The effect of growth hormone replacement on the thyroid axis in patients with hypopituitarism: in vivo and ex vivo studies.

OBJECTIVE: Alterations in the hypothalamic-pituitary-thyroid axis have been reported following growth hormone (GH) replacement. The aim was to examine the relationship between changes in serum concentration of thyroid hormones and deiodinase activity in subcutaneous adipose tissue, before and after GH replacement. DESIGN: A prospective, observational study of patients receiving GH replacement as part of routine clinical care. PATIENTS: Twenty adult hypopituitary men. MEASUREMENTS: Serum TSH, thyroid hormones - free and total thyroxine (T4) and triiodothyronine (T3) and reverse T3, thyroglobulin and thyroid-binding globulin (TBG) levels were measured before and after GH substitution. Changes in serum hormone levels were compared to the activity of deiodinase isoenzymes (DIO1, DIO2 and DIO3) in subcutaneous adipose tissue. RESULTS: The mean daily dose of growth hormone (GH) was 0·34 ± 0·11 mg (range 0·15-0·5 mg). Following GH replacement, mean free T4 levels declined (-1·09 ± 1·99 pmol/l, P = 0·02). Reverse T3 levels also fell (-3·44 ± 1·42 ng/dl, P = 0·03) and free T3 levels increased significantly (+0·34 ± 0·15 pmol/l, P = 0·03). In subcutaneous fat, DIO2 enzyme activity declined; DIO1 and DIO3 activities remained unchanged following GH substitution. Serum TSH, thyroglobulin and TBG levels were unaltered by GH therapy. CONCLUSIONS: In vitro analysis of subcutaneous adipose tissue from hypopituitary human subjects demonstrates that GH replacement is associated with significant changes in deiodinase isoenzyme activity. However, the observed variation in enzyme activity does not explain the changes in the circulating concentration of thyroid hormones induced by GH replacement. It is possible that deiodinase isoenzymes are differentially regulated by GH in other tissues including liver and muscle.

ATP synthase with its gamma subunit reduced to the N-terminal helix can still catalyze ATP synthesis.

ATP synthase uses a unique rotary mechanism to couple ATP synthesis and hydrolysis to transmembrane proton translocation. As part of the synthesis mechanism, the torque of the rotor has to be converted into conformational rearrangements of the catalytic binding sites on the stator to allow synthesis and release of ATP. The gamma subunit of the rotor, which plays a central role in the energy conversion, consists of two long helices inside the central cavity of the stator cylinder plus a globular portion outside the cylinder. Here, we show that the N-terminal helix alone is able to fulfill the function of full-length gamma in ATP synthesis as long as it connects to the rest of the rotor. This connection can occur via the epsilon subunit. No direct contact between gamma and the c ring seems to be required. In addition, the results indicate that the epsilon subunit of the rotor exists in two different conformations during ATP synthesis and ATP hydrolysis.

Septic shock non-thyroidal illness syndrome causes hypothyroidism and conditions for reduced sensitivity to thyroid hormone.

Non-thyroidal illness syndrome (NTIS) is part of the neuroendocrine response to stress, but the significance of this syndrome remains uncertain. The aim of this study was to investigate the effect of lipopolysaccharide (LPS)-induced NTIS on thyroid hormone (TH) levels and TH molecular targets, as well as the relationship between septic shock nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) activation and TH receptor ß (THRB) gene expression at a multi-tissue level in a pig model. Prepubertal domestic pigs were given i.v. saline or LPS for 48 h. Serum and tissue TH was measured by chemiluminescence and RIA. Expression of THRs and cofactors was measured by real-time PCR, and deiodinase (DIO) activity was measured by enzyme assays. Tissue NF-kB nuclear binding activity was evaluated by EMSA. LPS-treated pigs had decreased TH levels in serum and most tissues. DIO1 expression in liver and kidney and DIO1 activity in kidney decreased after LPS. No changes in DIO2 activity were observed between groups. LPS induced an increase in hypothalamus, thyroid, and liver DIO3 activity. Among the other studied genes, monocarboxylate transporter 8 and THRB were the most commonly repressed in endotoxemic pigs. LPS-induced NF-kB activation was associated with a decrease in THRB gene expression only in frontal lobe, adrenal gland, and kidney cortex. We conclude that LPS-induced NTIS in pigs is characterized by hypothyroidism and tissue-specific reduced TH sensitivity. The role of NF-kB in regulating THRB expression during endotoxemia, if any, is restricted to a limited number of tissues.

Identification and evaluation of constitutively active thyroid stimulating hormone receptor mutations.

Thyroid stimulating hormone receptor (TSHR) is a guanine nucleotide-binding protein-coupled seven-transmembrane-domain receptor that controls the differentiation, growth, and function of the thyroid gland through stimulation of adenylyl cyclase and phospholipase C pathways. Thyroid stimulating hormone (TSH) is the main TSHR ligand, and unliganded receptor remains silent due to the interaction of its large extracellular domain with the extracellular loops of the serpentine. The TSHR gene is highly mutagenic and constitutively active mutations have been extensively described. Naturally occurring TSHR-activating mutations can affect any part of the receptor, but most activating mutations affect the serpentine region, and the majority of these are located in the third intracellular loop or transmembrane domain six. We describe several simple and relatively cheap methods used in our laboratory to study constitutive TSHR mutations that include (1) screening of TSHR gene mutations in paraffin-embedded thyroid tissue samples, (2) measurement of TSHR constitutive activity in vitro, (3) measurement of TSHR expression at cell surface by flow cytometry analysis, (4) TSH binding to TSHR, and (5) TSHR phosphorylation analysis.