Metabolic features of rats resistant to a high-fat diet.

OBJECTIVE: We assessed the metabolic characteristics of high-fat-diet-resistant (DR) rats. METHODS: Body weight, energy intake, locomotor activity, oxygen consumption, plasma leptin and lipid levels, size of visceral-fat adipocytes, and mRNA levels of genes related to lipid metabolism were measured in control rats fed standard chow and in obesity-prone (high-fat-diet-induced obesity, DIO) and DR rats fed a high-fat diet. Glucose tolerance and insulin tolerance tests were also performed. RESULTS: DIO rats gained weight more rapidly than did DR and control rats; DR rats gained less weight than did DIO rats despite similar energy intake. Energy expenditure did not differ among the three groups. The diameter of visceral-fat adipocytes was similar in DR and control rats. mRNA levels of genes involved in lipogenesis, such as fatty acid synthase and acetyl-CoA carboxylase, tended to be lower in DR than in control and DIO rats, whereas those of carnitine palmitoyltransferase 1a, which is involved in fatty acid ß-oxidation, were greater in DR rats than in the other groups. DIO rats showed hyperinsulinemia and glucose intolerance, whereas DR rats had high sensitivity to insulin. CONCLUSION: DR rats show suppression of lipogenesis and acceleration of fatty acid ß-oxidation in the visceral fat. These characteristics likely contribute to the anti-obesity phenotype of DR rats.

Restricted expression of chromatin remodeling associated factor Chd3 during tooth root development.

BACKGROUND AND OBJECTIVE: The tooth root is one of the critical parts to maintain tooth function; however, the molecular mechanisms of root development remain unknown. We aimed to identify specific factors for root morphogenesis using a newly developed experimental system. MATERIAL AND METHODS: Tentative cementoblasts and periodontal ligament cells from mouse mandibular molars were isolated using laser capture microdissection. More than 500 cementoblasts and periodontal ligament cells were separately captured. After RNA extraction and amplification, mRNA expression in isolated cementoblasts was compared with that of periodontal ligament cells by cDNA microarray analysis. Then, putative cementoblast-specific genes were subjected to in situ hybridization analysis to confirm the results in mouse mandible. RESULTS: Approximately 2000 genes were differentially expressed between these tissues. Among those genes, zinc finger helicase (ZFH), also termed chromodomain-helicase-DNA-binding protein 3 (Chd3), was one of the highly expressed transcripts in tentative cementoblasts. In situ hybridization revealed that ZFH/Chd3 was strongly expressed in Hertwig's epithelial root sheath rather than in cementum. Moreover, its expression disappeared when root formation was advanced in the first molar. In contrast, Chd3 was continuously expressed in dental epithelial cells of the cervical loop, in which root extension is never terminated. CONCLUSION: These results suggest that ZFH/Chd3 might play an important role in tooth root development and subsequent cementogenesis.

Sex differences in energy metabolism in pre-pubescent, early pubescent and adult rats.

OBJECTIVE: This study was intended to investigate sex differences in response to a high fat (HF) diet at three stages, pre-puberty, early puberty, and adulthood. METHODS: Body weight, energy intake, glucose, insulin, and leptin concentrations were measured in male and female rats that were fed either a HF or a control chow during each stage of development. The sex hormones of adult rats were also examined. In addition, metabolic factors of male rats pair-fed with females were evaluated. RESULTS: At pre-puberty, the average body weight of pups born to a HF dam exceeded that of the control, whereas there were no significant differences in body weight between males and females. During early puberty and among 15-wk-old rats, males exhibited greater weight gain with higher energy intake than did females. During all three stages, HF rats exhibited significant increases in body weight, insulin and leptin concentrations. Estradiol levels of females were higher than those of males, and those of the HF groups were significantly lower than the control groups. Although the body weight gain in male rats pair-fed with females exceeded that of the females, the insulin and leptin levels of pair-fed HF males decreased to the control levels. CONCLUSION: HF male rats became obese earlier than HF females. This result may be the result of differences in estradiol levels between males and females. The decline of insulin and leptin levels in pair-fed male groups indicates that caloric restriction among male rats could reduce the incidence of metabolic diseases.

Microbial community of anammox bacteria immobilized in polyethylene glycol gel carrier.

Anaerobic ammonium oxidation (anammox) is a recently discovered microbial pathway in the biological nitrogen cycle and a new cost-effective way to remove ammonium from wastewater. We have so far developed new immobilization technique that anammox bacteria entrapped in polyethylene glycol (PEG) gel carrier. However, fate and behavior of anammox bacteria in a gel carrier is not well understood. In the present study, we focused on the population changes of anammox bacteria in a gel carrier. Three specific primer sets were designed for real-time PCR. For quantification of anammox bacteria in a gel carrier, real-time PCR was performed. The anammox bacteria related to HPT-WU-N03 clone were increased the rate in anammox population, and found to be a major population of anammox bacteria in a gel carrier. Furthermore, from the results of nitrogen removal performance and quantification of anammox bacteria, the correlation coefficient between copy numbers of anammox bacteria and nitrogen conversion rate was calculated as 0.947 in total anammox population. This is the first report that population changes of anammox bacteria immobilized in a gel carrier were evaluated.

Galanin-like peptide promotes feeding behaviour via activation of orexinergic neurones in the rat lateral hypothalamus.

Galanin-like peptide (GALP) is produced in neurones in the hypothalamic arcuate nucleus and is implicated in the neural control of feeding behaviour. Previously, we have reported that GALP immunoreactive fibres were in direct contact with orexin/hypocretin immunoreactive neurones in the rat lateral hypothalamus using double-immunofluorescence. Centrally administered GALP is known to stimulate feeding behaviour. However, the target neurones of this action have not been clarified. The present study aimed to determine features of the GALP-mediated neuronal feeding pathway in rat. Accordingly, at the ultrastructural level, GALP-immunoreactive axon terminals were found to make synapses on orexin/hypocretin immunoreactive cell bodies and dendritic processes in the lateral hypothalamus. c-Fos immunoreactivity was expressed in orexin/hypocretin-immunoreactive neurones but not in melanin concentrating hormone-immunoreactive neurones in the lateral hypothalamus at 90 min after the application of GALP by i.c.v. infusion. Furthermore, to determine whether GALP regulates feeding behaviour via orexin/hypocretin neurones, the feeding behaviour of rats was studied following GALP i.c.v. injection with or without anti-orexin A and B immunoglobulin (IgG) pretreatment. The anti-orexin IgGs markedly inhibited GALP-induced hyperphagia. These results suggest that orexin/hypocretin-containing neurones in the lateral hypothalamus are targeted by GALP, and that GALP-induced hyperphagia is mediated via orexin/hypocretin neurones in the rat hypothalamus.

Identification of hBD-3 in respiratory tract and serum: the increase in pneumonia.

Human beta-defensin (hBD)-3, a 45 amino acid antimicrobial peptide, was originally isolated from human skin. hBD-3 mRNA has also been detected in the airways by RT-PCR. While hBD-3 may be involved in antimicrobial defences within the respiratory tract, the presence of hBD-3 peptide in the respiratory system has not yet been confirmed. The antimicrobial activity of the synthesised hBD-3 peptide was measured by a radial diffusion assay and a colony count assay. The present authors confirmed the presence of hBD-3 peptide in homogenates of human lung and serum using reverse-phase HPLC coupled with a highly sensitive RIA. The localisation of the hBD-3 peptide was investigated by immunohistochemistry. In addition, the serum concentrations of hBD-3 were measured by RIA. hBD-3 exhibited a strong antimicrobial activity, which was unaffected by increasing salt concentrations. Immunohistochemically, the current authors observed the expression of hBD-3 in bronchial and bronchiolar epithelial cells. The mean+/-sd serum concentration of hBD-3 in patients with bacterial pneumonia was 239.4+/-17.8 pg x mL(-1) in the acute phase and, decreased to 159.3+/-20.1 pg x mL(-1) after the completion of therapy. In conclusion, these findings will help elucidate the role of human beta-defensin-3 in host immune responses and identify the pathophysiological significance of this molecule in respiratory infections.

Neuropeptide W is present in antral G cells of rat, mouse, and human stomach.

Neuropeptide W (NPW) is a 30-amino-acid peptide initially isolated from the porcine hypothalamus as an endogenous ligand for the G protein-coupled receptors GPR7 and GPR8. An intracerebroventricular administration of NPW increased serum prolactin and corticosterone concentrations, decreased dark-phase feeding, raised energy expenditure, and lowered body weight. Peripherally, GPR7 receptors are abundantly expressed throughout the gastrointestinal tract; the presence of NPW in the gastrointestinal endocrine system, however, remains unstudied. Using monoclonal and polyclonal antibodies raised against rat NPW, we studied the localization of NPW in the rat, mouse, and human stomach by light and electron microscopy. NPW-immunoreactive cells were identified within the gastric antral glands in all three species. Double immunohistochemistry and electron-microscopic immunohistochemistry studies in rats demonstrated that NPW is present in antral gastrin (G) cells. NPW immunoreactivity localized to round, intermediate-to-high-density granules in G cells. NPW-immunoreactive cells accounted for 90% chromagranin A- and 85% gastrin-immunoreactive endocrine cells in the rat gastric antral glands. Using reversed-phase HPLC coupled with enzyme immunoassays specific for NPW, we detected NPW30 and its C-terminally truncated form, NPW23, in the gastric mucosa. Plasma NPW concentration of the gastric antrum was significantly higher than that of the systemic vein, suggesting that circulating NPW is derived from the stomach. Plasma NPW concentration of the gastric antrum decreased significantly after 15-h fast and increased after refeeding. This is the first report to clarify the presence of NPW peptide in the stomachs of rats, mice, and humans. In conclusion, NPW is produced in gastric antral G cells; our findings will provide clues to additional mechanisms of the regulation of gastric function by this novel brain/gut peptide.

Familial leptomeningeal amyloidosis with a transthyretin variant Asp18Gly representing repeated subarachnoid haemorrhages with superficial siderosis.

OBJECTIVES: To report the clinical features of two Japanese brothers with familial leptomeningeal amyloidosis, showing a causative gene abnormality of a transthyretin (TTR) variant Asp18Gly, previously reported only in a Hungarian family. METHODS: The authors reported on a 42 year old man (patient 1) and his 45 year old brother (patient 2), both suffering from subarachnoid haemorrhage (SAH) without and with hydrocephalus, respectively. DNA sequences of the TTR gene were determined in both patients and the patients' clinical features described. A surgical biopsy of the leptomeninges was performed on patient 1. RESULTS: DNA sequence analyses demonstrated the glycine-for-aspartate substitution at position 18 of the TTR variant. Both patients revealed pyramidal tract signs and cerebellar ataxia. Audiometric studies showed bilateral, mild sensorineural hearing loss in the patients whose cerebrospinal fluid (CSF) protein levels increased. T1 weighted MRI after contrast administration showed diffuse leptomeningeal enhancement along the Sylvian fissures and over the surface of the brainstem, cerebellum, and spinal cord. Gradient echo T2* weighted MRI showed superficial siderosis mainly in the cerebellum. A biopsy of the leptomeninges was obtained from the spinal cord of patient 1. While performing the biopsy, the authors observed the varicose, elongating, and fragile veins on the dorsal surface of the spinal cord. Immunohistochemical study revealed marked deposits of TTR derived amyloid on his leptomeninges. CONCLUSIONS: This is the second report of familial leptomeningeal amyloidosis with an Asp18Gly TTR gene mutation, clinically causing only CNS symptoms. Repeated SAH from fragile veins on the dorsal surface of the spinal cord seemed to induce superficial siderosis of the CNS. So far, there have been two reliable hallmarks leading to the diagnosis of leptomeningeal amyloidosis: diffuse leptomeningeal enhancement on contrast MRI and greatly increased CSF protein content. This study has contributed a third hallmark: the presence of superficial siderosis is useful in diagnosing leptomeningeal amyloidosis.

Involvement of dendritic cells in sarcoidosis.

BACKGROUND: Sarcoidosis is a systemic granulomatous disease of unknown aetiology. It has been suggested that T helper type 1 (Th1) polarisation is associated with the pathophysiology of sarcoidosis, but the mechanism of skewing towards Th1 has not been elucidated. Dendritic cells (DCs) are known to regulate immune responses. This study was performed to determine whether DCs are involved in the aetiology of sarcoidosis. METHODS: The numbers of peripheral blood DCs in 24 patients with sarcoidosis were analysed and biopsy specimens from four patients were stained immunohistochemically using monoclonal antibodies. RESULTS: The numbers of both myeloid and lymphoid DC subsets were significantly decreased in the blood and mature DCs were found in the granulomas of patients with sarcoidosis. A number of interferon-gamma (IFN-gamma) producing T cells were also detected in the sarcoid granuloma, as well as many interleukin (IL)-4 producing T cells. Double staining of the biopsy specimen using anti-fascin and anti-CD3 antibodies showed an anatomical interaction between DCs and T cells. CONCLUSIONS: These findings suggest that the blood DC subsets may migrate into the affected tissues, contributing to the formation of the granulomas in sarcoidosis. It is hypothesised that the migrating DCs may regulate the T cell response in sarcoidosis, at least in the granulomatous lesions.

Ghrelin in neonatal rats: distribution in stomach and its possible role.

Ghrelin, a 28 amino acid peptide, has recently been isolated from the rat stomach as an endogenous ligand for the GH secretagogue receptor. The fact that administration of ghrelin, centrally or peripherally, stimulates both food intake and GH secretion suggests that stomach ghrelin has an important role in the growth of rats. We used immunohistochemistry and radioimmunoassay to determine the age at which ghrelin-immunostained cells begin to appear in the rat stomach. Ghrelin-immunoreactive cells were found to be expressed in the fetal stomach from pregnancy day 18. The number of ghrelin-immunoreactive cells in the fetal stomach increased as the stomach grew. The amount of ghrelin in the glandular part of the rat stomach also increased, in an age-dependent manner, from the neonatal stage to adult. Eight hours of milk restriction significantly decreased the ghrelin concentration in the stomachs of 1-week-old rats, and increased the ghrelin concentration in their plasma. Administration of ghrelin to 1- and 3-week-old rats increased plasma GH concentrations. The daily subcutaneous administration of ghrelin to pregnant rats from day 15 to day 21 of pregnancy caused an increase in body weight of newborn rats. In addition, daily subcutaneous administration of ghrelin to neonatal rats from birth advanced the day of vaginal opening from day 30.7+/-0.94 to day 27.9+/-0.05. These results suggest that ghrelin may be involved in neonatal development.

Immunocytochemical observation of ghrelin-containing neurons in the rat arcuate nucleus.

Ghrelin is a novel peptide that stimulates the release of growth hormone from the pituitary and is involved in hypothalamic feeding regulation. A pre-embedding immunostaining technique was used to study the ultrastructure and synaptic relationships of ghrelin-containing neurons in the rat arcuate nucleus (ARC). Ghrelin-like immunoreactive (ghrelin-LI) neurons were found in the ARC, and were especially abundant in its ventral part. At the electron microscopic level, ghrelin-LI neurons received afferent synapses from many unknown axon terminals. Ghrelin-LI products in the immunoreactive cell bodies, processes, and axon terminals were detected mainly in dense granular vesicles about 110 nm in diameter. Ghrelin-LI presynaptic axon terminals often made synapses with unknown immunonegative neurons. These results suggest that ghrelin acts to regulate food intake through synaptic connections in hypothalamic neuronal networks.

Elevated circulating level of ghrelin in cachexia associated with chronic heart failure: relationships between ghrelin and anabolic/catabolic factors.

BACKGROUND: Ghrelin is a novel growth hormone (GH)-releasing peptide, isolated from the stomach, that may also cause a positive energy balance by stimulating food intake and inducing adiposity. We sought to investigate the pathophysiology of ghrelin in the cachexia associated with chronic heart failure (CHF). METHODS AND RESULTS: Plasma ghrelin was measured in 74 patients with CHF and 12 control subjects, together with potentially important anabolic and catabolic factors, such as GH and tumor necrosis factor (TNF-alpha). Patients with CHF were divided into two groups, those with cachexia (n=28) and those without cachexia (n=46). Plasma ghrelin did not significantly differ between all CHF patients and controls (181+/-10 versus 140+/-14 fmol/mL, P=NS). However, plasma ghrelin was significantly higher in CHF patients with cachexia than in those without cachexia (237+/-18 versus 147+/-10 fmol/mL, P<0.001). Circulating GH, TNF-alpha, norepinephrine, and angiotensin II were also significantly higher in CHF patients with cachexia than in those without cachexia. Interestingly, plasma ghrelin correlated positively with GH (r=0.28, P<0.05) and TNF-alpha (r=0.31, P<0.05) and negatively with body mass index (r=-0.35, P<0.01). CONCLUSIONS: Plasma ghrelin was elevated in cachectic patients with CHF, associated with increases in GH and TNF-alpha and a decrease in body mass index. Considering ghrelin-induced positive energy effects, increased ghrelin may represent a compensatory mechanism under catabolic-anabolic imbalance in cachectic patients with CHF.

Uroguanylin level in umbilical cord blood.

BACKGROUND: Uroguanylin is a novel natriuretic and diuretic peptide originally isolated from urine. METHODS: To determine whether uroguanylin has a physiologic role during the perinatal period, uroguanylin levels in umbilical cord plasma obtained at the time of delivery were measured by radioimmunoassay and compared with cord serum osmolality. RESULTS: Mean (+/- SD) cord plasma uroguanylin concentrations (8.8 +/- 2.1 fmol/mL) were higher compared with normal adult values. The extent of maturity, mode of delivery and gender did not appear to influence cord uroguanylin levels. The uroguanylin concentration had a significant positive correlation with cord serum osmolality. CONCLUSION: These findings support some regulatory role of this peptide in perinatal renal and cardiovascular adaptation.

Nucleotide sequence and expression of rat beta-defensin-1: its significance in diabetic rodent models.

beta-Defensins are epithelium-derived antimicrobial peptides that function in the host's innate defense. We identified the first member of the rat beta-defensin family, beta-defensin-1 (BD-1), in the kidney and determined its nucleotide sequence. It was predicted to be a 37-amino-acid peptide. Rat BD-1 mRNA was expressed most abundantly in the kidney, next in skin, tongue, esophagus, and uterus, followed (at low levels) by brain, trachea, stomach, urinary bladder, and ovary. BD-1 gene expression in rat kidney was not increased by lipopolysaccharide administration. BD-1 gene expressions in the kidneys of diabetic rodent models, cholecystokinin-insensitive Otsuka Long-Evans Tokushima Fatty rats, leptin-insensitive obese (fa/fa) Wistar rats, and db/db mice, were significantly lower than those of their lean littermates. BD-1 reduction may be in part responsible for the high incidence of urinary tract infections in diabetes mellitus.

[A role for orexins and melanin-concentrating hormone in the central regulation of feeding behavior].

The hypothalamus is the most important region in the control of food intake and body weight. The ventromedial 'satiety center' and lateral hypothalamic 'feeding center' have been implicated in the regulation of feeding and energy homeostasis by various studies of brain lesions. Orexins(orexin A and orexin B) and melanin-concentrating hormone (MCH), whose intracerebroventricular injections increase food intake, are localized in the lateral hypothalamus and provide diffuse projections throughout the brain. Orexins and MCH neurons have a coextensive distribution, but are not colocalized. Orexins and MCH may affect feeding behavior through distinct neuronal pathways. We here describe the effect of orexins and MCH on feeding behavior from the physiological, neuroanatomical and molecular studies.

A case of late onset cardiac amyloidosis with a new transthyretin variant (lysine 92).

A new transthyretin (TTR) variant (lysine 92), which causes late onset cardiac amyloidosis, is described in a 71-year-old man. The patient at first had syncope due to ventricular tachycardia and was admitted our hospital. Typical findings of cardiac amyloidosis were observed by echocardiography, and a diagnosis of systemic amyloidosis was made by rectal biopsy. The man died approximately 3 years and 6 months after first admission, with gradually worsening congestive heart failure. Pathological examination showed prominent amyloid deposits in the heart and the vascular wall of many organs including the liver, pancreas, kidney, lung, and gastrointestinal tracts. Amyloid protein of transthyretin type was indicated by immunohistochemical study, and DNA sequencing identified a novel mutation in the transthyretin gene encoding 92 glutamine --> lysine. A polymerase chain reaction-induced mutation restriction analysis with a mismatched antisense primer showed that the patient was heterozygous for the TTR Lys92 allele.

Upregulation of Ghrelin expression in the stomach upon fasting, insulin-induced hypoglycemia, and leptin administration.

Ghrelin is a novel gut-brain peptide that binds to the growth hormone secretagogue receptor (GHS-R), thereby functioning in the regulation of growth hormone (GH) release and food intake. Ghrelin-producing cells are most abundant in the oxyntic glands of the stomach. The regulatory mechanism that governs the biosynthesis and secretion of ghrelin has not been clarified. We report that ghrelin mRNA expression in the gastric fundus was increased, but that ghrelin peptide content decreased after a 48-h fast. Both values returned to control levels after refeeding. The ghrelin plasma concentration in the gastric vein and systemic venous blood increased after 24- and 48-h fasts. Furthermore, des-octanoylated ghrelin and n-octanoylated ghrelin were found in rat stomach, with the ratio of des-octanoylated ghrelin to n-octanoylated ghrelin markedly increased after fasting. The ghrelin mRNA level in the stomach also increased after administration of insulin and leptin. Conversely, db/db mice, which are deficient in the leptin receptor, had lower ghrelin mRNA levels than control mice. These findings suggest that this novel gastrointestinal hormone plays a role in the regulation of energy balance.

A role for ghrelin in the central regulation of feeding.

Ghrelin is an acylated peptide that stimulates the release of growth hormone from the pituitary. Ghrelin-producing neurons are located in the hypothalamus, whereas ghrelin receptors are expressed in various regions of the brain, which is indicative of central-and as yet undefined-physiological functions. Here we show that ghrelin is involved in the hypothalamic regulation of energy homeostasis. Intracerebroventricular injections of ghrelin strongly stimulated feeding in rats and increased body weight gain. Ghrelin also increased feeding in rats that are genetically deficient in growth hormone. Anti-ghrelin immunoglobulin G robustly suppressed feeding. After intracerebroventricular ghrelin administration, Fos protein, a marker of neuronal activation, was found in regions of primary importance in the regulation of feeding, including neuropeptide Y6 (NPY) neurons and agouti-related protein (AGRP) neurons. Antibodies and antagonists of NPY and AGRP abolished ghrelin-induced feeding. Ghrelin augmented NPY gene expression and blocked leptin-induced feeding reduction, implying that there is a competitive interaction between ghrelin and leptin in feeding regulation. We conclude that ghrelin is a physiological mediator of feeding, and probably has a function in growth regulation by stimulating feeding and release of growth hormone.

Ghrelin acts in the central nervous system to stimulate gastric acid secretion.

Ghrelin is a novel acylated peptide that functions in the regulation of growth hormone release and energy metabolism. It was isolated from rat stomach as an endogenous ligand for growth hormone secretagogue receptor. Ghrelin is also localized in the arcuate nucleus of rat hypothalamus. Intracerebroventricular (ICV) administration increases food intake and body weight. We examined the effect of ghrelin on gastric acid secretion in urethane-anesthetized rats and found that ICV administration of ghrelin increased gastric acid output in a dose-dependent manner. Vagotomy and administration of atropine abolished the gastric acid secretion induced by ghrelin. ICV administration of ghrelin also induced c-fos expression in the neurons of the nucleus of the solitary tract and the dorsomotor nucleus of the vagus, which are key sites in the central nervous system for regulation of gastric acid secretion. Our results suggest that ghrelin participates in the central regulation of gastric acid secretion by activating the vagus system.