Effects of maternal levels of thyroid hormone (TH) on the hypothalamus-pituitary-thyroid set point: studies in TH receptor beta knockout mice.

A level of thyroid hormone (TH) in agreement with the tissue requirements is essential for vertebrate embryogenesis and fetal maturation. In this study we evaluate the immediate and long-term effects of incongruent intrauterine TH levels between mother and fetus using the TH receptor (TR) beta(-/-) knockout mouse as a model. We took advantage of the fact that the TRbeta(-/-) females have elevated serum TH but are not thyrotoxic due to resistance to TH. We used crosses between heterozygotes with wild-type phenotype (TRbeta(+/-)) males and TRbeta(-/-) females, with a hyperiodothyroninemic (high T(4) and T(3) levels) intrauterine environment (TH congruent with the TRbeta(-/-) fetus and excessive for the TRbeta(+/-) fetus), and reciprocal crosses between TRbeta(-/-) males and TRbeta(+/-) females, providing a euiodothyroninemic intrauterine environment. We found that TRbeta(-/-) dams had reduced litter sizes and pups with lower birth weight but preserved the mendelian TRbeta(-/-) to TRbeta(+/-) ratio at birth, indicating that the incongruous TH levels did not decrease intrauterine survival of a specific genotype. The results of studies in newborns demonstrate that TRbeta(+/-) pups born to TRbeta(-/-) dams have persistent suppression of serum TSH without a peak. On the other hand, TRbeta(-/-) pups born to TRbeta(+/-) dams have lower serum TSH at birth and a tendency to peak higher, compared with TRbeta(-/-) pups born to TRbeta(-/-) dams. The studies in the adult progeny demonstrate that TRbeta(+/-) mice born to TRbeta(-/-) dams and, thus, exposed to higher intrauterine TH levels, have greater resistance to TH at the level of the pituitary when stimulated with TRH. On the other hand, TRbeta(-/-) mice born to TRbeta(+/-) dams and, thus, deprived of TH in uterine life, were more sensitive to TH when similarly stimulated with TRH. Thus, TH exposure in utero has an effect on the regulatory set point of the hypothalamus-pituitary-thyroid axis, which can be seen early in life and persists into adulthood.

Pituitary-thyroid setpoint and thyrotropin receptor expression in consomic rats.

The genetic basis for differences in TSH sensitivity between two rat strains was examined using consomic rats generated from original strains salt-sensitive Dahl (SS) (TSH 1.8 +/- 0.1 ng/ml; free T(4) index 4.9 +/- 0.4) and Brown Norwegian (BN) (TSH 5.5 +/- 0.6 ng/ml, P < 0.05; free T(4) index 4.3 +/- 0.1, P not significant). Consomic rats SSBN6 [BN chromosome (CH) 6 placed in SS rat] and SSBN2 (BN CH 2 placed in SS rat) have TSH concentrations intermediate between pure SS and BN strains (2.9 +/- 0.3 and 3.1 +/- 0.3 ng/ml, respectively; P < 0.05). Candidate genes on rat CH 2 included TSH beta-subunit and on CH 6 the TSH receptor (TSHR). TSH from sera of BN, SS, SSBN6, and SSBN2 strains had similar in vitro bioactivity suggesting that the cause for the variable TSH concentrations was not due to an altered TSH. Physiological response to TSH was measured by changes in serum T(4) concentrations upon administration of bovine TSH (bTSH). Rat strain SS had a greater T(4) response to bTSH than BN (change in T(4), 1.3 +/- 0.1 vs. 0.4 +/- 0.1 microg/dl, P < 0.005), suggesting reduced thyrocyte sensitivity to TSH in BN. Sequencing of the TSHR coding region revealed an amino acid difference in BN (Q46R). This substitution is unlikely to contribute to the strain difference in serum TSH because both TSHR variants were equally expressed at the cell surface of transfected cells and responsive to bTSH. Given similar TSH activity and similar TSHR structure, TSHR mRNA expression in thyroid tissue was quantitated by real-time PCR. BN had 54 +/- 5% the total TSHR expression compared to SS (100 +/- 7%, P < 0.0001), when corrected for GAPDH expression, a difference confirmed at the protein level. Therefore, the higher TSH level in the BN strain appears to reflect an adjustment of the feedback loop to reduced thyrocyte sensitivity to TSH secondary to reduced TSHR expression. These strains of rat provide a model to study the cis- and trans-acting factors underlying the difference in TSHR expression.

Serum Concentrations of IgG4 in the Spanish Adult Population: Relationship with Age, Gender, and Atopy.

BACKGROUND AND AIM: Serum IgG4 concentrations are commonly measured in clinical practice. The aim of this study was to investigate serum IgG4 concentrations in adults and their potential relationship with demographic, lifestyle, metabolic, and allergy-related factors. METHODS: Serum IgG4 concentrations were measured with a commercial assay in 413 individuals (median age 55 years, 45% males) who were randomly selected from a general adult population. RESULTS: Median IgG4 concentration was 26.8 mg/dL. Five out of the 413 individuals (1.2%) exhibited IgG4 concentrations >135 mg/dL, and 17 out of 411 (4.1%) exhibited an IgG4/total IgG ratio >8%. Serum IgG4 concentrations were significantly higher in males than in females and decreased with age. After adjusting for age and sex, serum IgG4 concentrations were not significantly influenced by alcohol consumption, smoking or common metabolic abnormalities (obesity and the related metabolic syndrome). Serum IgG4 concentrations were not significantly correlated with serum concentrations of proinflammatory cytokines and inflammation markers. Serum IgG4 concentrations were significantly correlated with IgE concentrations. Serum IgG4 concentrations tended to be higher in atopics (individuals with IgE-mediated sensitization to aeroallergens) than in non-atopics, particularly among atopics without respiratory symptoms. Serum IgG4 concentrations were not significantly correlated with total eosinophil blood count. Cases of IgG4-related disease were neither present at baseline nor detected after a median of 11 years of follow-up. CONCLUSIONS: Studies aimed at defining reference IgG4 values should consider partitioning by age and sex. Further studies are needed to confirm the potential influence of atopy status on serum IgG4 concentrations.

Angiotensin II type 1 receptor blocker irbesartan ameliorates vascular function in spontaneously hypertensive rats regardless of oestrogen status.

BACKGROUND: Angiotensin II type 1 (AT1) receptor overexpression may play a decisive role in endothelial dysfunction during oestrogen deficiency in spontaneously hypertensive rats (SHRs). Similarly, exaggerated production of angiotensin II and enhanced expression of AT1 receptor have been reported in vessels of SHRs compared with normotensive rats. OBJECTIVE: To test the hypothesis that antihypertensive treatment with the AT1 receptor antagonist, irbesartan, could not only decrease blood pressure but also ameliorate endothelial dysfunction associated with both hypertension and oestrogen deficiency. METHODS: Ovariectomized and sham-ovariectomized SHRs were treated with 50 mg/kg irbesartan per day, administered with chow for 30 weeks. Sham-ovariectomized and ovariectomized rats receiving no treatment were used as control groups. At the end of the treatment period, the vascular reactivity of aortic rings was studied. RESULTS: In the irbesartan-treated groups, vasoconstriction induced by Nomega-nitro-l-arginine methyl ester (l-NAME) was increased and the response to phenylephrine exhibited greater potentiation in the presence of l-NAME, demonstrating a greater availability of basal nitric oxide in these groups. In addition, chronic treatment with irbesartan similarly enhanced the responsiveness of aortic rings from ovariectomized or sham-ovariectomized rats to acetylcholine and sodium nitroprusside. Incubation with indomethacin did not significantly alter acetylcholine- and sodium nitroprusside-induced relaxations in the irbesartan-treated rats. However, relaxations induced by acetylcholine and sodium nitroprusside in aortic rings from non-treated rats were significantly greater in the presence of indomethacin. CONCLUSION: Our data suggest that irbesartan enhances basal nitric oxide availability and ameliorates vascular relaxations in SHRs, by decreasing the production of cyclooxygenase-dependent contracting factors in smooth muscle cells, regardless of oestrogen status.

Determinants of serum concentrations of lipopolysaccharide-binding protein (LBP) in the adult population: the role of obesity.

BACKGROUND AND AIM: Assessment of serum concentration of lipopolysaccharide (LPS)-binding protein (LBP) has been suggested as a useful biomarker to indicate activation of innate immune responses to microbial products. We investigated LBP concentrations and associations with demographics, lifestyle factors, and common metabolic abnormalities in adults. We also examined if LBP concentrations were associated with common polymorphisms in genes coding for LBP (rs2232618), CD14 (rs2569190), and TLR4 (rs4986790), the molecules responsible for the innate immune response to LPS, or serum levels of soluble CD14 (sCD14) and proinflammatory cytokines. METHODS: Serum LBP was measured with a commercial immunoassay in a random sample of the adult population (n = 420, 45% males, age 18-92 years) from a single municipality. RESULTS: Serum LBP concentrations increased with age (P<0.001) and were higher in individuals who were overweight or obese than in normal-weight individuals (P<0.001). Similarly, LBP concentrations were higher in individuals with metabolic syndrome than in individuals without it (P<0.001). Among metabolic syndrome components, LBP concentrations were independently associated with abdominal obesity (P = 0.002) and low concentrations of HDL-cholesterol (P<0.001). Serum LBP concentrations tended to be independently associated with smoking (P = 0.05), but not with alcohol consumption. Likewise, there was not significant association between LBP concentrations and gene polymorphisms. Concentrations of LBP significantly correlated with serum levels of proinflammatory cytokines (IL-6 and IL-8), sCD14, and with liver enzymes. CONCLUSIONS: Serum LBP concentrations increased with age. Overweight, obesity, and having metabolic syndrome (particularly, low HDL cholesterol levels) were associated with higher LBP concentrations. These findings are consistent with microbial exposure playing a role in these inflammatory, metabolic abnormalities.

Thyroid hormone receptor α and regulation of type 3 deiodinase.

Mice deficient in thyroid hormone receptor α (TRα) display hypersensitivity to thyroid hormone (TH), with normal serum TSH but diminished serum T(4). Our aim was to determine whether altered TH metabolism played a role in this hypersensitivity. TRα knockout (KO) mice have lower levels of rT(3), and lower rT(3)/T(4) ratios compared with wild-type (WT) mice. These alterations could be due to increased type 1 deiodinase (D1) or decreased type 3 deiodinase (D3). No differences in D1 mRNA expression and enzymatic activity were found between WT and TRαKO mice. We observed that T(3) treatment increased D3 mRNA in mouse embryonic fibroblasts obtained from WT or TRßKO mice, but not in those from TRαKO mice. T(3) stimulated the promoter activity of 1.5 kb 5'-flanking region of the human (h) DIO3 promoter in GH3 cells after cotransfection with hTRα but not with hTRß. Moreover, treatment of GH3 cells with T(3) increased D3 mRNA after overexpression of TRα. The region necessary for the T(3)-TRα stimulation of the hD3 promoter (region -1200 to -1369) was identified by transfection studies in Neuro2A cells that stably overexpress either TRα or TRß. These results indicate that TRα mediates the up-regulation of D3 by TH in vitro. TRαKO mice display impairment in the regulation of D3 by TH in both brain and pituitary and have reduced clearance rate of TH as a consequence of D3 deregulation. We conclude that the absence of TRα results in decreased clearance of TH by D3 and contributes to the TH hypersensitivity.

In vivo interaction of steroid receptor coactivator (SRC)-1 and the activation function-2 domain of the thyroid hormone receptor (TR) beta in TRbeta E457A knock-in and SRC-1 knockout mice.

The activation function-2 (AF-2) domain of the thyroid hormone (TH) receptor (TR)-beta is a TH-dependent binding site for nuclear coactivators (NCoA), which modulate TH-dependent gene transcription. In contrast, the putative AF-1 domain is a TH-independent region interacting with NCoA. We determined the specificity of the AF-2 domain and NCoA interaction by evaluating thyroid function in mice with combined disruption of the AF-2 domain in TRbeta, due to a point mutation (E457A), and deletion of one of the NCoAs, steroid receptor coactivator (SRC)-1. The E457A mutation was chosen because it abolishes NCoA recruitment in vitro while preserving normal TH binding and corepressor interactions resulting in resistance to TH. At baseline, disruption of SRC-1 in the homozygous knock-in (TRbeta(E457A/E457A)) mice worsened the degree of resistance to TH, resulting in increased serum T(4) and TSH. During TH deprivation, disruption of AF-2 and SRC-1 resulted in a TSH rise 50% of what was seen when AF-2 alone was removed, suggesting that SRC-1 was interacting outside of the AF-2 domain. Therefore, 1) during TH deprivation, SRC-1 is necessary for activating the hypothalamic-pituitary-thyroid axis; 2) ligand-dependent repression of TSH requires an intact AF-2; and 3) SRC-1 may interact with the another region of the TRbeta or the TRalpha to regulate TH action in the pituitary. This report demonstrates the dual interaction of NCoA in vivo: the TH-independent up-regulation possibly through another domain and TH-dependent down-regulation through the AF-2 domain.

Mitochondrial A-kinase anchoring protein 121 binds type II protein kinase A and enhances steroidogenic acute regulatory protein-mediated steroidogenesis in MA-10 mouse leydig tumor cells.

The expression of the steroidogenic acute regulatory protein (STAR) is regulated by PKA in response to trophic hormone stimulation through the second messenger cAMP. However, in steroidogenic cells, the concentrations of hormone necessary to maximally induce cAMP synthesis and PKA activity are often significantly higher than is necessary to achieve maximum steroidogenesis. One general mechanism believed to make PKA signaling more effective is the use of A-kinase anchoring proteins (AKAPs) to recruit PKA to discrete subcellular compartments, which coordinates and focuses PKA action with respect to its substrates. The characterization of AKAP121 has suggested that it enhances the posttranscriptional regulation of STAR by recruiting both Star mRNA and PKA to the mitochondria, thereby permitting more effective translation and phosphorylation of STAR. Testing this hypothesis revealed that cAMP-induced STAR expression and steroidogenesis closely followed AKAP121 abundance when this AKAP was silenced or overexpressed in MA-10 cells but that these changes were effected posttranscriptionally. Moreover, silencing AKAP121 expression in these cells specifically altered the localization of type II PKA regulatory subunit alpha (PKAR2A) at the mitochondria but did not affect its relative expression within the cell. Affinity purification experiments showed that PKAR2A preferentially associated with AKAP121, and cAMP analogs that activate type II PKA induced STAR phosphorylation more efficiently than analogs stimulating type I PKA. This suggests that AKAP121 and PKAR2A serve to enhance steroidogenesis by directing the synthesis and activation of STAR at the mitochondria in response to cAMP.

Localization of a negative vitamin D response sequence in the human growth hormone gene.

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2) D(3)] exerts its biological effects by binding to the vitamin D receptor (VDR), which binds in turn to the vitamin D response elements located in the target gene's promoter. We have previously demonstrated that VDR binds in vitro with high affinity to the 5'-flanking sequence of the human growth hormone (hGH) gene. In this study, we analyzed the response to 1,25(OH)(2) D(3) of hGH-promoter constructs introduced by transfection into the MCF-7 human adenocarcinoma cell line. We found that the transcriptional activity of some of these constructs was markedly reduced by 1,25(OH)(2) D(3). Deletion analyses revealed that a 34-bp sequence located between positions -62 and -29 upstream of the transcription start site is sufficient for this repressive response. This conclusion was also confirmed by gel mobility shift assays. Our results indicate that vitamin D inhibits hGH gene transcription, directly or by interference with other transcription factors.