Thermal Effect on Heat Shock Protein 70 Family to Prevent Atherosclerotic Cardiovascular Disease.

Heat shock protein 70 (HSP70) is a chaperone protein induced by various stresses on cells and is involved in various disease mechanisms. In recent years, the expression of HSP70 in skeletal muscle has attracted attention for its use as a prevention of atherosclerotic cardiovascular disease (ASCVD) and as a disease marker. We have previously reported the effect of thermal stimulation targeted to skeletal muscles and skeletal muscle-derived cells. In this article, we reported review articles including our research results. HSP70 contributes to the improvement of insulin resistance as well as chronic inflammation which are underlying pathologies of type 2 diabetes, obesity, and atherosclerosis. Thus, induction of HSP70 expression by external stimulation such as heat and exercise may be useful for ASCVD prevention. It may be possible to induce HSP70 by thermal stimulus in those who have difficulty in exercise because of obesity or locomotive syndrome. It requires further investigation to determine whether monitoring serum HSP70 concentration is useful for ASCVD prevention.

Branched-chain amino acids protect against dexamethasone-induced soleus muscle atrophy in rats.

We investigated the utility of branched-chain amino acids (BCAA) in dexamethasone-induced muscle atrophy. Dexamethasone (600 microg/kg, intraperitoneally) and/or BCAA (600 mg/kg, orally) were administered for 5 days in rats, and the effect of BCAA on dexamethasone-induced muscle atrophy was evaluated. Dexamethasone decreased total protein concentration of rat soleus muscles. Concomitant administration of BCAA reversed the decrease. Dexamethasone decreased mean cross-sectional area of soleus muscle fibers, which was reversed by BCAA. Dexamethasone increased atrogin-1 expression, which has been reported to play a pivotal role in muscle atrophy. The increased expression of atrogin-1 mRNA was significantly attenuated by BCAA. Furthermore, dexamethasone-induced conversion from microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II, which is an indicator of autophagy, was blocked by BCAA. These findings suggest that BCAA decreased protein breakdown to prevent muscle atrophy. BCAA administration appears to be useful for prevention of steroid myopathy.

Development of disaster pamphlets based on health needs of patients with chronic illnesses.

The aim of this research was to develop a pamphlet that would enable patients with diabetes, rheumatic diseases, chronic respiratory disease, and dialysis treatment to be aware of changes in their physical conditions at an early stage of a disaster, cope with these changes, maintain self-care measures, and recover their health. Illness-specific pamphlets were produced based on disaster-related literature, news articles, surveys of victims of the Great Hanshin-Awaji Earthquake Disaster and Typhoon Tokage, and other sources. Each pamphlet consisted of seven sections-each section includes items common to all illnesses as well as items specific to each illness. The first section, "Physical Self-Care", contains a checklist of 18 common physical symptoms as well as symptoms specific to each illness, and goes on to explain what the symptoms may indicate and what should be done about them. The main aim of the "Changes in Mental Health Conditions" section is to detect posttraumatic stress disorder (PTSD) at an early stage. The section "Preventing the Deterioration of Chronic Illnesses" is designed to prevent the worsening of each illness through the provision of information on cold prevention, adjustment to the living environment, and ways of coping with stress. In the sections, "Medication Control" and "Importance of Having Medical Examinations", spaces are provided to list medications currently being used and details of the hospital address, in order to ensure the continued use of medications. The section, "Preparing for Evacuations" gives a list of everyday items and medical items needed to be prepared for a disaster. Finally, the "Methods of Contact in an Emergency" section provides details of how to use the voicemail service. The following content-specific to each illness also was explained in detail: (1) for diabetes, complications arising from the deterioration of the illness, attention to nutrition, and insulin management; (2) for rheumatic diseases, a checklist of factors indicating the worsening of the illness and methods of coping with stress; (3) for chronic respiratory disease, prevention of respiratory infections and management of supplemental oxygen; and (4) for patients requiring dialysis, conditions of dialysis (such as dry weight, dialyzer, number of dialysis treatments, and dialysis hours) and what to do if a disaster occurs during dialysis. It is expected that these pamphlets will be useful to patients with chronic illnesses, and will be used to prepare for disasters, thereby helping the patients cope with the unusual situation that during a disaster and recover as soon as possible.

Branched-chain amino acids and arginine suppress MaFbx/atrogin-1 mRNA expression via mTOR pathway in C2C12 cell line.

The effect of amino acid on muscle protein degradation remains unclear. Recent studies have elucidated that proteolysis in catabolic conditions occurs through ubiquitin-proteasome proteolysis pathway and that muscle-specific ubiquitin ligases (atrogin-1 and MuRF1) play an important role in protein degradation. In the present study, we examined the direct effect of 5 mM amino acids (leucine, isoleucine, valine, glutamine and arginine) on atrogin-1 and MuRF1 levels in C2C12 muscle cells and the involved intracellular signal transduction pathway. Leucine, isoleucine and valine suppressed atrogin-1 and MuRF1 mRNA levels (approximately equal to 50%) at 6 and 24 h stimulations. Arginine showed a similar effect except at 24 h-treatment for atrogin-1 mRNA. However, glutamine failed to reduce atrogin-1 and MuRF1 mRNA levels. The inhibitory effect of leucine, isoleucine or arginine on atrogin-1 mRNA level was reversed by rapamycin, although wortmannin did not reverse the effect. PD98059 and HA89 reduced basal atrogin-1 level without influencing the inhibitory effects of those amino acids. The inhibitory effect of leucine, isoleucine or arginine on MuRF1 mRNA levels was not reversed by rapamycin. Taken together, these findings indicated that leucine, isoleucine and arginine decreased atrogin-1 mRNA levels via mTOR and that different pathways were involved in the effect of those amino acids on MuRF1 mRNA levels.

Difference between Japanese and Caucasian populations in the allelic frequency of growth hormone receptor polymorphisms.

BACKGROUND: Polymorphisms in the growth hormone receptor (GHR) gene were reported in Caucasian populations. However, the frequency of those in other ethnic backgrounds remains unclear. AIM: We investigated the presence of polymorphisms in the GHR gene in a Japanese population and compared the frequencies with those reported in Caucasian populations. POPULATION: We selected 30 children with idiopathic short stature and 30 adult Japanese of normal height. METHODS: The sequences of exons 6 and 10 in the GHR gene were determined by direct sequencing by polymerase chain reaction (PCR). The genomic deletion of exon 3 (GHR-d3) was investigated by multiplex PCR. RESULTS: The frequency of the GGG genotype at codon 168 was significantly higher than that reported in Caucasian populations. The frequency of GHR-d3 in Japanese was significantly lower than that in Caucasian populations. CONCLUSIONS: The frequencies of the G168G polymorphism and GHR-d3 in Japanese are different from those in Caucasians.

Identification and analysis of prophet of Pit-1-binding sites in human Pit-1 gene.

Prophet of Pit-1 (Prop1) is a transcription factor that regulates Pit-1 gene expression. Because Pit-1 regulates the differentiation of pituitary cells and the expressions of GH, prolactin and TSHbeta genes, Prop1 mutation results in combined pituitary hormone deficiency in humans. However, Prop1-binding sites in human Pit-1 gene and the mechanism leading to combined pituitary hormone deficiency have remained unclear. In this study, we identified and analyzed Prop1-binding elements of the human Pit-1 gene. Prop1 stimulated the expression of the reporter plasmid containing Pit-1 gene from translation start site to -1340 dose dependently in GH3 cells. The activation by Prop1 was observed in GH3 and TtT/GF cells but not COS7, HeLa, JEG3, and HuH7 cells. Deletion analysis of Pit-1 gene showed that the Prop1-responsive elements were present within the -257-bp region. Within the -257-bp region, there are four elements similar to consensus sequence of paired-like transcription factors. Because Prop1 is a member of paired-like transcription factors, we assessed the elements. EMSA and transient transfection assay using the mutation of the elements revealed that the element from -63 to -53 (the proximal Prop1 binding element) was essential to Prop1-binding and Prop1-induced activation of Pit-1 reporter plasmid. A region at -8kb of human Pit-1 gene is similar to the distal region containing Prop1-binding elements in mouse Pit-1 gene. We showed the region functioned as an enhancer. Furthermore, chromatin immunoprecipitation assay showed that the proximal element could bind Prop1 in vivo cultured cells. Taken together, these findings indicated the novel functioning binding elements of Prop1 in human Pit-1 gene.

Chemerin enhances insulin signaling and potentiates insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

To explore a novel adipokine, we screened adipocyte differentiation-related gene and found that TIG2/chemerin was strongly induced during the adipocyte differentiation. Chemerin was secreted by the mature 3T3-L1 adipocytes and expressed abundantly in adipose tissue in vivo as recently described. Intriguingly, the expression of chemerin was differently regulated in the liver and adipose tissue in db/db mice. In addition, serum chemerin concentration was decreased in db/db mice. Chemerin and its receptor/ChemR23 were expressed in mature adipocytes, suggesting its function in autocrine/paracrine fashion. Finally, chemerin potentiated insulin-stimulated glucose uptake concomitant with enhanced insulin signaling in the 3T3-L1 adipocytes. These data establish that chemerin is a novel adipokine that regulates adipocyte function.

Involvement of mPOU (Brn-5), a class VI POU protein, in the gene expression of Pit-1 as well as PRL.

PRL is mainly expressed in the pituitary and its gene expression is regulated by a variety of transcription factors including Pit-1. Brn-5 is a transcription factor that binds to Pit-1 binding elements and stimulates PRL reporter gene expression. In this study, the role of Brn-5 was examined. RNA interference (RNAi) against Brn-5 leaded to reduction in PRL content of GH3 cells, indicating endogenous Brn-5 may play a role in PRL gene expression. Furthermore Brn-5 RNAi decreased Pit-1 mRNA. Transfection of expression vectors for mPOU (human ortholog of Brn-5) modestly but significantly stimulated activities of PRL-Luc and Pit-1-Luc reporter genes in GH3 and HEK 293 cells. In addition, mPOU showed synergistic action with Pit-1 and CBP on PRL-Luc expression. mPOU-FL, a splicing variant of mPOU, showed weaker activity than mPOU. Chip assay suggested binding of mPOU to PRL and Pit-1 promoters of genomic DNA. Taken together, these results suggest that mPOU (Brn-5) enhances PRL gene expression directly in association with Pit-1 and CBP, and indirectly via the activation of Pit-1 gene expression.

GHRP-2, a GHS-R agonist, directly acts on myocytes to attenuate the dexamethasone-induced expressions of muscle-specific ubiquitin ligases, Atrogin-1 and MuRF1.

Recent reports suggest that Atrogin-1 and MuRF1, E3 ubiquitin ligases, play a pivotal role in muscle atrophy. In the present study, effect of Growth Hormone Releasing Peptide-2 (GHRP-2), a GH secretagogue receptor (GHS-R) agonist, on the expressions of Atrogin-1 and MuRF1 in vivo rat muscles was examined. Dexamethasone administration increased Atrogin-1 mRNA level in rat soleus muscle. The increased mRNA level of Atrogin-1 was significantly attenuated by GHRP-2. In addition, GHRP-2 decreased MuRF1 mRNA level irrespective of the presence of dexamethasone. Although IGF-I is a well-known protective factor for muscle atrophy, GHRP-2 did not influence plasma IGF-I levels and IGF-I mRNA levels in muscles. To clarify a direct effect of GHRP-2, differentiated C2C12 myocytes were used. Ten micrometer dexamethasone increased both Atrogin-1 and MuRF1 mRNA levels in C2C12 cells. GHRP-2 attenuated dexamethasone-induced expression of them dose-dependently and decreased the basal level of MuRF1 mRNA. The suppressive effect on the expressions of Atrogin-1 and MuRF1 by GHRP-2 was blocked by [D-Lys(3)]-GHRP-6, a GHS-R1a blocker, suggesting the effect of GHRP-2 was mediated through GHS-R1a. Taken together, GHRP-2 directly attenuates Atrogin-1 and MuRF1 mRNA levels through ghrelin receptors in myocytes.

Low serum IGF-I/GH ratio is associated with abnormal glucose tolerance in acromegaly.

BACKGROUND/AIMS: Acromegaly is frequently accompanied with impaired glucose tolerance (IGT) and diabetes mellitus (DM). It remains unclear which factors determine the abnormal glucose tolerance status in acromegaly. In addition, diverse actions of GH and IGF-I in regulating glucose metabolism in acromegaly have not yet been well elucidated. The aim of this study was to investigate the factors associated with abnormal glucose tolerance in acromegaly. SUBJECTS AND STUDY DESIGN: We conducted a retrospective cross-sectional study that included 48 patients with active acromegaly. The subjects were divided into two groups by the results of 75 g OGTT: normal glucose tolerance (NGT) group (n = 19) and IGT+DM group (n = 29). RESULTS: Systolic blood pressure (SBP) was significantly higher in the IGT+DM than in the NGT group. Homeostasis model assessment of beta-cell function (HOMA-beta) was significantly decreased in the IGT+DM group compared with the NGT group. Although serum GH or IGF-I levels were not different between the two groups, the IGF-I/GH ratio in the IGT+DM group was significantly lower than that in the NGT group. CONCLUSIONS: We have shown that a low serum IGF-I/GH ratio was associated with abnormal glucose tolerance in acromegaly. We propose that the IGF-I/GH ratio is a useful marker to understand the metabolic status in acromegaly.

Hepatic failure and enhanced oxidative stress in mitochondrial diabetes.

Mitochondrial diabetes is characterized by diabetes and hearing loss in maternal transmission with a heteroplasmic A3243G mutation in the mitochondrial gene. In patients with the mutation, it has been reported that hepatic involvement is rarely observed. We demonstrated a case of hypertrophic cardiomyopathy and hepatic failure with mitochondrial diabetes. To clarify the pathogenesis we analyzed the mitochondrial ultrastructure in the myocytes, the reactive oxygen species (ROS) production in the liver and the status of heteroplasmy of the mitochondrial A3243G mutation in the organs involved. In cardiomyocytes and skeletal muscle, electron microscopic analysis demonstrated typical morphological mitochondrial abnormalities. Immunohistochemical analysis demonstrated enhanced ROS production associated with marked steatosis in the liver, which is often associated with mitochondrial dysfunction. Analysis of the A3243G mutation revealed a substantial ratio of heteroplasmy in these organs including the liver. The presence of steatosis and enhanced oxidative stress in the liver suggested that hepatic failure was associated with mitochondrial dysfunction.

CXCL14 enhances insulin-dependent glucose uptake in adipocytes and is related to high-fat diet-induced obesity.

Accumulating evidence suggests an association between obesity and adipose tissue inflammation. Chemokines are involved in the regulation of inflammation status. Chemokine (C-X-C motif) ligand 14 (CXCL14) is known to be a chemoattractant for monocyte and dendritic cells. Recently, it was reported that CXCL14-deficient mice show resistance to high-fat diet-induced obesity. In this study, we identified CXCL14 as a growth hormone (GH)-induced gene in HepG2 hepatoma cells. Substantial in vivo expression of CXCL14 was detected in the adipose tissue and liver. Its expression and secretion were strikingly increased by insulin administration and high-fat diet. Intriguingly, incubation of 3T3-L1 adipocytes with CXCL14 stimulated insulin-dependent glucose uptake. Further, this effect was associated with enhanced insulin signaling. CXCL14 enhanced the insulin-induced tyrosine phosphorylation of insulin receptors and insulin receptor substrate-1. These results suggest that CXCL14 plays a causal role in high-fat diet-induced obesity.

Neither intravenous nor intracerebroventricular administration of obestatin affects the secretion of GH, PRL, TSH and ACTH in rats.

To examine the effect of obestatin, a recently identified peptide derived from preproghrelin, on pituitary hormone secretion, obestatin was administered in anesthetized male rats. Intravenous administration of obestatin did not show any effect on plasma GH, PRL, ACTH and TSH levels. Since obestatin has been reported to have opposite effects of ghrelin in regulating food intake, gastric emptying and intestinal contractility, GH suppressive effect, which is opposite effect of ghrelin, was tested. Intravenous administration of GHRH or GHRP-2, a ghrelin receptor ligand, resulted in a marked plasma GH elevation. However obestatin did not show any effect on GHRH- or GHRP-2-induced GH rise. Furthermore intracerebroventricular administration of obestatin also did not influence plasma GH, PRL, ACTH and TSH levels. These findings suggest that obestatin has no effect on pituitary hormone secretions despite the presence of GPR39, a receptor for obestatin, in the pituitary.

Diverse regulation of full-length and truncated growth hormone receptor expression in 3T3-L1 adipocytes.

Two truncated forms of growth hormone (GH) receptor (GHR), 1-277 and 1-279, were reported to be normally produced in human tissues by alternative splicing in exon 9 and its boundary. We found previously that GHR-277 exerts a dominant-negative effect on full-length GHR (GHR-fl)-mediated GH signaling causing short stature. The existence of truncated GHRs (hGHR-tr) in normal tissues suggests that hGHR-tr may play a physiological role in regulation of GH action at the cellular level. To clarify the physiological significance of GHR-tr and the regulation mechanism of GHR-tr expression, we examined the expression of mouse GHR-tr (mGHR-tr) mRNA in mouse adipocyte 3T3-L1 cells, comparing with that of mouse GHR-fl (mGHR-fl). The mRNAs of two mGHR-tr, mGHR-282 and mGHR-280, were detected by RT-PCR methods using specific primers. Although the mGHR-282 and mGHR-280 mRNA levels were approximately 100 times lower than that of mGHR-fl in mature 3T3-L1 cells, quantitative analysis by competitive RT-PCR methods revealed that the mRNA levels of mGHR-280 in 3T3-L1 cells were transiently reduced and thereafter increased during differentiation from preadipocyte to adipocyte. In contrast, the mRNA levels of mGHR-fl were increased in parallel with the progress of differentiation. Stimulation by GH of differentiated 3T3-L1 mature adipocytes resulted in dose-dependent increases of the mRNA of both mGHR-fl and mGHR-282, whereas it caused a paradoxical decrease of the mRNA of mGHR-280 stimulated by high concentration of GH. These findings suggest that the expressions of truncated mGHRs were regulated in a different manner from that of mGHR-fl, thereby modulating GH action in murine adipocytes.

Cloning of a protein binding to the most proximal Pit-1 binding element of prolactin gene from human pituitary cDNA library.

A human pituitary cDNA library was screened using a yeast one-hybrid system to find a factor binding Pit-1 binding elements in the PRL gene other than Pit-1. Beside colonies containing Pit-1 or Oct-1 cDNA, three colonies contained mPOU cDNA, a member of the POU protein family. Immunohistochemical analysis showed mPOU-like immunoreactivity was present in human PRL-producing pituitary tumors but not in non-functioning pituitary tumors. Mobility shift analysis revealed that mPOU bound to Pit-1 binding elements of the PRL gene, 1P and 3P. mPOU activated the expression of 0.6 k PRL and 7x1P reporter genes in the presence of Pit-1 and cAMP, although it did not enhance Pit-1-induced expression of 7x3P reporter gene. These findings suggest that mPOU is involved in the activation of the PRL gene by cAMP through 1P in the presence of Pit-1.

Direct causes of death in Japanese patients with hypopituitarism as analyzed from a nation-wide autopsy database.

OBJECTIVES: The direct causes of death in Japanese patients with hypopituitarism remain unclear. In this study, the direct causes of death were investigated and compared between Japanese patients with hypopituitarism from a nation-wide autopsy database and an age- and gender-matched control population from national reports. SUBJECTS: Three hundred and ninety-one subjects with hypopituitarism who had died were selected from a nation-wide autopsy database (1984-1993). The ratios of each cause of death among the age- and gender-matched control population were derived from national reports. RESULTS: In subjects with hypopituitarism, an increased relative frequency of death from cerebrovascular diseases (male; 2.02 (95% confidence interval (CI) 1.45-2.82), female; 1.73 (95% CI 1.18-2.52)) was found. In particular, the relative frequency of death from cerebral hemorrhage was 4.60 (95% CI 2.95-7.17) in male and 4.80 (95% CI 2.90-7.94) in female subjects with hypopituitarism. Unexpectedly, a decreased relative frequency of death from all heart diseases (male; 0.439 (95% CI 0.277-0.696), female; 0.267 (95% CI 0.149-0.478)) was found in subjects with hypopituitarism, although there was no difference between subjects with hypopituitarism and controls in the frequency of death from ischemic heart disease. CONCLUSIONS: These results provide useful information for the long-term care of Japanese patients with hypopituitarism.

A study of carotid intima-media thickness in GH-deficient Japanese adults during onset among adults and children.

OBJECTIVES: Increased carotid intima-media thickness (IMT) has been reported among Caucasian adult GH-deficient (AGHD) patients, but not Japanese. Also, it is known that the clinical and biochemical characteristics of AGHD patients are somewhat different based on the onset of the disease in either childhood or adult life. Nevertheless, there has been no study comparing the magnitude of the deviation of their IMT from normal subjects between child-onset (CO) and adult-onset (AO) patients in terms of Z score. The aim of this study, therefore, was first to examine whether Japanese AGHD patients have a risk of early development of atherosclerosis similar to Caucasian patients and secondly to assess the difference in the onset and in progression of atherosclerosis. DESIGN AND SUBJECTS: Thirty-four patients (17 CO-AGHD, age 29+/-7 Years, body mass index (BMI) 24+/-3.8 kg/m(2) and 17 AO-AGHD, age 48+/-12 Years, BMI 23+/-3.6 kg/m(2)) and 34 age- and sex-matched healthy controls (17 CO controls and 17 AO controls) were enrolled in the present study. Blood samples were taken for measurements of lipids, lipoproteins and IGF-I. Subsequently, patients underwent IMT assessment. RESULTS: CO patients were significantly younger than AO patients. The duration of GH-deficiency in CO patients was significantly longer than that in AO patients. Serum triglyceride (TG) was significantly higher in CO patients than in CO controls (P<0.05). Serum total cholesterol and TG were significantly higher in AO patients than in AO controls (P<0.01). The IMT was significantly greater in CO and AO patients (0.82+/-0.08 and 0.79+/-0.03 mm) than in CO and AO controls (0.59+/-0.02 and 0.68+/-0.03 mm, P<0.01 and P<0.01 respectively). There was no significant difference in raw values of IMT between CO and AO patients. However, the Z score of IMT calculated using normal Japanese IMT values was significantly higher in CO than in AO patients (2.07+/-0.68 vs 0.35+/-0.48, P<0.05). CONCLUSIONS: These findings suggest that GH deficiency appears to increase an atherosclerotic risk in Japanese AGHD patients, as with Caucasians, and to cause more extensive IMT thickening in CO-AGHD than AO-AGHD patients.

The R271W mutant form of Pit-1 does not act as a dominant inhibitor of Pit-1 action to activate the promoters of GH and prolactin genes.

OBJECTIVE: Genetic abnormalities of the pituitary specific transcription factor, Pit-1, have been reported in several patients with GH, prolactin (PRL) and TSH deficiencies. The most common is a mutation altering an arginine to a tryptophan in codon 271 (R271W) in one allele of the Pit-1 gene. According to the previous in vitro expression study, R271W acted as a dominant negative inhibitor of the wild type to activate the GH promoter. However, healthy carriers with this mutation, who should be affected by the dominant negative effect of R271W, have also been reported. The aim of this study was to clarify in more detail the function of this mutant form of Pit-1. METHODS: Transcriptional activity of R271W for the expression of Pit-1-associated genes was investigated in COS7 cells with the aid of transient transfection assays. The 1.8 kb rat GH, 0.6 kb rat PRL or 1.9 kb rat PRL 5'-flanking regions were inserted upstream of the luciferase reporter gene and were used for functional analysis of R271W. Another reporter gene containing seven Pit-1 responsive elements was also used. The same experiments were also performed using JEG3 and CHO cells. RESULTS: We could not confirm the dominant negative effect of R271W on wild type Pit-1. Furthermore, our expression study revealed that R271W could activate the promoters of GH and PRL genes to levels similar to the wild type. CONCLUSION: Taken together with the evidence that phenotypically normal cases have been reported with this mutation, our results deny the relationship between R271W and combined pituitary hormone deficiency.

Intravenous administration of ghrelin stimulates growth hormone secretion in vagotomized patients as well as normal subjects.

OBJECTIVE: Ghrelin is a potent peptide stimulating GH secretion. Besides its direct action on the pituitary, ghrelin has been reported to stimulate GH release via the vagal afferent nerve in rats. To examine the involvement of vagal nerve in ghrelin-induced GH secretion in humans, GH responses to ghrelin were compared between vagotomized patients with gastrectomy and normal subjects. METHODS: Ghrelin (0.2 microg/kg) or GHRH (1 microg/kg) was administered intravenously in vagotomized patients and normal subjects on separate days, and plasma GH responses to the stimuli were examined. RESULTS: Ghrelin caused a significant plasma GH rise in both vagotomized patients and normal subjects. Peak GH levels in vagotomized patients (37.5+/-16.9 ng/ml) were not different from those in normal subjects (29.9+/-23.1 ng/ml). The areas under the curve of GH response to ghrelin did not differ between the two groups. GHRH also increased GH levels, and peak GH levels and areas under the curve after GHRH stimulation were also comparable between vagotomized patients and normal subjects. CONCLUSIONS: In the present study, the involvement of the afferent vagal nerve in ghrelin-induced GH secretion was not confirmed in humans.

A novel heterozygous T51I mutation of growth hormone receptor is not associated with short stature.

A novel missense mutation of the growth hormone receptor (GHR) gene was identified in a Japanese short boy with GH insensitivity. The analysis of the GHR gene revealed a novel heterozygous T51I mutation in exon 4. To clarify the effect of this mutation on GH signaling, the mutant GHR was expressed in CHO cells and its functional properties were investigated. The signal transducer and activator of transcription 5 (STAT5)-mediated transcriptional activation in mutant GHR (GHR-T51I)-expressing cells was significantly reduced as compared with wild-type GHR (GHR-wt)-expressing cells. The CHO cells co-expressing the GHR-T51I and GHR-wt, however, revealed no functional differences as compared with cells expressing GHR-wt alone, indicating that the T51I mutation does not exert the dominant negative effect. In addition, the mother and the elder brother of the proband, whose heights were normal, possessed the same heterozygous mutation. These results suggest that the heterozygous T51I mutation of GHR does not inhibit the signal transduction of GH and is not responsible for GH insensitivity.