Hiromi Abe

Leptin Induces Mitogen-activated Protein Kinase- dependent Proliferation of C3H10T1/2 Cells

Leptin, secreted by adipocytes, regulates satiety and energy expenditure. Several forms of leptin receptors produced by alternative mRNA splicing are found in many tissues, including the hypothalamus, liver, lung, kidney, hematopoietic cells, and gonads, suggesting that leptin exerts effects in these tissues. In accordance with the distribution of leptin receptors, there is accumulating evidence that leptin plays various roles in reproduction, hematopoiesis, and the immune systems in addition to the regulation of food intake and energy expenditure. In the present study, we examined the in vitro effects of leptin on proliferation of a mouse embryonic cell line, C3H10T1/2, and its mechanism of action. Leptin caused a dose- and time-dependent increase in mitogen-activated protein kinase (MAPK) activity that was accompanied by an increase in C3H10T1/2 cell number. The MAPK kinase-1-specific inhibitor PD98059 completely blocked the increases in both MAPK activity and cell proliferation caused by leptin. These findings indicate that leptin stimulates the proliferation of C3H10T1/2 cells via the MAPK cascade.

Short Stature Caused by a Mutant Growth Hormone

The causes of growth hormone–dependent short stature are primary pituitary disease, pituitary deficiency due to hypothalamic dysfunction, and, less often, insensitivity to growth hormone. The prototypical syndrome of growth hormone insensitivity is Laron-type dwarfism, which is characterized by absent or defective growth hormone receptors. Kowarski et al. described two children with growth retardation resulting from biologically inactive growth hormone1; additional cases were reported subsequently.2–7 This disorder is characterized by high serum concentrations of immunoreactive growth hormone, low serum concentrations of insulin-like growth factor I (IGF-I), and increases in both serum IGF-I and linear growth after the administration of .xa0.xa0.

Biologically inactive growth hormone caused by an amino acid substitution.

Short stature caused by biologically inactive growth hormone (GH) is characterized by lack of GH action despite high immunoassayable GH levels in serum and marked catch-up growth to exogenous GH administration. We found a heterozygous single-base substitution (A-->G) in exon 4 of the GH-1 gene of a girl with short stature, clinically suspected to indicate the presence of bioinactive GH and resulting in the substitution of glycine for aspartic acid at codon 112. We confirmed the presence of mutant GH in the serum using isoelectric focusing analysis. The locus of mutation D112G was found within site 2 of the GH molecule in binding with GH receptor (GHR)/GH binding protein (GHBP). The expressed recombinant mutant GH tended to form a 1:1 instead of the 1:2 GH-GHBP complex normally produced by wild-type GH. The formation of a 1:2 GH-GHBP complex is compatible with the dimerization of GHRs by GH, a crucial step in GH signal transduction. Mutant GH was less potent than wild-type GH not only in phosphorylation of tyrosine residues in GHR, janus kinase 2 (JAK2), and signal transducers and activators of transcription 5 (STAT5) in IM-9 cells, but also in metabolic responses of BaF/GM cells, a stable clone transfected with cDNA of the chimera of the extracellular domain of human GHR, the transmembrane and the cytoplasmic domain of the human thrombopoietin receptor. These results indicate that the D112G mutation in the GH-1 gene causes production of bioinactive GH, which prevents dimerization of GHR and is therefore responsible for the patients short stature.

Growth hormone and prolactin release by substance P in rats

Abstract Intravenous injection of synthetic Substance P resulted in a significant and dose-related increase in plasma growth hormone (GH) and prolactin (PRL) in urethane-anesthetized rats. Increases in plasma GH induced by Substance P were significantly suppressed by the simultaneous administration of either l-dopa or nicotine, whereas plasma PRL responses to Substance P were blunted by l-dopa but not by nicotine. Substance P also raised plasma GH and PRL in rats with extensive hypothalamic destruction. L-dopa significantly suppressed plasma PRL responses to Substance P in rats with hypothalamic destruction. However, plasma GH responses to Substance P were not significantly affected by l-dopa nor by nicotine in animals with hypothalamic ablation. These results suggest that Substance P stimulates rat GH and PRL secretion possibly acting on the anterior pituitary and that l-dopa and nicotine affect GH and PRL release induced by Substance P in different ways.

Bone metabolism and body composition in Japanese patients with active acromegaly.

OBJECTIVE Skeletal involvement is a common clinical feature in acromegalic patients. Although several recent reports are available concerning bone mineral density (BMD) in acromegaly, the controversy still exists as to whether BMD of acromegalic patients is increased or not. The present study was performed to examine biochemical bone metabolic indices and BMD as well as body composition in 26 Japanese patients with active acromegaly and 26 control subjects matched for age, sex, race and height in a cross‐sectional study.

Cloning and Characterization of the 5′-Flanking Region of the Human Growth Hormone-releasing Hormone Receptor Gene

We cloned the 5′-flanking region of the human growth hormone-releasing hormone receptor (GHRH-R) gene and determined the nucleotide sequence of 2.7 kilobases upstream from the translation start site. RNase protection analysis showed the major transcription start site is 122 base pairs upstream from the translation start site. The 5′-end of the longest product of 5′-rapid amplification of cDNA ends was close to the site. There were no typical TATA homologies but several putative regulatory elements including Pit-1-binding site-like element. Transient transfection studies using a luciferase reporter gene demonstrated that 5′-flanking region had promoter activity in GH3 cells (derived from rat pituitary tumor) but not in nonpituitary cells, BeWo and HeLa cells. However, co-transfection of Pit-1 expression vector increased luciferase activity in BeWo cells. Deletion study showed that the regions from −310 to −130 and from −130 to −120 were important for the GHRH-R gene expression in GH3 cells, although the latter contributed less to the gene expression. In BeWo cells co-transfected with Pit-1 expression vector, the region from −310 to −130 was essential for the Pit-1-dependent expression of GHRH-R gene. The region from −310 to −120 has two putative Pit-1-binding sites, P1 and P2, located from −129 to −123 and from −171 to −160, respectively. Both mobility shift assay and DNase-I footprint analysis showed that P2 had much higher Pit-1 binding affinity than P1. Mutation of P2 decreased GHRH-R gene expression in GH3 cells. These findings were consistent with the results that the region from −310 to −130 is an important element for Pit-1-dependent expression of GHRH-R gene.

Calcitonin gene-related peptide-like immunoreactivity in the central nervous system and peripheral organs of rats

The concentrations of rat calcitonin gene-related peptide-like immunoreactivity (rCGRP-LI) in various organs of male rats as well as the molecular heterogeneity of rCGRP-LI in tissue extracts was examined using a specific radioimmunoassay (RIA) for rCGRP and gel-filtration chromatography. rCGRP-LI was high in extracts of the spinal cord (202 +/- 22.6 pg/mg wet wt. of tissue; mean +/- S.E.M.) and of the thyroid (229 +/- 62.3 pg/mg). rCGRP-LI was detectable in the brainstem, hypothalamus, stomach, duedenum, pancreas and kidney. The elution pattern of the extracts on a Sephadex G-50 column showed 3 peaks of rCGRP-LI irrespective of organs and tissues. The first peak corresponded to authentic rCGRP-(1-37). The second and third rCGRP-LI peaks probably consisted of C-terminal fragments of rCGRP, because they had a lower molecular weight than rCGRP-(1-37) and because our antiserum cross-reacts with a synthetic C-terminal fragment. The ratio of 3 rCGRP-LI molecules, however, differed between neural tissue extracts and others. The main component of rCGRP-LI in neural tissue was authentic rCGRP-(1-37), while the smaller fragments of rCGRP were chiefly contained in other tissues like the stomach, pancreas and thyroid. The relative ratio of rCGRP-LI molecules with different size in respective tissue extracts was not changed after leaving the dissected tissues for 2 h at room temperature. These findings indicate that rCGRP-LI is abundantly present in the thyroid as well as the spinal cord and it is detected in lower amounts in the alimentary tract and central nervous system. rCGRP-LI in the extracts consists of 3 different components, the proportions of which vary from one tissue to another, probably reflecting tissue-specific differences in the processing of CGRP.

Somatostatin release from isolated perfused rat stomach.

Abstract Gastric somatostatin release from the isolated rat stomach was studied using a perfusion technique. Somatostatin released from the isolated perfused rat stomach was found to be identical in molecular size and immunoreactively with synthetic somatostatin. Infusion of glucagon (10−7 M) caused biphasic increase of gastric somatostatin release. Gastric somatostatin release was also stimulated by infusion of theophylline (10−3 M) and dibutyryl cyclic AMP (10−3 M). These results indicate the possible involvement of adenylate cyclase-cyclic AMP system in the regulatory mechanism of gastric somatostatin release.

Effect of Calcitonin on prolactin release in rats

Abstract Injection of [Asu 1,7 ]-eel calcitonin (CT) (0.1–2.5μg) into the lateral ventricle resulted in a significant and dose-related increase of plasma prolactin (PRL) levels in urethane-anesthetized male rats. Naloxone failed to block [Asu 1,7 ]-eel CT induced PRL release. Salmon CT, human CT and porcine CT were similarly effective to stimulate PRL release when injected intraventricularly. Intravenous administration of [Asu 1,7 ]-eel CT(20 μg) failed to cause any significant changes in plasma PRL levels, while this peptide (10 −8 −10 −6 M) possesed a mild stimulating activity of PRL release from the anterior pituitary cells cultured in vitro . These results suggest that CT stimulates rat PRL secretion mainly through the central nervous system like one of the neurotransmitters, though it may also act directly on the pituitary.

Adrenocorticotrophin-independent macronodular adrenal hyperplasia in a patient with lysine vasopressin responsiveness but insensitivity to gastric inhibitory polypeptide

The aetiology of ACTH‐independent macronodular adrenal hyperplasia (AIMAH) is uncertain. We examined a 55 year old man with Cushings syndrome due to AIMAH, whose cortisol levels increased after stimulation with lysine‐8‐vasopressin (LVP) in vitro as well as in vivo. Abdominal MRI revealed nodular enlargement of both adrenal glands. No adenoma was evident on pituitary MRI. 131I‐adosterol scintigraphy exhibited marked uptake into both adrenal glands. Although baseline plasma cortisol levels were within normal limits, urinary free cortisol excretion was 3‐fold higher than the upper limit of the normal range. Plasma ACTH levels were undetectable. Oral dexamethasone failed to suppress plasma cortisol levels irrespective of dose, and administration of corticotrophin releasing hormone failed to increase plasma ACTH and cortisol levels. LVP injection failed to increase plasma ACTH levels, but elicited an increase in plasma cortisol levels. The direct stimulatory effect of LVP on cortisol secretion was confirmed in vitro in cultured adrenocortical cells from macronodules obtained at surgery. Food intake, gastric inhibitory polypeptide (GIP), or octreotide administration, which were reported to regulate cortisol release in patients with AIMAH, failed to affect plasma cortisol levels. In conclusion, plasma cortisol responsiveness to LVP, GIP, and octreotide is heterogeneous in patients with AIMAH.