Principles of the prolactin/vasoinhibin axis.

The hormonal family of vasoinhibins, which derive from the anterior pituitary hormone prolactin, are known for their inhibiting effects on blood vessel growth, vasopermeability, and vasodilation. As pleiotropic hormones, vasoinhibins act in multiple target organs and tissues. The generation, secretion, and regulation of vasoinhibins are embedded into the organizational principle of an axis, which integrates the hypothalamus, the pituitary, and the target tissue microenvironment. This axis is designated as the prolactin/vasoinhibin axis. Disturbances of the prolactin/vasoinhibin axis are associated with the pathogenesis of retinal and cardiac diseases and with diseases occurring during pregnancy. New phylogenetical, physiological, and clinical implications are discussed.

Risk profiles and penetrance estimations in multiple endocrine neoplasia type 2A caused by germline RET mutations located in exon 10.

Multiple endocrine neoplasia type 2 is characterized by germline mutations in RET. For exon 10, comprehensive molecular and corresponding phenotypic data are scarce. The International RET Exon 10 Consortium, comprising 27 centers from 15 countries, analyzed patients with RET exon 10 mutations for clinical-risk profiles. Presentation, age-dependent penetrance, and stage at presentation of medullary thyroid carcinoma (MTC), pheochromocytoma, and hyperparathyroidism were studied. A total of 340 subjects from 103 families, age 4-86, were registered. There were 21 distinct single nucleotide germline mutations located in codons 609 (45 subjects), 611 (50), 618 (94), and 620 (151). MTC was present in 263 registrants, pheochromocytoma in 54, and hyperparathyroidism in 8 subjects. Of the patients with MTC, 53% were detected when asymptomatic, and among those with pheochromocytoma, 54%. Penetrance for MTC was 4% by age 10, 25% by 25, and 80% by 50. Codon-associated penetrance by age 50 ranged from 60% (codon 611) to 86% (620). More advanced stage and increasing risk of metastases correlated with mutation in codon position (609→620) near the juxtamembrane domain. Our data provide rigorous bases for timing of premorbid diagnosis and personalized treatment/prophylactic procedure decisions depending on specific RET exon 10 codons affected.

Decreased Expression of the Human Urea Transporter SLC14A1 in Bone is Induced by Cytokines and Stimulates Adipogenesis of Mesenchymal Progenitor Cells.

The human urea transporter SLC14A1 (HUT11/UT-B) has been suggested as a marker for the adipogenic differentiation of bone cells with a relevance for bone diseases. We investigated the function of SLC14A1 in different cells models from bone environment. SLC14A1 expression and cytokine production was investigated in bone cells obtained from patients with osteoporosis. Gene and protein expression of SLC14A1 was studied during adipogenic or osteogenic differentiation of human mesenchymal progenitor cells (hMSCs) and of the single-cell-derived hMSC line (SCP-1), as well as in osteoclasts and chondrocytes. Localization was determined by histochemical methods and functionality by urea transport experiments. Expression of SLC14A1 mRNA was lower in cells from patients with osteoporosis that produced high levels of cytokines. Accordingly, when adding a combination of cytokines to SCP-1 SLC14A1 mRNA expression decreased. SLC14A1 mRNA expression decreased after both osteogenic and more pronounced adipogenic stimulation of hMSCs and SCP-1 cells. The highest SLC14A1 expression was determined in undifferentiated cells, lowest in chondrocytes and osteoclasts. Downregulation of SLC14A1 by siRNA resulted in an increased expression of interleukin-6 and interleukin-1 beta as well as adipogenic markers. Urea influx through SLC14A1 increased expression of osteogenic markers, adipogenic markers were suppressed. SLC14A1 protein was localized in the cell membrane and the cytoplasm. Summarizing, the SLC14A1 urea transporter affects early differentiation of hMSCs by diminishing osteogenesis or by favoring adipogenesis, depending on its expression level. Therefore, SLC14A1 is not unequivocally an adipogenic marker in bone. Our findings suggest an involvement of SLC14A1 in bone metabolism and inflammatory processes and disease-dependent influences on its expression.

Cytokines, osteoprotegerin, and RANKL in vitro and histomorphometric indices of bone turnover in patients with different bone diseases.

Cytokines are supposed to play an essential role in the regulation of the bone metabolic unit. However, information on cytokine production of primary human osteoblasts from patients with metabolic bone disease is scarce, and few attempts have been made to correlate such data to histomorphometric parameters of individual patients. We investigated 11 patients with metabolic bone disease referred to our outpatient department for bone biopsy and analyzed interleukin (IL)-1, IL-6, and TNF-alpha protein release and gene expression in primary osteoblast cultures. Compared with four controls, five patients showed normal cytokine protein release, whereas six patients showed much higher levels of interleukin-6 (26-fold) and TNF-alpha (84-fold). All three cytokines were strongly correlated concerning gene expression and/or protein levels (r = 0.72-0.96). Histomorphometric analysis of the bone samples showed that eroded surface (ES/BS) as a parameter of bone resorption was significantly associated with TNF-a. In addition, RANKL gene expression was positively associated with ES/BS and osteoclast surface (Oc.S/BS). Finally, the formation parameters osteoid volume and osteoid surface were negatively associated with TNF-alpha. In conclusion, in an in vitro-ex vivo model of bone cells obtained from a group of 11 patients with different forms of metabolic bone disease, cytokine release in conditioned medium was significantly associated with bone resorption and bone formation, as quantified by histomorphometry. TNF-alpha seemed to be the more important cytokine; its effect on bone resorption could be mediated by RANKL.

A polymorphism within the vitamin D-binding protein gene is associated with Graves' disease but not with Hashimoto's thyroiditis.

Graves' disease and Hashimoto's thyroiditis are common autoimmune thyroid disorders. Experimentally, 1,25(OH)(2) D(3) prevents Hashimoto's thyroiditis. Vitamin D serum levels in Graves' disease were found to be significantly lower than in nonautoimmune hyperthyroidism. The polymorphic vitamin D-binding protein (DBP) greatly facilitates vitamin D actions, and DBP alleles differ regarding their affinity for 1,25(OH)(2) D(3). Therefore, we investigated polymorphisms of the DBP gene for an association with thyroid autoimmunity. Families with an offspring affected by Graves' disease (95 pedigrees) or by Hashimoto's thyroiditis (92 pedigrees) encompassing 561 individuals of Caucasian origin were genotyped for three DBP polymorphisms [(TAAA)(N) in intron 8; StyI; and HaeIII in exon 11]. Indirect haplotyping and (extended) transmission disequilibrium testing were performed. There was a significant transmission disequilibrium of the intron 8 polymorphism in patients with Graves' disease (P < 0.03) but not of the exon 11 polymorphism. In contrast, neither the intron 8 nor the exon 11 polymorphism was associated with Hashimoto's thyroiditis. Maternal and paternal transmission as well as allele frequencies in DQ2(+) and DQ2(-) patients did not differ in either disease. Therefore, allelic variants of the DBP gene confer susceptibility to Graves' disease but not to Hashimoto's thyroiditis in our population. These findings support a role of the vitamin D endocrine system in thyroid autoimmunity.

Effects of glucagon-like peptide 1 on counterregulatory hormone responses, cognitive functions, and insulin secretion during hyperinsulinemic, stepped hypoglycemic clamp experiments in healthy volunteers.

Glucagon-like peptide 1 (GLP-1) and analogues are being evaluated as a new therapeutic principle for the treatment of type 2 diabetes. GLP-1 suppresses glucagon secretion, which could lead to disturbances of hypoglycemia counterregulation. This has, however, not been tested. Nine healthy volunteers with normal oral glucose tolerance received infusions of regular insulin (1 mU x kg(-1) x min(-1)) over 360 min on two occasions in the fasting state. Capillary glucose concentrations were clamped at plateaus of 4.3, 3.7, 3.0, and 2.3 mmol/liter for 90 min each (stepwise hypoglycemic clamp); on one occasion, GLP-1 (1.2 pmol x kg(-1) x min(-1)) was administered i.v. (steady-state concentration, approximately 125 pmol/liter); on the other occasion, NaCl was administered as placebo. Glucagon, cortisol, GH (immunoassays), and catecholamines (radioenzymatic assay) were determined, autonomous and neuroglucopenic symptoms were assessed, and cognitive function was tested at each plateau. Insulin secretion rates were estimated by deconvolution (two-compartment model of C-peptide kinetics). At insulin concentrations of approximately 45 mU/liter, glucose infusion rates were similar with and without GLP-1 (P = 0.26). Only during the euglycemic plateau (4.3 mmol/liter), GLP-1 suppressed glucagon concentrations (4.1 +/- 0.4 vs. 6.5 +/- 0.7 pmol/liter; P = 0.012); at all hypoglycemic plateaus, glucagon increased similarly with GLP-1 or placebo, to maximum values greater than 20 pmol/liter (P = 0.97). The other counterregulatory hormones and autonomic or neuroglucopenic symptom scores increased, and cognitive functions decreased with decreasing glucose concentrations, but there were no significant differences comparing experiments with GLP-1 or placebo, except for a significant reduction of GH responses during hypoglycemia with GLP-1 (P = 0.04). GLP-1 stimulated insulin secretion only at plasma glucose concentrations of at least 4.3 mmol/liter. In conclusion, the suppression of glucagon by GLP-1 does occur at euglycemia, but not at hypoglycemic plasma glucose concentrations (< or = 3.7 mmol/liter). GLP-1 does not impair overall hypoglycemia counterregulation except for a reduction in GH responses, which is in line with other findings demonstrating pituitary actions of GLP-1. Below plasma glucose concentrations of 4.3 mmol/liter, the insulinotropic action of GLP-1 is negligible.

Vitamin D 1alpha-hydroxylase (CYP1alpha) polymorphism in Graves' disease, Hashimoto's thyroiditis and type 1 diabetes mellitus.

OBJECTIVE: The vitamin D endocrine system plays a role in the regulation of (auto)immunity and cell proliferation. Vitamin D 1alpha-hydroxylase (CYP1alpha) is one of the key enzymes regulating both systemic and tissue levels of 1,25-dihyroxyvitamin D(3) (1,25(OH)(2)D(3)). Administration of 1,25(OH)(2)D(3), whose serum levels were found to be reduced in type 1 diabetes and thyroid autoimmunity, prevents these diseases in animal models. We therefore investigated a recently reported CYP1alpha polymorphism for an association with type 1 diabetes mellitus, Graves' disease and Hashimoto's thyroiditis. DESIGN AND METHODS: Four hundred and seven Caucasian pedigrees with one offspring affected by either type 1 diabetes (209 families), Graves' disease (92 families) or Hashimoto's thyroiditis (106 families) were genotyped for a C/T polymorphism in intron 6 of the CYP1alpha gene on chromosome 12q13.1-13.3 and transmission disequilibrium testing (TDT) was performed. Subsets of affected offspring stratified for HLA-DQ haplotype were compared using chi(2) testing. RESULTS: There was no deviation from the expected transmission frequency in either type 1 diabetes mellitus (P=0.825), Graves' disease (P=0.909) or Hashimoto's thyroiditis (P=0.204). However, in Hashimoto's thyroiditis the CYP1alpha C allele was significantly more often transmitted to HLA-DQ2(-) patients (27 transmitted vs 14 not transmitted; TDT: P=0.042) than expected. The C allele was less often transmitted to HLA-DQ2(+) patients (9 transmitted vs 12 not transmitted; TDT: P=0.513), although the difference was not significant (chi(2) test: P=0.143). A similar difference was observed in type 1 diabetes between offspring with high and low risk HLA-DQ haplotypes (chi(2) test: P=0.095). CONCLUSIONS: The CYP1alpha intron 6 polymorphism appears not to be associated with type 1 diabetes mellitus, Graves' disease and Hashimoto's thyroiditis. A potential association in subsets of patients with type 1 diabetes and Hashimoto's thyroiditis should be further investigated as well as its functional implications.

Persistent disturbance of the hypothalamic-pituitary-gonadal axis in abstinent alcoholic men.

AIMS: Testosterone synthesis in chronic alcoholics is affected by a variety of mechanisms. Little is known about the reversibility of these changes upon abstinence and available data on circulating hormone levels are incomplete and inconsistent. METHODS: Serum concentrations of free testosterone, total testosterone, and luteinizing hormone (LH) were determined in 18 male non-cirrhotic chronic alcoholics on days 2, 22, 82 and 127 of strictly controlled abstinence, as well as in a group of 20 healthy age-matched controls. RESULTS: Higher total testosterone concentrations were found in alcoholics on the second day of abstinence, as compared to controls (7.1 +/- 1.9 vs 5.6 +/- 1.4 ng/ml) and throughout the whole observation period. Correspondingly, free testosterone concentrations were increased over control levels on day 2 (40.0 +/- 12.1 vs 29.7 +/- 8.1 pg/ml) and stayed elevated in the presence of augmented concentrations of LH [4.5 U/l (range 1.6-9.5 U/l) vs 2.0 U/l (range 0.8-8.1 U/l)] for up to 127 days of strictly controlled abstinence. CONCLUSIONS: Sustained increases in serum free and total testosterone levels in the presence of inadequately raised LH concentrations point towards persisting disturbances of the hypothalamic-pituitary-gonadal axis in male alcoholics upon cessation of drinking.

Polysomnographic findings in five adult patients with pituitary insufficiency before and after cessation of human growth hormone replacement therapy.

OBJECTIVE: We observed the new onset of severe obstructive sleep apnoea syndrome (OSAS) in an adult male patient during human growth hormone (hGH) replacement therapy. This prompted us to evaluate the potential influence of hGH substitution therapy on sleep in middle-aged men. DESIGN: A longitudinal study. SUBJECTS: Five male patients (aged 44-56 years, median age 54 years) with postoperative pituitary insufficiency given hGH replacement therapy for 1-2 years (median dose 2.0 U/day; median IGF-I serum concentration 351 microg/l) and 6 months after cessation of hGH treatment (median IGF-I level 77 microg/l - 1 microg/l = 0.131 nmol/l). MEASUREMENTS: Polysomnographic studies were performed, and the following parameters were determined: time in bed (TIB), sleep period time (SPT), total sleep time (TST), sleep efficiency (SE = TST/TIB), sleep stage 1 onset latency (SL), different sleep stages [W (wake), S1, S2, SWS (slow wave sleep = S3 + S4) and REM; % of SPT], stage shifts per hour of SPT (SS/h), stage shifts to W/h of SPT [A/h (awakening)], index of apnoea and hypopnoea events per hour of TST (AH/h), arousals from apnoea and hypopnoea per hour of TST (Ar/h), index of obstructive (OAH/h), central (CAH/h) and mixed (MAH/h) events of apnoea and hypopnoea per hour of TST and minimal desaturation (MD). RESULTS: Median baseline results were: TIB, 479 min; SPT, 465 min; TST, 405 min; SE, 77%; SL, 8.5 min; W, 18.9%; S1, 8.2%; S2, 52.7%; REM, 13.5%; SS/h, 17.7; A/h, 2.8; AH/h, 11.9; Ar/h, 4.4; MD, 80%. These parameters did not change significantly after cessation of hGH treatment. In contrast, median SWS decreased significantly from 33 min (7.1%) to 7.5 min (1.8%; P = 0.03). Median OAH/h decreased significantly from 4.4 to 0.1 (P = 0.03) whereas CAH/h increased from 6.3 to 14.6 (P = 0.03) after cessation of hGH. Correspondingly, one patient with OSAS improved markedly whereas another patient developed new and asymptomatic central SAS after cessation of hGH. CONCLUSION: This study showed that hGH replacement therapy influenced sleep reaction in a complex way in middle-aged men; cessation of treatment was associated with a significant decrease in slow wave sleep and a shift from obstructive to central apnoea and hypopnoea.

Differential regulation of Cbfa1/Runx2 and osteocalcin gene expression by vitamin-D3, dexamethasone, and local growth factors in primary human osteoblasts.

Core binding factor alpha 1 (Cbfa1) is an osteoblast-specific transcription factor essential to develop a mature osteoblast phenotype. However, its exact role in the signaling of various osteotropic-differentiating agents is still unclear. In this study, we assessed the effects of 1,25-(OH)(2)-D3 (D3), ascorbic acid, bone morphogenetic protein-2 (BMP-2), dexamethasone (Dex), and transforming growth factor-beta (TGF-beta) on Cbfa1 and osteocalcin (OCN) mRNA steady state levels (by semiquantitative RT-PCR) in an in vitro model of osteoblast differentiation. TGF-beta increased Cbfa1 mRNA levels in normal primary human osteoblasts (pHOB) by 2.6-fold in a time-dependent fashion with maximum effect on day 28 (P < 0.001). Similarly, the glucocorticoid Dex enhanced Cbfa1 gene expression by pHOB in a time-dependent fashion by up to 4.6-fold (P < 0.001). In contrast, Dex inhibited OCN gene expression levels by 68% (P < 0.01). Treatment with BMP-2 resulted in an earlier enhancement of Cbfa1 and led to a 4.2-fold increase with a maximum on day 21 (P < 0.001). Ascorbic acid did not modulate Cbfa1 and OCN gene expression. The effect of vitamin D (D3) on Cbfa1 mRNA expression was influenced by the duration of treatment, being inhibitory after 1 h and having a stimulatory effect after 48 h. Time course experiments indicated a stimulatory effect of D3 on Cbfa1 mRNA levels (by 2.5-fold after 48 h; P < 0.01). Analysis of the late cellular differentiation marker osteocalcin revealed that D3 increased OCN gene expression by 14-fold (P < 0.001). In conclusion, in normal primary human osteoblasts, the rapid and pronounced increase of OCN after treatment with D3 seems not to be mediated by Cbfa1. These data imply that Cbfa1 gene expression is differentially regulated by various osteoblastic differentiating agents and is dependent on the stage of maturation.

Prolonged culture of HOS 58 human osteosarcoma cells with 1,25-(OH)2-D3, TGF-beta, and dexamethasone reveals physiological regulation of alkaline phosphatase, dissociated osteocalcin gene expression, and protein synthesis and lack of mineralization.

Cultured rodent osteoblastic cells reiterate the phenotypic differentiation and maturation of osteoblasts seen in vivo. As previously shown, the human osteosarcoma cell line HOS 58 represents a differentiated stage of osteoblast development. The potential of HOS 58 for still further in vitro differentiation suggests the line can serve as a model of osteoblast maturation. Using this cell line, we have investigated the influence of 1,25-(OH)2-D3 (D3), TGF-beta and Dexamethasone (Dex) on proliferation and on the protein and mRNA levels of alkaline phosphatase (AP), procollagen 1 (Col 1), and osteocalcin (Oc), as well as mineralization during 28 days in culture. AP mRNA and protein were highly expressed throughout the culture period with further increase of protein AP activity at constant gene expression levels. A differentiation inhibiting effect of either TGF-beta or Dex was seen. Col 1 was investigated without the use of ascorbic acid and showed only minor changes during culture time or stimulation. The gene expression for Oc increased continually whereas protein synthesis peaked at confluence and decreased thereafter. TGF-beta and Dex treatments decreased Oc mRNA and protein levels. Stimulation by D3 was maximal at day 7 with a decrease thereafter. HOS 58 cells showed no mineralization capacity when stimulated with different agents, as measured by energy-dispersive X-ray microanalysis. This was not due to absence of Cbfa1 expression. In conclusion, the HOS 58 osteosarcoma cell line represents a differentiated cell line with highly expressed and physiologically regulated AP expression during further differentiation in culture. We observed a dissociation between osteocalcin gene expression and protein secretion which may contribute to the lack of mineralization in this cell line.