Insulin-like growth factor-II (IGF-II) is an important regulator of

embryonic growth and differentiation, but its function in postnatal life

is unclear. To address this point, we generated transgenic mice harboring

fusion genes in which a human IGF-II complementary DNA is

placed under the transcriptional control of the rat phosphoenolpyruvate

carboxykinase promoter. Transgene-specific messenger RNA was detected

in liver, kidney, and several parts of the gut. Serum IGF-II levels

in transgenic mice were 2-3 times higher than those in controls and

increased after starvation. Circulating IGF-I correlated negatively and

IGF-binding protein-2 (IGFBP-2) positively with IGF-II levels, suggesting

that IGF-I is displaced from IGFBPs by IGF-II and that IGFII

is a major regulator of IGFBP-2. Serum levels of IGFBP-3 and

IGFBP-4 tended to be higher in phosphoenolpyruvate carboxykinase-

IGF-II transgenic mice than in controls, as evaluated by ligand blot

analysis. Starvation reduced serum IGF-I, but increased IGFBP-2 in

transgenic mice more markedly than in controls. Fasting insulin levels

were significantly reduced in transgenic mice, whereas glucose levels

were not influenced by elevated IGF-II. The body growth of 4- and 12-

week-old mice was not significantly influenced by elevated IGF-II, but

transgenic mice displayed increased kidney and testis weight at the age

of 4 weeks, and increased adrenal weight at the age of 12 weeks. Our

results demonstrate that elevated IGF-II in postnatal life has multiple

endocrine consequences and subtle time-specific effects on organ

growth.

Patients with glucocorticoid-remediable aldosteronism (GRA) possess a chimeric gene resulting from fusion of the genes encoding steroid aldosterone synthase and 11 beta-hydroxylase. In the adrenal zona fasciculata, this may lead to ectopic expression under ACTH control of aldosterone synthase activity and increased formation of cortisol C18 oxidation products. We assessed mineralocorticoid and glucocorticoid pathways in three patients with GRA. Baseline plasma progesterone, 17 alpha-hydroxyprogesterone, corticosterone, and cortisol were normal in all patients, whereas 11-deoxycorticosterone, aldosterone, and 11-deoxycortisol were above normal. The ratios of both corticosterone/11-deoxycorticosterone and cortisol/11-deoxycortisol were abnormally low, and decreased further 60 min after administration of ACTH-(1-24) (250 micrograms) as an i.v. bolus. A low corticosterone/11-deoxycorticosterone ratio is consistent with an increased aldosterone synthase activity forming aldosterone by corticosterone. Similarly, a decreased cortisol/11-deoxycortisol ratio could reflect enhanced cortisol C18 oxidation. Our findings are in agreement with a hyperfunction of the 11 beta-hydroxylase/aldosterone synthase complex in the adrenal zona fasciculata of GRA induced by the new chimeric gene.

We have compared the expression of atria1 natriuretic peptide (ANP) and brain natriuretic peptide (BNP) genes in various human tissues using a quantitative polymerase chain reaction technique. Tissues of three human subjects, obtained at autopsy, were analyzed. BNP transcripts could be detected in the central nervous system, lung, thyroid, adrenal, kidney, spleen, small intestine, ovary, uterus, and striated muscle. ANP transcripts could also be demonstrated in various human extracardiac tissues including several endocrine organs. In all periphera1 tissues, the level of both natriuretic peptide transcripts was approximately l-2 orders of magnitude lower than in cardiac ventricular tissues. This distribution is in marked contrast to the much lower level of ANP and BNP transcripts present in extracardiac rat tissues (generally less than l/1000 of ventricles). These data suggest differential expression of the two natriuretic peptide genes in cardiac and extracardiac tissues in man. Furthermore, the presence of local synthesis of ANP and BNP in various peripheral organs su gests paracrine and/or autocrine function of these natriuretic peptides.

The synthetic hexapeptide GH-releasing peptide (GHRP; His-D-Trp-Ala-Trp-D-Phe-Lys-NH2) specifically stimulates GH secretion in humans in vivo and in animals in vitro and in vivo via a still unknown receptor and mechanism. To determine the effect of GHRP on human somatotroph cells in vitro, we stimulated cell cultures derived from 12 different human somatotroph adenomas with GHRP alone and in combination with GH-releasing hormone (GHRH), TRH, and the somatostatin analog octreotide. GH secretion of all 12 adenoma cultures could be stimulated with GHRP, whereas GHRH was active only in 6 adenoma cultures. In GHRH-responsive cell cultures, simultaneous application of GHRH and GHRP had an additive effect on GH secretion. TRH stimulated GH release in 4 of 7 adenoma cultures; in TRH-responsive cell cultures there was also an additive effect of GHRP and TRH on GH secretion. In 5 of 9 adenoma cultures investigated, octreotide inhibited basal GH secretion. In these cell cultures, GHRP-induced GH release was suppressed by octreotide. In 5 of 5 cases, the protein kinase-C inhibitor phloretin partly inhibited GHRP-stimulated GH release, but not basal GH secretion. In summary, GH secretion was stimulated by GHRP in all somatotroph adenomas investigated, indicating that its unknown receptor and signaling pathway are expressed more consistently in somatotroph adenoma cells than those for GHRH, TRH, and somatostatin. Our data give further evidence that GHRP-stimulated GH secretion is mediated by a receptor different from that for GHRH or TRH, respectively, and that protein kinase-C is involved in the signal transduction pathway. Because human somatotroph adenoma cell cultures respond differently to various neuropeptides (GHRH, TRH, somatostatin, and others), they provide a model for further investigation of the mechanism of action of GHRP-induced GH secretion.

hCG is a putative thyroid stimulator. The present studies were undertaken to examine its interaction and that of its desialylated variant asialo-hCG with recombinant human TSH (hTSH) receptor (hTSHr). To this end, we transfected a human thyroid carcinoma cell line (HTC) lacking endogenous TSHr with the full-length cDNA of the hTSHr. Unlike the wild type, the transfected cells, termed HTC-TSHr cells, were able to bind bovine TSH (bTSH) with high affinity and increase cAMP production in response to bTSH stimulation. Of the hCG forms, intact hCG displayed a weak activity to inhibit [125I] bTSH binding to HTC-TSHr cells, with 100 mg/L (2.6 x 10(-6) mol/L) producing maximally a 20% inhibition, whereas asialo-hCG achieved half-maximum binding inhibition at a concentration of 8 mg/L (2.3 x 10(-7) mol/L). The inhibitory constant (Ki) of asialo-hCG for recombinant hTSHr was calculated from saturation experiments in the presence of variable doses of bTSH and a fixed concentration of asialo-hCG to be approximately 8 x 10(-8) mol/L. The interaction of asialo-hCG with TSHr was further assessed by studies of the direct binding of the radioactively labeled hormone to both HTC and HTC-TSHr cells. [125I]Asialo-hCG binding to HTC-TSHr cells was 4.7%, compared to 1.5% in the wild-type cells lacking TSHr and was displaceable by bTSH (0.1-100 IU/L), indicating specific binding of the tracer to TSHr. Functionally, hCG (up to 100 mg/L; 2.6 x 10(-6) mol/L) proved unable to evoke any significant cAMP response over basal values in HTC-TSHr cells, as did asialo-hCG. Asialo-hCG, but not hCG, inhibited bTSH-stimulated adenylate cyclase activity in the cells in a dose-dependent manner. In conclusion, the present data show that intact hCG binds only weakly to HTC-TSHr cells and produces no significant cAMP stimulation, which is at variance with data obtained in FRTL-5 and Chinese hamster ovary-TSHr cells, but in good accord with previous findings in human thyroid membranes. Asialo-hCG, on the other hand, strongly binds to recombinant TSHr and inhibits the cAMP response to bTSH in HTC-TSHr cells, indicating that the desialylated hCG variant directly interacts with the receptor and truly is an antagonist of the hTSHr.

Orbital and pretibial fibroblasts are targets of autoimmune attack in Graves' ophthalmopathy (GO) and pretibial dermopathy (PTD). The fibroblast autoantigen involved in these peripheral manifestations of Graves' disease and the reason for the association of GO and PTD with hyperthyroidism are unknown. RNA encoding the full-length extracellular domain of the TSH receptor has been demonstrated in orbital and dermal fibroblasts from patients with GO and normal subjects, suggesting a possible antigenic link between fibroblasts and thyrocytes. RNA was isolated from cultured orbital, pretibial, and abdominal fibroblasts obtained from patients with severe GO (n = 22) and normal subjects (n = 5). RNA was reverse transcribed, and the resulting cDNA was amplified by the polymerase chain reaction, using primers spanning overlapping regions of the entire extracellular domain of the TSH receptor. Nucleotide sequence analysis showed an A for C substitution in the first position of codon 52 in 2 of the patients, both of whom had GO, PTD, and acropachy. Genomic DNA isolated from the 2 affected patients, and not from an additional 12 normal subjects, revealed the codon 52 mutation by direct sequencing and AciI restriction enzyme digestions. In conclusion, we have demonstrated the presence of a genomic point mutation, leading to a threonine for proline amino acid shift in the predicted peptide, in the extracellular domain of the TSH receptor in two patients with severe GO, PTD, acropachy, and high thyroid-stimulating immunoglobulin levels. RNA encoding this mutant product was demonstrated in the fibroblasts of these patients. We suggest that the TSH receptor may be an important fibroblast autoantigen in GO and PTD, and that this mutant form of the receptor may have unique immunogenic properties.