Mastication stimuli regulate the heartbeat rate through rhythmic regulation by the hypothalamic-autonomic system; molecular and telemetric studies in weaning-stage rats.

Mastication stimuli have been demonstrated to affect memory function and autonomic nerve activity; however, this process has not been well studied during weaning compared to old age. Previously, we conducted molecular analyses of the thalamus and hippocampus to elucidate the mechanisms underlying this memory-enhancing effect in weaning-stage rats. In this study, we aimed to evaluate the effect of masticatory stimuli on the regulation of heartbeat rate (HR) through the hypothalamic-autonomic system. Three-week-old male rats were administered a powdered diet (P group) or chow-diet (C group) for 10 days. Thereafter, transcriptome analysis was performed. Vasopressin, cocaine-amphetamine-regulated transcript prepropeptide, corticotropin-releasing hormone, and thyrotropin-releasing hormone, which are involved in sympathetic activation of heart rate, were downregulated in the C group. Electrocardiograms were recorded continuously for 12 days under the same condition. Interestingly, rats in the C group had a significantly lower HR than those in the P group on day 11. We checked several parameters representing the autonomic regulation of HR. The C group had higher values for the high-frequency band integration of the HR power spectrum (parasympathetic marker) and root mean square successive difference of R-wave intervals (parasympathetic marker) relative to the P group. Such findings provide a molecular and physiological basis for understanding the regulation of cardiovascular function in response to masticatory stimuli in the autonomic nervous system.

Mastication stimuli enhance the learning ability of weaning-stage rats, altering the hippocampal neuron transcriptome and micromorphology.

Mastication stimuli are known to relieve senile dementia in human and animal studies. However, few studies have focused on its effect on weaning-stage animals and the underlying molecular processes. In this study, 3-week-old male rats were raised on a powdered (P-group) or chow (C-group) diet for 8 days, and their behavior was examined using the Y-maze and novel object recognition tests. In the Y-maze test, the C-group rats showed a larger alternation ratio than the P-group rats. In the novel object recognition test, the C-group rats exhibited a significantly larger discrimination index for novel objects than for familiar objects, but the P-group rats did not. We then compared the hippocampal neuron morphology and transcriptome between the groups. C-group rats exhibited larger dendrite branch numbers in the apical dendrites of pyramidal cells in the cornu ammonis 1 (CA1) region and a larger spine density in the basal dendrites of CA1 neurons than the P-group rats. Using DNA microarray analysis, we identified 621 (P < C) and 96 (P > C) genes that were differentially expressed between the groups. These genes were enriched in functional terms related to dendrite growth and included the Igf2, RhoA, and Rho GEF genes, most of which were upregulated in the C-group. These results suggest that the mastication stimuli during the weaning period can enhance the learning ability of rats by increasing the dendrite branches of hippocampal CA1 neurons and by regulating genes related to dendrite growth.

Safety and efficacy of Melissa officinalis extract containing rosmarinic acid in the prevention of Alzheimer's disease progression.

We conducted a randomized placebo-controlled double-blind 24-week trial using Melissa officinalis (M. officinalis) extract richly containing rosmarinic acid (RA) on patients with mild dementia due to Alzheimer's disease (AD) with the aim to examine the safety and tolerability (primary endpoint) of RA (500 mg daily) and its clinical effects and disease-related biomarker changes (secondary endpoints). Patients (n = 23) diagnosed with mild dementia due to probable AD were randomized to either the placebo or M. officinalis extract group. No differences in vital signs or physical and neurologic examination results were detected between the M. officinalis and placebo groups. No serious adverse events occurred. There were no significant differences in cognitive measures; however, the mean Neuropsychiatric Inventory Questionnaire (NPI-Q) score improved by 0.5 points in the M. officinalis group and worsened by 0.7 points in the placebo group between the baseline and 24-week visit, indicating a significant difference (P = 0.012). No significant differences were apparent in disease-related biomarkers between the groups. M. officinalis extract containing 500 mg of RA taken daily was safe and well-tolerated by patients with mild dementia due to AD. Our results suggest that RA may help prevent the worsening of AD-related neuropsychiatric symptoms.Trial registration: The registration number for this clinical trial is UMIN000007734 (16/04/2012).

Short-term mastication after weaning upregulates GABAergic signalling and reduces dendritic spine in thalamus.

Mastication enhances brain function and mental health, but little is known about the molecular mechanisms underlying the effects of mastication on neural development in early childhood. Therefore, we analysed the gene expression in juvenile neural circuits in rats fed with a soft or chow diet immediately after weaning. We observed that the gene expression patterns in the thalamus varied depending on the diet. Furthermore, gene ontology analysis revealed that two terms were significantly enhanced: chemical synaptic transmission and positive regulation of dendritic spine morphogenesis. With respect to chemical synaptic transmission, glutamate decarboxylase and GABA receptors were upregulated in the chow diet group. The related genes, including vesicular GABA transporter, were also upregulated, suggesting that mastication activates GABAergic signalling. With respect to dendritic spine morphogenesis, Ingenuity Pathway Analysis predicted fewer extension of neurites and neurons and fewer number of branches in the chow diet group. The numbers of spines in the ventral posterolateral and posteromedial regions were significantly decreased. These results suggest that mastication in the early developing period upregulates GABAergic signalling genes, with a decrease of spines in the thalamus.

Transcriptomic responses of the liver and adipose tissues to altered carbohydrate-fat ratio in diet: an isoenergetic study in young rats.

BACKGROUND: To elucidate the effects of altered dietary carbohydrate and fat balance on liver and adipose tissue transcriptomes, 3-week-old rats were fed three kinds of diets: low-, moderate-, and high-fat diets (L, M, and H) containing a different ratio of carbohydrate-fat (C-F) (65:15, 60:20, and 35:45 in energy percent, respectively). METHODS: The rats consumed the diets for 9 weeks and were subjected to biochemical and DNA microarray analyses. RESULTS: The rats in the H-group exhibited lower serum triacylglycerol (TG) levels but higher liver TG and cholesterol content than rats in the L-group. The analysis of differentially expressed genes (DEGs) between each group (L vs M, M vs H, and L vs H) in the liver revealed about 35% of L vs H DEGs that were regulated in the same way as M vs H DEGs, and most of the others were L- vs H-specific. Gene ontology analysis of these L vs H DEGs indicated that those related to fatty acid synthesis and circadian rhythm were enriched. Interestingly, about 30% of L vs M DEGs were regulated in a reverse way compared with L vs H and M vs H DEGs. These reversed liver DEGs included M-up/H-down genes (Sds for gluconeogenesis from amino acids) and M-down/H-up genes (Gpd2 for gluconeogenesis from glycerol, Agpat9 for TG synthesis, and Acot1 for beta-oxidation). We also analyzed L vs H DEGs in white (WAT) and brown (BAT) adipose tissues and found that both oxidation and synthesis of fatty acids were inhibited in these tissues. CONCLUSIONS: These results indicate that the alteration of dietary C-F balance differentially affects the transcriptomes of metabolizing and energy-storing tissues.

Pharmacokinetics, Safety and Tolerability of Melissa officinalis Extract which Contained Rosmarinic Acid in Healthy Individuals: A Randomized Controlled Trial.

The aim of this study was to evaluate the safety, tolerability and pharmacokinetics of single dose of Melissa officinalis extract which contained rosmarinic acid, including food-effects in healthy individuals. A total of eleven healthy individuals were randomly assigned to treatment arms in the two studies [Study 1 (fasted state) and Study 2 (fed state)]. Rosmarinic acid in serum was measured by a coulometric detection method using High-Performance Liquid Chromatography electrochemical detector. The serum concentration of total rosmarinic acid peaked at 1 hour after administration of Melissa officinalis extract containing 500mg rosmarinic acid in fasted state, with a maximum serum concentration 162.20 nmol/ L. The area under the curve for intact rosmarinic acid was calculated from the serum concentration-time profile to be 832.13 nmol • hour/ L. Food intake increases area under the curve and delayed time at which the maximum serum concentration. Rosmarinic acid supplementation did not affect liver, kidney, or blood cell function parameters. No adverse event was reported by any of the participants due to the study treatment. Single dose of Melissa officinalis extract containing 500 mg rosmarinic acid appears to be safe and tolerable in healthy individuals. Food intake increased the exposure of rosmarinic acid and delayed absorption of rosmarinic acid in healthy individuals.

Transport mechanisms for soy isoflavones and microbial metabolites dihydrogenistein and dihydrodaidzein across monolayers and membranes.

Isoflavone data concerning the metabolism and permeability on intestinal epithelial cells are scarce, particularly for microbial isoflavone metabolites. This study evaluates the absorption mechanisms for the isoflavones, genistein and daidzein, and their microbial metabolites, dihydrogenistein (DHG) and dihydrodaidzein (DHD). The permeability characteristics of isoflavones were compared by using the Caco-2 human colon adenocarcinoma cell line for a parallel artificial membrane permeability assay, and comparing their physicochemical properties. The data suggest that genistein, DHG and DHD were efficiently transported by passive diffusion according to the pH-partition hypothesis. Genistein was conjugated by phase II metabolizing enzymes and acted as a substrate of the breast cancer resistance protein (BCRP). Daidzein was not conjugated but did act as a substrate for BCRP, multidrug resistance-associated proteins, and P-glycoprotein. In contrast, DHG and DHD were markedly more permeable than their parent isoflavones; they were therefore difficult to transport by the efflux effect, and glucuronidation/sulfation was limited by the flux time.

Transport mechanisms of flavanone aglycones across Caco-2 cell monolayers and artificial PAMPA membranes.

OBJECTIVES: We recently reported that flavanone aglycones (hesperetin, naringenin and eriodictyol) are efficiently absorbed via proton-coupled active transport, in addition to transcellular passive diffusion, in Caco-2 cells. Here, we aimed to evaluate in detail the absorption mechanisms of these flavanones, as well as homoeriodictyol and sakuranetin. METHODS: We evaluated the absorption mechanisms of the above compounds by means of in vitro studies in Caco-2 cells in parallel with an artificial membrane permeation assay (PAMPA) under pH-gradient and iso-pH conditions. KEY FINDINGS: Comparison of the permeability characteristics of flavanones in Caco-2 cells and in PAMPA under these conditions, as well as a consideration of the physicochemical properties, indicated that hesperetin, naringenin, eriodictyol and homoeriodictyol were efficiently transported by passive diffusion according to the pH-partition hypothesis, except in the case of sakuranetin. However, transport of all flavanones were remarkably temperature-dependent, and was significantly reduced when Caco-2 cells were treated with amino acid-modifying reagents. CONCLUSIONS: Our data confirm that both passive diffusion and an active transport mechanism contribute to flavanone absorption through human intestinal epithelium.

pH-Dependent inhibition of the human bitter taste receptor hTAS2R16 by a variety of acidic substances.

Some acidic peptides are known to reduce bitterness, but the detailed mechanism underlying this effect remains to be elucidated. In this study, we analyzed the effects of acidic dipeptides on the inhibition of the human bitter taste receptor hTAS2R16. Calcium imaging analysis of HEK293T cells expressing hTAS2R16 revealed that their response to the bitter tastant salicin was reduced in the presence of acidic dipeptides. A similar inhibitory effect was observed in a variety of other acids. The inhibition depended on the pH values resulting from the addition of acids but not on their concentrations. Our results suggest that the inhibition of the bitter taste receptors can be attributed to the bitterness-masking effect of the acidic dipeptides and that acidic pH may be one of the critical factors responsible for this sensory event.

Characterization of ligands for fish taste receptors.

Recent progress in the molecular biology of taste reception has revealed that in mammals, the heteromeric receptors T1R1/3 and T1R2/3 respond to amino acids and sweeteners, respectively, whereas T2Rs are receptors for bitter tastants. Similar taste receptors have also been characterized in fish, but their ligands have not been identified yet. In the present study, we conducted a series of experiments to identify the fish taste receptor ligands. Facial nerve recordings in zebrafish (Danio rerio) demonstrated that the fish perceived amino acids and even denatonium, which is a representative of aversive bitter compounds for mammals and Drosophila. Calcium imaging analysis of T1Rs in zebrafish and medaka fish (Oryzias latipes) using an HEK293T heterologous expression system revealed that both T1R1/3 and a series of T1R2/3 responded to amino acids but not to sugars. A triple-labeling, in situ hybridization analysis demonstrated that cells expressing T1R1/3 and T1R2/3s exist in PLCbeta2-expressing taste bud cells of medaka fish. Functional analysis using T2Rs showed that zfT2R5 and mfT2R1 responded to denatonium. Behavior observations confirmed that zebrafish prefer amino acids and avoid denatonium. These results suggest that, although there may be some fish-specific way of discriminating ligands, vertebrates could have a conserved gustatory mechanism by which T1Rs and T2Rs respond to attractive and aversive tastants, respectively.

Two families of candidate taste receptors in fishes.

Vertebrates receive tastants, such as sugars, amino acids, and nucleotides, via taste bud cells in epithelial tissues. In mammals, two families of G protein-coupled receptors for tastants are expressed in taste bud cells-T1Rs for sweet tastants and umami tastants (l-amino acids) and T2Rs for bitter tastants. Here, we report two families of candidate taste receptors in fish species, fish T1Rs and T2Rs, which show significant identity to mammalian T1Rs and T2Rs, respectively. Fish T1Rs consist of three types: fish T1R1 and T1R3 that show the highest degrees of identity to mammalian T1R1 and T1R3, respectively, and fish T1R2 that shows almost equivalent identity to both mammalian T1R1 and T1R2. Unlike mammalian T1R2, fish T1R2 consists of two or three members in each species. We also identified two fish T2Rs that show low degrees of identity to mammalian T2Rs. In situ hybridization experiments revealed that fish T1R and T2R genes were expressed specifically in taste bud cells, but not in olfactory receptor cells. Fish T1R1 and T1R2 genes were expressed in different subsets of taste bud cells, and fish T1R3 gene was co-expressed with either fish T1R1 or T1R2 gene as in the case of mammals. There were also a significant number of cells expressing fish T1R2 genes only. Fish T2R genes were expressed in different cells from those expressing fish T1R genes. These results suggest that vertebrates commonly have two kinds of taste signaling pathways that are defined by the types of taste receptors expressed in taste receptor cells.