Biomedical subjects
S Melmed
Publications and source records attributed to S Melmed.
The role of growth factors in the pituitary.
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Cardiac physiology, biochemistry and morphology in response to excess growth hormone in the rat.
The cardiac effects of excess growth hormone (GH) were studied in the intact adult rat and in tissues prepared from the rat. Female Wistar-Furth rats were inoculated with a clonal cell line of pituitary cells which secrete GH. Five weeks later, heart weight had increased 37% compared to control (P less than 0.01) due to concomitant increases in left and right ventricular weight. Hemodynamic measurements in the anesthetized rat showed that GH stimulated rats had a decrease in blood pressure and heart rate and a small increase of left ventricular end-diastolic pressure (P less than 0.05). Measurement of left ventricular contractility and relaxation, and response to beta-adrenergic stimulation were decreased in GH compared to control (P less than 0.05). Contractile protein biochemistry showed an 18% reduction in Ca2(+)-myosin ATPase activity of the left ventricle (P less than 0.05) and non-denaturing pyrophosphate gels of purified myosin demonstrated a significant shift of isoforms from the exclusive V1 pattern to both V1 and V3 isomyosins in both ventricles (P less than 0.05). In contrast to the physiological and protein biochemistry adaptations, left ventricular morphology by light microscopy and ultrastructure by electron microscopy were normal in the GH stimulated heart. There were no significant changes in myofibril fraction, in the myofibril to mitochondria ratio or in the capillary numerical density of the hypertrophied left ventricle (P = N.S.). This study demonstrates that under prolonged and extreme stimulation by GH, the heart undergoes considerable growth/hypertrophy. Although cardiac morphology remains normal during this growth, there are alterations of the isomyosins such that ATPase activity is diminished and ventricular function is decreased.
An insulin-induced DNA-binding protein for the human growth hormone gene.
The control of gene transcription is usually mediated by transacting transcriptional factors that bind to upstream regulatory elements. As insulin regulates transcription of the growth hormone (GH) gene, we tested nuclear extracts from unstimulated and insulin-stimulated Chinese hamster ovarian (CHO) cells for binding to four human GH (hGH) gene promoter oligonucleotide fragments identified as target-binding sequences by DNAse I footprinting. Using a mobility shift assay, an insulin-induced DNA-binding protein was identified. This protein binds to two upstream overlapping oligonucleotide sequences. Binding activity is present at low levels in unstimulated CHO cells and is stimulated by insulin treatment with a time course suggesting that protein synthesis is required. Incubation of the cells with cycloheximide and puromycin confirmed that de novo protein synthesis is necessary for the increased binding activity. Competition with excess unlabeled specific competitor oligonucleotides prevented binding, while unrelated similar-sized oligonucleotides failed to compete for binding, indicating that the observed DNA-protein complex formation is specific. A protein of approximately 70-80 kD was detected by gradient gel electrophoresis. In conclusion, insulin-mediated DNA-protein binding has been identified on the upstream hGH promoter, suggesting a trans-active role for insulin in mediating polypeptide hormone gene expression.
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.
Clonal origin of pituitary adenomas.
As the pathogenesis of pituitary adenomas remains unclear, the tumor clonal composition of these common neoplasms was studied. Clonality was determined in female patients by analysis of restriction fragment length polymorphisms of the X-chromosome genes hypoxanthine phosphoribosyl transferase and phosphoglycerate kinase in conjunction with their respective methylation patterns. Peripheral lymphocyte DNA was screened from 62 female patients undergoing transsphenoidal surgery for pituitary adenoma. Eleven patients were heterozygous for the BglI site on PGK, 4 for the BamHI site on HPRT, and 1 patient for both sites. Of these 16 patients, 3 had acromegaly, 4 had Cushing's disease, 7 had hyperprolactinemia, and 2 were clinically nonfunctional. After surgery, morphological study, including immunohistochemistry and electron microscopy of the pathological specimens, allowed a direct comparison between clonality and tumor cell type. Control fresh normal pituitary tissue was found to be polyclonal. The following tumors were monoclonal: all 3 somatotroph adenomas, 4 of 4 lactotroph tumors, 3 of 4 corticotroph cell adenomas, a gonadotroph adenoma, and a nonsecretory adenoma. A mixed plurihormonal adenoma was polyclonal, as were 2 tumors consisting of adenomatous lactotrophs interspersed with nontumorous adenohypophyseal pituitary tissue and one corticotroph adenoma mixed with normal pituitary tissue. Functional pituitary adenomas derived from somatotrophs, corticotrophs, or lactotrophs and nonsecretory tumors are monoclonal in nature, suggesting that somatic cell mutations precede clonal expansion of these cells and play a major role in pituitary tumorigenesis.
In vivo regulation of hepatic insulin-like growth factor-1 messenger ribonucleic acids with thyroid hormone.
Insulin-like growth factor-1 (IGF-1) is synthesized primarily by the liver in response to growth hormone (GH). Thyroid hormone plays a major role in mediating pituitary GH secretion. In order to clarify the effect of thyroid hormone on IGF-1 gene expression, we measured hepatic IGF-1 mRNA levels in rats with thyroid dysfunction. Female Wistar rats were rendered hypothyroid by surgical thyroidectomy or hyperthyroid by daily injections of thyroxine (12 micrograms/day) for 2 weeks. Northern gel analysis of hepatic poly (A) RNA revealed the multiple sizes of the RNA transcripts ranging from 1.6 to 9.0 kb. After 4 weeks, hepatic IGF-1 mRNA levels were suppressed in hypothyroid rats, to less than 20% of control euthyroid animals. These suppressed mRNA levels were restored to euthyroid levels by thyroid hormone replacement for 2 weeks. Hyperthyroid rats, however, did not contain altered levels of hepatic IGF-1 mRNA as compared to euthyroid rats. The gamma-actin mRNA hybridization signal was not altered in hypothyroid or hyperthyroid rats. These results suggest that thyroid hormone regulates the in vivo expression of hepatic IGF-1 mRNA, probably through the mechanism of GH regulation.
Insulin-like growth factor-I action on growth hormone secretion and messenger ribonucleic acid levels: interaction with somatostatin.
Insulin-like growth factor I (IGF-I) suppresses GH gene transcription and GH secretion, while somatostatin (SRIF) only suppresses GH secretion. The interaction of these inhibitors of GH was, therefore, tested in primary rat pituitary cells grown in serum-free medium. Maximal inhibition of GH secretion (to 30% of the control value) was achieved by 13 nM IGF-I, while 5 nM SRIF suppressed GH to 36% of control secretion. The respective ED50 values for IGF-I and SRIF inhibition of GH secretion were similar (approximately 2.5 nM). Treatment of cells with the two agents together resulted in a further inhibition of basal GH secretion to 18% of control untreated cells (P less than 0.005). Increasing doses of SRIF (2.5-10 nM) in the presence of IGF-I caused a dose-dependent suppression of GH secretion. PRL levels were not altered by these treatments, indicating a selective effect on GH. GRH-induced GH secretion was further attenuated by combined IGF-I and SRIF treatment compared to the effect of either of these two agents alone. Northern analysis showed that IGF-I suppressed GH mRNA transcripts, while SRIF did not alter GH mRNA levels. The results indicate that physiological concentrations of both IGF-I and SRIF suppress long term basal GH secretion. Only IGF-I alters GH mRNA levels. These two peptides, therefore, appear to attenuate in vitro GH secretion by different mechanisms.
Retinoic acid selectively stimulates growth hormone secretion and messenger ribonucleic acid levels in rat pituitary cells.
Hydrocortisone (HCT) is known to stimulate rat GH gene expression. As the retinoic acid receptor cDNA bears a 15% homology to the hydrocortisone receptor cDNA, the effects of retinoic acid were tested in GH3 rat pituitary cells. Retinoic acid selectively stimulated GH secretion after 24 h. Maximal induction of basal GH secretion (220% of controls) was achieved with 1 microM retinoic acid after 72 h. HCT (50 nM) alone stimulated GH secretion 3.5-fold during 72-h incubation, and retinoic acid (1 microM) with HCT caused a further 7-fold increase in GH secretion. The stimulating effects of retinoic acid on GH were observed when cells were incubated in defined serum-free medium, 4% fetal calf serum, or Dulbecco's Modified Eagle's Medium containing HCT. Northern gel hybridization showed that steady state levels of GH mRNA transcripts in these cells were also selectively induced by retinoic acid. PRL secretion and mRNA levels, however, were not appreciably altered by retinoic acid. The results show that retinoic acid selectively stimulates basal and HCT-induced GH secretion and mRNA levels in these cells in a dose- and time-dependent manner. This selective action of retinoic acid appears to occur independently of the presence of serum in the culture medium.
Pituitary insulin-like growth factor-I gene expression: regulation by triiodothyronine and growth hormone.
Most of the growth-promoting effects of GH are mediated through insulin-like growth factor-I (IGF-I). Pituitary GH gene expression is, in turn, inhibited by IGF-I. Since rat pituitary tissue and GH3 pituitary tumor cells express both the GH and the IGF-I genes, we have attempted to clarify their potential interactions in the somatotroph by examining hormonal factors involved in the regulation of pituitary IGF-I gene expression. IGF-I mRNA was measured in GH3 cells by a solution hybridization/RNase protection assay, using riboprobes to differentially protect the IGF-I variant mRNAs arising by alternative splicing at both the 5' untranslated (UT) and 3' ends of the primary transcript. GH3 cells contained both class A and class C 5' UT variant mRNAs, with a relative abundance similar to that found in the liver. Sixty-five percent of the total IGF-I mRNA in GH3 cells was processed at the 3' end to IGF-Ia, and 35% to IGF-Ib mRNAs, whereas in the liver the proportions were 85% and 15%, respectively. GH3 cells grown in thyroid hormone-depleted medium for 4 days contained low levels of IGF-I mRNA. T3 and human (h) GH induced total IGF-I mRNA content in thyroid hormone-depleted cells, with both 5' and 3' alternative transcripts regulated coordinately, an effect that was maximal at 48-72 h. T3 stimulation of GH3 IGF-I mRNA over 48 h was dose dependent (0.01-5 nM). Similarly, hGH (0.5-10 micrograms/ml) evoked a dose-dependent induction of IGF-I mRNA in the thyroid hormone-deficient GH3 cells. The effects of T3 (5 nM) and hGH (10 micrograms/ml) on IGF-I mRNA were not additive. Furthermore, the effects of both T3 and hGH were selective for IGF-I mRNA, as neither of these treatments stimulated PRL mRNA, and treatment with hGH decreased GH3 cell GH mRNA content. This model does not discriminate whether T3 has an independent effect on IGF-I gene expression or if its action is mediated solely through induction of GH. In conclusion, IGF-I mRNA transcripts are present in GH3 cells and are modulated by T3 and GH. Local paracrine or autocrine interactions may, therefore, be involved in the feedback control of GH secretion.
Identification of growth hormone at the myocardial cell surface.
Growth hormone immunoreactivity has been demonstrated in a variety of normal human tissues, and ectopic production has been documented in a number of malignant tumors. However, myocardium has not previously been reported to contain growth hormone. Monkey anti-rat growth hormone antiserum was used in a sensitive immunoperoxidase staining method to histologically localize growth hormone in myocardium obtained from normal rats and rats harboring growth hormone-secreting tumors. Immunoreactive growth hormone was localized to the myocardial cell surface and was not seen in vascular endothelial cells of small arteries, veins, or capillaries. No intracellular staining of myocytes was evident. Specific staining was abolished by neutralization with purified growth hormone. Specific staining was abolished by neutralization with purified growth hormone. Myocardial cells did not stain with anti-triiodothyronine or anti-thyroxine immune serum. The cell surface staining is consistent with the binding of growth hormone to the myocardial cell surface, as it is unlikely that the myocardium synthesizes growth hormone polypeptide. These findings suggest that growth hormone may have a direct growth promoting or metabolic effect on cardiac tissue.
Reversible shrinkage of a growth hormone-secreting pituitary adenoma by a long-acting somatostatin analogue, octreotide.
Octreotide acetate (SMS-201-995), a somatostatin analogue, was used to treat an acromegalic patient harboring a growth hormone-secreting pituitary macroadenoma. Intermittent subcutaneous administration of octreotide suppressed growth hormone and insulin-like growth factor-I (IGF-I) levels and ameliorated clinical symptoms. Magnetic resonance imaging performed after 16 weeks revealed a 70% shrinkage of the pituitary mass, with a resultant partially empty sella turcica. To document that this shrinkage occurred as a result of octreotide treatment and not spontaneous tumor infarction, the medication was withdrawn for 4 weeks. A second magnetic resonance image disclosed regrowth of the tumor accompanied by rebound of growth hormone and IGF-I secretion. Subsequent biochemical remission has been sustained with preservation of anterior pituitary function since the drug was reinitiated. These findings suggest that intermittent subcutaneous administration of octreotide may provide potent medical ablation of growth hormone-secreting macroadenomas.
Bromocriptine and pleuropulmonary disease.
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Insulin regulates expression of the human growth hormone gene in transfected cells.
Insulin has been shown to inhibit rat growth hormone (GH) gene transcription. The effects of insulin were therefore tested on the expression of a transfected human GH gene. A 2.6-kilobase EcoRI fragment of the human GH gene was propagated in pUC18 and transfected by calcium-phosphate shock into HeLa and GC cells, respectively. Transfected cells grown in serum-free medium for 72 h expressed human GH measured by specific radioimmunoassay, incorporation of [35S] methionine into newly synthesized GH, and the presence of the appropriately sized protected transcripts seen after RNase protection assay. Immunoprecipitation analysis showed that insulin (0.7-7 nM) suppressed both the basal as well as the hydrocortisone (100 nM)-stimulated expression of newly synthesized 22-kDa GH in a dose-dependent fashion. Insulin (7 nM) also suppressed the basal and hydrocortisone-stimulated GH mRNA transcripts in these cells. Control nontransfected cells did not express human GH. Cells transfected with the truncated pOGH gene and pTKGH gene failed to respond to insulin treatment, whereas the human GH promoter was able to confer insulin responsiveness to the chloramphenicol acetyltransferase reporter gene. cis-Acting regulatory sequences residing on the 497-base pair 5'-flanking region of the human GH gene therefore appear to be a requirement for human GH gene response to the insulin signal.
Regulation of pituitary insulin-like growth factor-I messenger ribonucleic acid levels in rats harboring somatomammotropic tumors: implications for growth hormone autoregulation.
Insulin-like growth factor-I (IGF-I) is produced in multiple tissues and is believed to exert its action in a paracrine or autocrine fashion. We have investigated the GH dependency of tissue IGF-I gene expression in rats harboring implanted somatomammotropic tumors. Circulating GH and radioimmunoassayable IGF-I levels were markedly elevated in these animals. Hepatic IGF-I messenger RNA (mRNA) transcripts were induced about 5-fold in the tumor-bearing rats relative to control animals, and about 50-fold relative to the hypophysectomized liver IGF-I mRNA. IGF-I mRNA content was also modestly induced in the heart, muscle, and kidney of the tumor-bearing animals. IGF-I mRNA transcripts of 8.0, 6.2, 3.8, 2.4, 1.3, and 1.0 kilobases were markedly stimulated in the pituitaries of the GH-tumor bearing rats relative to control pituitaries. Pituitary IGF-I mRNA induction was maximal between 2 and 6 weeks after tumor implantation, a time at which circulating GH levels were also increasing sharply. As previously reported, pituitary GH mRNA was inhibited in tumor-bearing rats. The stimulation of pituitary IGF-I gene expression in these animals, therefore, appears to be dependent on circulating, and not local, pituitary GH concentrations. These data support the GH dependency of pituitary IGF-I gene expression. IGF-I is known to decrease GH production in vitro. High circulating levels of IGF-I present in these animals may account for the observed inhibition of GH gene expression. These data raise the alternative possibility that induction of locally produced IGF-I may contribute to this phenomenon.
Insulin regulates prolactin secretion and messenger ribonucleic acid levels in pituitary cells.
The regulation of PRL production in GH3 rat pituitary tumor cells and normal rat pituitary cells exposed to insulin was studied. Cells were treated with semisynthetic human insulin for up to 5 days. Insulin (0.7 nM) stimulated PRL production by 30%, and cortisol (100 nM) suppressed it by 80% in GH3 cells. Insulin (3.5 nM) partially blocked the suppression of PRL secretion induced by up to 100 nM cortisol. Equimolar doses of insulin-like growth factor I failed to consistently stimulate either basal PRL secretion into the medium or cortisol-suppressed PRL in GH3 cells. A 65 nM concentration of insulin-like growth factor I was required to stimulate basal PRL secretion in GH3 cells. Relative levels of PRL mRNA sequences in both GH3 cells and normal rat pituitary cells were stimulated by insulin. When cells were incubated with cortisol (10 nM), PRL mRNA levels were suppressed to 40% of control values. Simultaneous incubation of cells with insulin (3.5 nM) partially reversed the cortisol-induced suppression of PRL mRNA to 60% of control values. The results show that insulin antagonizes cortisol-induced suppression of PRL. Physiological doses of insulin stimulate PRL production and PRL mRNA levels, suggesting a direct effect of insulin on either PRL gene transcription or stabilization of PRL mRNA in these cells.
Medical management of acromegaly due to ectopic production of growth hormone-releasing hormone by a carcinoid tumor.
A 59-yr-old woman with a disseminated carcinoid tumor was evaluated for acromegaly. She had previously undergone a hypophysectomy for acromegaly and an enlarged pituitary, with a reduction in her serum GH levels from 100 to 4 micrograms/L. Recurrence of acromegalic symptoms 2 yr later was accompanied by elevated serum GH (16 micrograms/L) and insulin-like growth factor I (IGF-I; 528 micrograms/L) and plasma GHRH levels (12 micrograms/L; normal, less than 30 ng/L). Computed tomographic scan did not reveal pituitary enlargement. Metastatic carcinoid tissue in bone removed at biopsy contained GHRH (100 pg/mg tissue). High performance liquid chromatography of plasma GHRH revealed predominantly GHRH-(3-40)-OH, a biologically inactive GHRH metabolite, along with mature GHRH forms, while carcinoid tissue contained both GHRH-(1-40)-OH and GHRH-(1-44)-NH2. Treatment with pergolide initially resulted in reduction in serum GH and IGF-I levels and amelioration of symptoms of acromegaly. However, after 14 months of pergolide therapy, serum GH levels increased despite administration of up to 1000 micrograms pergolide/day. Plasma GHRH levels remained elevated throughout the treatment period. Subsequent treatment with SMS 201-995, a long-acting somatostatin analog, for over 1 yr resulted in sustained reductions of ectopic GHRH secretion, GH hypersecretion, and IGF-I levels. Plasma GHRH levels correlated with simultaneously measured serum GH levels in response to acute SMS 201-995 administration. SMS 201-995 was an effective medical treatment for acromegaly caused by ectopic GHRH production in this patient.
Regulation of human growth hormone gene expression by insulin-like growth factor I in transfected cells.
Insulin-like growth factor I (IGF-I), a target hormone for growth hormone (GH) action, has been shown to regulate rat GH gene transcription. We further investigated its direct action on the human GH gene transfected in human choriocarcinoma cells (JEG3) which possess abundant IGF-I receptors. The 2.6 kilobase (kb) hGH gene (EcoRI fragment) in pUC18 (phGH) was transfected into cells by calcium-phosphate-dimethyl sulfoxide shock. The cells were subsequently treated for 72 h, when GH gene expression was measured. 8-Bromo-cAMP (Br-cAMP, 2.5 mM) and hydrocortisone (100 nM), respectively, stimulated GH secretion in transfected cells by 50% over unstimulated control cells, while combined treatment with these two agents caused a 340% increase of GH secretion as measured by radioimmunoassay. IGF-I (6.5 nM) did not suppress the basal level of Gh secretion but did inhibit the stimulated GH secretion by over 50%. Immunoprecipitation analysis showed that 8-Br-cAMP + hydrocortisone markedly increased newly synthesized 22-kDa [35S]GH. This de novo GH synthesis was inhibited by IGF-I. Northern gel analysis of poly(A) RNA showed that 8-Br-cAMP + hydrocortisone induced a 1.0-kb mRNA species as detected with 32P-labeled hGH-cDNA. IGF-I suppressed the GH mRNA induced by cAMP + hydrocortisone as well as suppressing a larger 2.2-kb GH mRNA precursor. The results show that the transfected human GH gene was expressed and regulated in homologous cells. The 2.6-kb hGH fragment therefore contains non-tissue-specific cis-acting regulatory elements responsive to cAMP, hydrocortisone, and IGF-I. Specific IGF-I-responsive GH DNA sequences may therefore reside in the 0.5-kb 5'-flanking region, or in an intron, as described for hydrocortisone.