Masatomo Mori

Identification of nesfatin-1 as a satiety molecule in the hypothalamus.

The brain hypothalamus contains certain secreted molecules that are important in regulating feeding behaviour. Here we show that nesfatin, corresponding to NEFA/nucleobindin2 (NUCB2), a secreted protein of unknown function, is expressed in the appetite-control hypothalamic nuclei in rats. Intracerebroventricular (i.c.v.) injection of NUCB2 reduces feeding. Rat cerebrospinal fluid contains nesfatin-1, an amino-terminal fragment derived from NUCB2, and its expression is decreased in the hypothalamic paraventricular nucleus under starved conditions. I.c.v. injection of nesfatin-1 decreases food intake in a dose-dependent manner, whereas injection of an antibody neutralizing nesfatin-1 stimulates appetite. In contrast, i.c.v. injection of other possible fragments processed from NUCB2 does not promote satiety, and conversion of NUCB2 to nesfatin-1 is necessary to induce feeding suppression. Chronic i.c.v. injection of nesfatin-1 reduces body weight, whereas rats gain body weight after chronic i.c.v. injection of antisense morpholino oligonucleotide against the gene encoding NUCB2. Nesfatin-1-induced anorexia occurs in Zucker rats with a leptin receptor mutation, and an anti-nesfatin-1 antibody does not block leptin-induced anorexia. In contrast, central injection of α-melanocyte-stimulating hormone elevates NUCB2 gene expression in the paraventricular nucleus, and satiety by nesfatin-1 is abolished by an antagonist of the melanocortin-3/4 receptor. We identify nesfatin-1 as a satiety molecule that is associated with melanocortin signalling in the hypothalamus.

Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles

We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS97xS99). Surprisingly, serine 97 is not appreciably phosphorylated, whereas serine 99 is only a specific substrate for Akt2 but not Akt1 or Akt3. Although wild-type Synip (WT-Synip) undergoes an insulin-stimulated dissociation from Syntaxin4, the Synip serine 99 to phenylalanine mutant (S99F-Synip) is resistant to Akt2 phosphorylation and fails to display insulin-stimulated Syntaxin4 dissociation. Furthermore, overexpression of WT-Synip in 3T3L1 adipocytes had no effect on insulin-stimulated recruitment of glucose transporter 4 (GLUT4) to the plasma membrane, whereas overexpression of S99F-Synip functioned in a dominant-interfering manner by preventing insulin-stimulated GLUT4 recruitment and plasma membrane fusion. These data demonstrate that insulin activation of Akt2 specifically regulates the docking/fusion step of GLUT4-containing vesicles at the plasma membrane through the regulation of Synip phosphorylation and Synip–Syntaxin4 interaction.

Dominant Role of Thyrotropin-releasing Hormone in the Hypothalamic-Pituitary-Thyroid Axis

Hypothalamic thyrotropin-releasing hormone (TRH) stimulates thyroid-stimulating hormone (TSH) secretion from the anterior pituitary. TSH then initiates thyroid hormone (TH) synthesis and release from the thyroid gland. Although opposing TRH and TH inputs regulate the hypothalamic-pituitary-thyroid axis, TH negative feedback is thought to be the primary regulator. This hypothesis, however, has yet to be proven in vivo. To elucidate the relative importance of TRH and TH in regulating the hypothalamic-pituitary-thyroid axis, we have generated mice that lack either TRH, the β isoforms of TH receptors (TRβ KO), or both (double KO). TRβ knock-out (KO) mice have significantly higher TH and TSH levels compared with wild-type mice, in contrast to double KO mice, which have reduced TH and TSH levels. Unexpectedly, hypothyroid double KO mice also failed to mount a significant rise in serum TSH levels, and pituitary TSH immunostaining was markedly reduced compared with all other hypothyroid mouse genotypes. This impaired TSH response, however, was not due to a reduced number of pituitary thyrotrophs because thyrotroph cell number, as assessed by counting TSH immunopositive cells, was restored after chronic TRH treatment. Thus, TRH is absolutely required for both TSH and TH synthesis but is not necessary for thyrotroph cell development.

Cross-talk between Thyroid Hormone Receptor and Liver X Receptor Regulatory Pathways Is Revealed in a Thyroid Hormone Resistance Mouse Model

Hypercholesterolemia is found in patients with hypothyroidism and resistance to thyroid hormone. In this study, we examined cholesterol metabolism in a thyroid hormone receptor β (TR-β) mutant mouse model of resistance to thyroid hormone. Whereas studies of cholesterol metabolism have been reported in TR-β knock-out mice, generalized expression of a non-ligand binding TR-β protein in this knock-in model more fully recapitulates the hypothyroid state, because the hypothyroid effect of TRs is mediated by the unliganded receptor. In the hypothyroid state, a high cholesterol diet increased serum cholesterol levels in wild-type animals (WT) but either did not change or reduced levels in mutant (MUT) mice relative to hypothyroidism alone. 7α-Hydroxylase (CYP7A1) is the rate-limiting enzyme in cholesterol metabolism and mRNA levels were undetectable in the hypothyroid state in all animals. triiodothyronine replacement restored CYP7A1 mRNA levels in WT mice but had minimal effect in MUT mice. In contrast, a high cholesterol diet markedly induced CYP7A1 levels in MUT but not WT mice in the hypothyroid state. Elevation of CYP7A1 mRNA levels and reduced hepatic cholesterol content in MUT animals are likely because of cross-talk between TR-β and liver X receptor α (LXR-α), which both bind to a direct repeat + 4(DR+4) element in the CYP7A1 promoter. In transfection studies, WT but not MUT TR-β antagonized induction of this promoter by LXR-α. Electromobility shift analysis revealed that LXR/RXR heterodimers bound to the DR+4 element in the presence of MUT but not WT TR-β. A mechanism for cross-talk, and potential antagonism, between TR-β and LXR-α is proposed.

Chop-deficient Mice Showed Increased Adiposity But No Glucose Intolerance

Objective: CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP)‐10/growth arrest and DNA damage 153 is a dominant‐negative member of the C/EBP transcription family and inhibits adipogenesis in vitro. The study was undertaken to determine the role of CHOP in obesity in vivo.

Inhibition of appetite by nasal leptin administration in rats

OBJECTIVE:Leptin inhibits appetite and reduces body weight. However, subcutaneous leptin administration is not very effective on weight reduction. The present studies were undertaken to test the hypotheses that nasally administered leptin effectively accesses to the brain and inhibits appetite.METHODS:Recombinant leptin (0.5 mg/rat) was administered into the bilateral nasal spaces of rats (i.n.). Changes in serum immunoreactive leptin (IRL) and cerebrospinal fluid (CSF)-IRL concentrations after i.n. leptin administration were compared after intraperitoneal (i.p.) administration. The influence of 0.1 or 0.5% lysophosphatidylcholine (LPC) as an optimizer of leptin absorption was examined. The anorexic effects of i.n. leptin were compared with i.p. leptin in ad libitum fed rats.RESULTS:The i.n. leptin increased CSF-IRL concentrations, although serum IRL concentrations of rats administered leptin i.n. were lower than those administered i.p. The addition of 0.1 and 0.5% LPC dose-dependently increased serum IRL concentrations, but did not modify CSF-IRL concentrations in i.n. leptin-treated rats. The i.n. leptin inhibited dark-time food consumption at 0–1 h and 3–6 h in ad libitum fed rats. In contrast, i.p. leptin reduced food consumption only for an hour. Phosphorylated signal transducer and activator of transcription (STAT) 3 immunoreactive cells increased in the arcuate nucleus (ARC) of the hypothalamus at 3 h only following i.n. leptin.CONCLUSION:The present study demonstrated that i.n. leptin caused longer inhibition of appetite and phosphorylation of STAT3 in ARC. It is concluded that the trans-nasal route may be useful for the selective access of leptin to the brain in obese people.

Troglitazone inhibits isolated cell proliferation, and induces apoptosis in isolated rat mesangial cells

Background/Aims: Troglitazone is one of thiazolidinedione derivatives as a high affinity ligand for peroxisome proliferator-activated receptor-gamma (PPAR-γ). The in vivo studies demonstrated that troglitazone ameliorated microalbuminuria. There have been few reports about direct effect of thiazolidinedione derivatives on mesangial cell function. We determined the effect of troglitazone on isolated rat mesangial cell proliferation. Methods: We determined PPAR-γ mRNA expression in isolated rat mesangial cells. Chronic effects of 10–6 to 10–4 mol/l troglitazone on mesangial cell proliferation and mitogen-activated protein (MAP) kinase activity were also determined. The effects of troglitazone on apoptosis were investigated in rat mesangial cells. Results: Rat PPAR-γ mRNA was detected in isolated rat mesangial cells. Living cell number, assessed by colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay, was significantly decreased with 10–4 mol/l troglitazone. The addition of 10–6 to 10–4 mol/l troglitazone dose-dependently inhibited 5-bromo-2′-deoxyuridine (BrdU) uptake into isolated rat mesangial cells. The addition of 10–4 and 10–5 mol/l troglitazone significantly reduced MAP kinase activity. Troglitazone at the concentrations of 10–6 to 10–4 mol/l dose-dependently increased DNA fragmentation rates, indicating that troglitazone may cause apoptosis in rat mesangial cells. Bax and Bcl-xL proteins were not changed, although Bcl-2 proteins increased with troglitazone. Conclusions: The present data demonstrated that troglitazone inhibits cell proliferation, and induces apoptosis in rat mesangial cells, raising a possibility that it directly affects renal function.

Relationship between hyperinsulinemia and pulse wave velocity in moderately hyperglycemic patients

AIM Arterial stiffness assessed by pulse wave velocity (PWV) reflects early stage arteriosclerosis. The influence of hyperinsulinemia on peripheral vascular disease (PVD) is still unknown. We determined the influences of hyperinsulinemia on PVD assessed by PWV in moderately hyperglycemic patients. METHODS Thirty-six moderately hyperglycemic, outcoming patients were recruited in this study. All subjects were divided into two groups by fasting immunoreactive insulin (F-IRI) concentrations; group A; F-IRI> or =5 microU/ml, group B; F-IRI<5 microU/ml. Both hbPWV (from heart to brachial artery) and baPWV (brachial to artery to ankle) were evaluated by using Form PWV/ABI, in addition to ankle-brachial pressure index (ABPI). RESULTS In group A, both hbPWV and baPWV showed significantly higher values than in group B. ABPIs were not different between two groups. Although age, FPG, plasma HbA1c, serum total-cholesterol, HDL-cholesterol concentrations, and systolic and diastolic blood pressure were at same levels in group A as group B, body mass index, HOMA-R, serum triglyceride concentrations were significantly higher in group A, indicating the existence of insulin resistance in group A. CONCLUSION Hyperinsulinemia may be involved in the development of PVD in moderately hyperglycemic patients.

Thyrotropin-releasing Hormone (TRH) specific interaction between amino terminus of P-Lim and CREB binding protein (CBP)

P-Lim (Lhx3a) is a LIM homeodomain transcription factor essential for pituitary development and motor neuron specification in mice. The Lhx3 gene encodes two isoforms, which differ in their amino (N) termini, Lhx3a and 3b. The P-Lim DNA binding site on the glycoprotein hormone alpha subunit (alpha-GSU) gene promoter is conserved in mammals. P-Lim plays a pivotal role in mediating thyrotropin-releasing hormone (TRH) signaling by binding CREB binding protein (CBP), as we have reported previously. Here, we demonstrate that P-Lim (Lhx3a) but not Lhx3b can mediate TRH signaling and bind to CBP. Moreover, TRH specifically induces P-Lim-CBP binding through the N-termini of P-Lim. We also found that the protein kinase C (PKC) phosphorylation site within the N-terminus of P-Lim is responsible for the P-Lim-CBP binding suggesting that the TRH signaling pathway phosphorylates P-Lim. These studies have elucidated the molecular mechanism by which TRH stimulates alpha-GSU gene expression.

Allelic losses on chromosome 3p are accumulated in relation to morphological changes of lung adenocarcinoma

We performed allelotyping analysis at nine regions on chromosome 3p using 56 microdissected samples from 23 primary lung adenocarcinomas to examine the process of progression within individual lung adenocarcinoma with various grades of differentiation. Identical allelic patterns among various grades of differentiation were found in eight cases. Accumulation of allelic losses from high to lower differentiated portions was found in seven cases and accumulation of allelic losses from low to higher differentiated portions was found in five cases. Various allelic patterns among various grades of differentiation were found in three cases. These results suggested that allelic losses on 3p play an important role in morphological changes of lung adenocarcinomas. We also investigated the relationship between allelic losses on 3p and histological subtypes of lung adenocarcinoma. The frequencies of allelic losses at 3p14.2 and telomeric region of 3p21.3 were higher in papillary type tumour (nine out of 14, 64% and 11 out of 15, 73%) than in bronchioloalveolar carcinoma-type tumour (one out of 8, 13%; P=0.031 and four out of 12, 33%; P = 0.057). These results indicated that allelic losses at 3p14.2 and telomeric region of 3p21.3 are related to pattern of the proliferation of lung adenocarcinoma.