delta. 1139 Mar 99

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) affects glycemia due to reduced gluconeogenesis; when combined with a reduction in feed intake, this culminates in decreased body weight. We investigated the effects of steady-state levels of TCDD (loading dose rates of 0.0125, 0.05, 0.2, 0.8, and 3.2 microg/kg) or approximately isoeffective dose rates of 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) (loading dose rates of 0.3125, 1.25, 5, 20, and 80 microg/kg) on body weight, phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression and activity, and circulating concentrations of insulin, glucose, and insulin-like growth factor-I (IGF-I), and expression of hepatic phosphorylated AMP kinase-alpha (p-AMPK) protein in female Sprague-Dawley rats (approximately 250 gm) at 2, 4, 8, 16, 32, 64, and 128 days after commencement of treatment. At the 0.05 and 1.25 microg/kg loading dose rates of TCDD and HxCDD, respectively, there was a slight increase in body weight as compared to controls, whereas at the 3.2 and 80 microg/kg loading dose rates of TCDD and HxCDD, respectively, body weight of the rats was significantly decreased. TCDD and HxCDD also inhibited PEPCK activity in a dose-dependent fashion, as demonstrated by reductions in PEPCK mRNA and protein. Serum IGF-I levels of rats treated initially with 3.2 microg/kg TCDD or 80 microg/kg HxCDD started to decline at day 4 and decreased to about 40% of levels seen in controls after day 16, remaining low for the duration of the study. Eight days after initial dosing, hepatic p-AMPK protein was increased in a dose-dependent manner with higher doses of TCDD and HxCDD. There was no effect with any dose of TCDD or HxCDD on circulating insulin or glucose levels. In conclusion, doses of TCDD or HxCDD that began to inhibit body weight in female rats also started to inhibit PEPCK, inhibited IGF-I, while at the same time inducing p-AMPK.
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PMID:2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) alter body weight by decreasing insulin-like growth factor I (IGF-I) signaling. 1570 65