Hirotoshi Nakamura

Fibroblast Growth Factor 21 Regulates Lipolysis in White Adipose Tissue But Is Not Required for Ketogenesis and Triglyceride Clearance in Liver

Fibroblast growth factors (Fgfs) are polypeptide growth factors with diverse functions. Fgf21, a unique member of the Fgf family, is expected to function as a metabolic regulator in an endocrine manner. Hepatic Fgf21 expression was increased by fasting. The phenotypes of hepatic Fgf21 transgenic or knockdown mice and high-fat, low-carbohydrate ketogenic diet-fed mice suggests that Fgf21 stimulates lipolysis in the white adipose tissue during normal feeding and is required for ketogenesis and triglyceride clearance in the liver during fasting. However, the physiological roles of Fgf21 remain unclear. To elucidate the physiological roles of Fgf21, we generated Fgf21 knockout (KO) mice by targeted disruption. Fgf21 KO mice were viable, fertile, and seemingly normal. Food intake, oxygen consumption, and energy expenditure were also essentially unchanged in Fgf21 KO mice. However, hypertrophy of adipocytes, decreased lipolysis in adipocytes, and decreased blood nonesterified fatty acid levels were observed when Fgf21 KO mice were fed normally. In contrast, increased lipolysis in adipocytes and increased blood nonesterified fatty acid levels were observed in Fgf21 KO mice by fasting for 24 h, indicating that Fgf21 stimulates lipolysis in the white adipose tissue during feeding but inhibits it during fasting. In contrast, unexpectedly, hepatic triglyceride levels were essentially unchanged in Fgf21 KO mice. In addition, ketogenesis in Fgf21 KO mice was not impaired by fasting for 24 h. The present results indicate that Fgf21 regulates lipolysis in adipocytes in response to the metabolic state but is not required for ketogenesis and triglyceride clearance in the liver.

A point mutation of the T3 receptor β1 gene in a kindred of generalized resistance to thyroid hormone

Mutations of the thyroid hormone receptor (TR) beta 1 gene have recently been detected in several unrelated families with generalized resistance to thyroid hormone (GRTH). We now report a novel point mutation in the TR beta 1 gene in a case of a Korean-Japanese kindred. The intracellular localization and the amount of TR proteins were considered to be normal by the immunocytochemical study of cultured skin fibroblasts from the patients using anti-T3 receptor antibody. The cDNA of the T3-binding domain of the TR beta 1 gene, synthesized from the total RNA of the patients fibroblasts, was amplified by the polymerase chain reaction, and was sequenced. A point mutation, A to G, in one allele at 1612 resulting in an amino acid substitution from lysine 438 to glutamic acid was detected. The same mutation was identified in one allele in each of the affected members. In vitro translation products of the mutant TR beta 1 gene showed decreased T3-binding activity. These data suggest that a TR mutation is predominantly responsible for GRTH, irrespective of ethnic background.

ANTI-TSH ANTIBODY WITH HIGH SPECIFICITY TO HUMAN TSH IN SERA FROM A PATIENT WITH GRAVES' DISEASE: ITS ISOLATION FROM, AND INTERACTION WITH, TSH RECEPTOR ANTIBODIES

A patient with thyrotoxic Graves disease had an apparent measurable level of serum TSH (2–5 μU/ml) by double‐antibody radioimmunoassay (RIA). The serum IgG bound with both [125I]human(h)TSH and [125I]bovine(b)TSH. The [125I]hTSH binding was more effectively displaced by human than bovine TSH, whereas [125I]bTSH binding was displaced exclusively by bTSH. Scatchard analyses revealed that [125I]hTSH binding showed two components, whereas [125I]bTSH binding had only one component. Serum TSH determined by RIA became undetectable 21 months after antithyroid drug treatment with a parallel decrease of [125I]hTSH binding IgG activity. Four thyrotrophin binding inhibitory immunoglobulins (TBII) from other patients did not interfere with the binding of the patients serum to [125I]h‐ or bTSH. Furthermore, the in‐vitro thyroid stimulating activities of three thyroid stimulating antibodies (TSAb) were not affected by the addition of this patients IgG. On the other hand, this patients Ig (3 mg/ml) abolished the in‐vitro thyroid stimulation by bTSH (100 /μU/ml), but did not affect that by hTSH (100 /μU/ml). The anti‐hTSH antibody, TSH receptor antibody and anti‐bTSH antibody in the serum, which contains TSAb as well as anti‐TSH antibodies, could be partially purified by hTSH‐agarose and subsequently by guinea pig fat cell membrane affinity absorptions. However, the anti‐hTSH antibody fraction obtained had both hTSH binding activity and thyroid stimulating activity, and this fraction did not show any inhibitory effect on the in‐vitro thyroid stimulation of autologous TSH receptor antibody or hTSH. The possible significance of anti‐TSH antibodies is discussed.

Recognition of rat liver and kidney nuclear T3 receptors by an antibody against c-erb A peptide

It has been reported that c-erb A encodes nuclear T3 receptors (NT3R). Based on the sequence of c-erb A cDNA, we synthesized a polypeptide consisting of 15 amino acids, the sequence of which has high homology between c-erb A alpha 1 and beta. The antibody against this c-erb A peptide not only immunoprecipitated rat liver and kidney NT3R but also inhibited T3 binding to NT3R. In a displacement study, the inhibition of [125I]T3-binding by the antibody was parallel to that by T3 in terms of the concentration of the competitor added in the incubation mixture. Scatchard analysis revealed that the antibody decreased the value for the association constant in a dose dependent manner. The antibody did not bind T3 itself. The results show that the antibody against c-erb A peptide recognizes rat liver and kidney NT3R and that the sequence encoding this peptide, the closest carboxyl-terminal of c-erb A may be critical or at least closely related to the hormone binding.

Role of Fgf receptor 2c in adipocyte hypertrophy in mesenteric white adipose tissue.

Fgf receptor 2c (Fgfr2c) was expressed in mature adipocytes of mouse white adipose tissue (WAT). To examine the role of Fgfr2c in mature adipocytes, we generated adipocyte-specific Fgfr2 knockout (Fgfr2 CKO) mice. The hypertrophy impairment of adipocytes in the mesenteric WAT but not in the subcutaneous WAT and decreased plasma free fatty acid (FFA) levels were observed in Fgfr2 CKO mice. Although the expression of genes involved in adipocyte differentiation and lipid metabolism in the mesenteric WAT was essentially unchanged, the expression of uncoupling protein 2 potentially involved in energy dissipation was significantly increased. Among potential Fgf ligands for Fgfr2c, Fgf9 was preferentially expressed in the mesenteric WAT. The present findings indicate that Fgfr2c potentially activated by Fgf9 plays a role in the adipocyte hypertrophy in the mesenteric WAT and FFA metabolism and/or energy dissipation in the mesenteric WAT might be involved in the hypertrophy impairment.

Efficacy of Bromocriptine Administration for Selective Pituitary Resistance to Thyroid Hormone

The relation between thyroid-stimulating hormone (TSH) and triiodothyronine (T3) was evaluated in a girl with the selective pituitary type of thyroid hormone resistance for more than 7 years to clarify whether bromocriptine was an effective treatment or not. Levels of T3 (before: 2.44 +/- 0.64 nmol/l, mean +/- SD) and TSH (4.81 +/- 2.52 mU/l) were significantly decreased during therapy (T3: 2.15 +/- 0.44 nmol/l; TSH: 1.59 +/- 0.78 mU/l). T3 x TSH, calculated as one of the indices of pituitary resistance, on bromocriptine therapy (3.229 +/- 1.255 mU/l x nmol/l) was significantly (p < 0.005) smaller than the product before the administration (11.298 +/- 5.891 mU/l x nmol/l). The results suggest that bromocriptine should be one of the agents initially considered for the treatment of pituitary resistance to thyroid hormone.

Triiodothyronine effects on RNA polymerase activities in isolated neuronal and glial nuclei of the mature rat brain cortex

Our previous study demonstrated that a high level of nuclear triiodothyronine receptors (NT3R), which are identical to the hepatic NT3R, exists in neuronal nuclei of the cerebral cortex from an adult rat brain. To investigate whether thyroid hormone acts through binding to nuclear receptors, we measured RNA polymerase activities in isolated neuronal and glial nuclei of cerebral cortices prepared from three groups of rats with different T3 levels: T3 (20 micrograms/100 g BW/d, for three days)-injected hyperthyroid rats, control normal rats, and thyroidectomized rats. The enzyme activities in both nuclear fractions were assayed under the condition of dose-response linearity. When RNA polymerase I activity in neuronal nuclei from control rats was expressed as 100%, the activities from T3-injected and hypothyroid rats were 112.3 +/- 3.4% (n = 5, P less than .05) and 86.9 +/- 3.5% (n = 5, P less than .05), respectively. The increase in the enzyme activities were parallel to the increase in T3 content in neuronal nuclei among the groups. Glial nuclear RNA polymerase I showed the same tendency in response to T3, although the enzyme activity was smaller than from neuronal nuclei. RNA polymerase II, however, showed no significant change in response to altered T3 levels. The existence of numerous receptors and an induction of increased RNA polymerase I activity by T3 in neuronal nuclei raise the possibility that thyroid hormone through a NT3R pathway in the cerebral cortex of even the mature rat brain.

Starvation-induced decrease in the maximal binding capacity for triiodothyronine of the thyroid hormone receptor is due to a decrease in the receptor protein.

Biological responses to thyroid hormones are mediated by the nuclear thyroid hormone receptor (TR). Alterations in the maximal triiodothyronine (T3)-binding capacity (Cmax) of TR measured using a ligand binding assay have been reported under some pathophysiological conditions. Northern blot analysis has indicated that TR mRNA concentrations do not necessarily correlate with Cmax levels. For example, although the decrease in Cmax in rat liver induced by prolonged fasting is well established, TR mRNA concentrations have been reported to be constant. In the present study, we examined starvation-induced changes in TR by Western blot with anti-TR(alpha 1 + beta)antiserum and by Scatchard plot analysis. Starvation of rats for 72 hours decreased Cmax in the liver to 72.5% of control levels. The 47- and 55-kd TR proteins detected in hepatic nuclear extract by Western blotting also decreased to 64% and 66% of control values, respectively. The starvation-induced changes in Cmax and TR protein levels paralleled the change in total hepatic nuclear protein concentration. These results suggest that the decrease in T3-binding activity of the TR is due to a reduction of the TR protein itself.

Clinical significance of elevated labeled TSH binding (LTB) activity in sera of patients with Graves' disease and other thyroid disorders

Labeled TSH binding (LTB) of individual serum samples was monitored simultaneously using the thyrotropin binding inhibitor immunoglobulin (TBII) assay. In 643 TBII determinations, 86 sera (13.4%) showed elevated LTB. The incidence of elevated LTB in active Graves’ patients (17.5%) was much higher than that of inactive Graves’ patients (6.7%). After TBII activities were corrected by LTB, 79% of the active Graves’ patients who had negative raw TBII were found to be positive. In patients with untreated active Graves’ disease, the detectability of TBII increased from 85% to 91% after LTB correction, while those in inactive Graves’ and other thyroid disorders did not increase so much (1.6 and 0%, respectively). Further, most of elevated LTB seen in other thyroid disorders were found to be different from those in Graves’ disease by heat stability experiment. Serial observations of LTB and TBII in 24 Graves’ patients showed 2 patterns. Parallel alterations were observed in 13 patients and reciprocal alterations in 11 patients. Patients showing parallel alteration had smaller goiter and were more sensitive to antithyroid drugs than those showing the latter pattern.

Negative Regulation of the Thyrotropin β Gene by Thyroid Hormone

Thyroid hormone (T3 and T4) is secreted from the thyroid gland, and is known to reduce the level of serum thyrotropin (thyroid-stimulating hormone, TSH) in the pituitary gland (Sarapura et al., 2002; Shupnik et al., 1989) (Fig. 1A). This is a typical example of negative feedback between the pituitary and endocrine organs, and is a key component of thyroid hormone homeostasis. TSH is one of the peptide hormones generated in the anterior pituitary, and is a heterodimer composed of an ┙ chain (┙-glycoprotein subunit, ┙GSU) and a ┚ chain (TSH┚) (Shupnik et al., 1989). While ┙GSU is common to follicle stimulating hormone (FSH), luteinizing hormone (LH) and chorionic gonadotropin (CG), TSH┚ is specific to TSH alone. Although the concentration of serum T4 is much higher than that of T3, T4 is converted to T3 by deiodinase (Dio) in the TSH-producing cells (thyrotrophs) of the pituitary (Christoffolete et al., 2006), and T3 exhibits biological activity as a thyroid hormone (Gereben et al., 2008). T3 inhibits expression of both TSH┚ and ┙GSU at the transcriptional level (Shupnik et al., 1989). The magnitude of T3-induced repression of the TSH┚ gene is greater than that of ┙GSU. Here, we provide an overview of the molecular mechanisms involved in T3-induced negative regulation of the TSH┚ gene and its related genes.