Production of free glutamate in milk requires the leucine transporter LAT1.

The concentration of free glutamate (Glu) in rat's milk is ∼10 times higher than that in plasma. Previous work has shown that mammary tissue actively transports circulatory leucine (Leu), which is transaminated to synthesize other amino acids such as Glu and aspartate (Asp). To investigate the molecular basis of Leu transport and its conversion into Glu in the mammary gland, we characterized the expression of Leu transporters and [(3)H]Leu uptake in rat mammary cells. Gene expression analysis indicated that mammary cells express two Leu transporters, LAT1 and LAT2, with LAT1 being more abundant than LAT2. This transport system is sodium independent and transports large neutral amino acids. The Leu transport system in isolated rat mammary cells could be specifically blocked by the LAT1 inhibitors 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid (BCH) and triiodothyronine (T3). In organ cultures, Glu secretion was markedly inhibited by these LAT1 inhibitors. Furthermore, the profiles of Leu uptake inhibition by amino acids in mammary cells were similar to those reported for LAT1. In vivo, concentrations of free Glu and Asp increased in milk by oral gavage with Leu at 6, 12, and 18 days of lactation. These results indicate that the main Leu transporter in mammary tissue is LAT1 and the transport of Leu is a limiting factor for the synthesis and release of Glu and Asp into milk. Our studies provide the bases for the molecular mechanism of Leu transport in mammary tissue by LAT1 and its active role on free Glu secretion in milk, which confer umami taste in suckling pups.

Amino acid sensing in the gastrointestinal tract.

Rapid progress in gastroenterology during the first part of the last century has shown that gastrointestinal (GI) function is regulated by neuroendocrine, paracrine and endocrine signals. However, recent advances in chemical sensing, especially in the last decade, have revealed that free L-amino acids (AA), among other nutrients, play a critical role in modifying exocrine and endocrine secretion, modulating protein digestion, metabolism and nutrient utilization, and supporting the integrity and defense of the GI mucosa. Many of the mechanisms by which AAs elicit these functions in the GI has been linked to the traditional concept of hormone release and nervous system activation. But most these effects are not direct. AAs appear to function by binding to a chemical communication system such as G protein-coupled receptors (GPCRs) that activate signaling pathways. These intracellular signals, although their molecular bases are not completely elucidated yet, are the ones responsible for the neuronal activity and release of hormones that in turn regulate GI functions. This review aims to describe the distribution of the known GPCRs from the class 3 superfamily that bind to different kinds of AA, especially from the oropharyngeal cavity to the stomach, what kind of taste qualities they elicit, such as umami, bitter or sweet, and their activity in the GI tract.

New frontiers in gut nutrient sensor research: luminal glutamate-sensing cells in rat gastric mucosa.

Dietary free glutamate is known to elicit umami, one of the five basic tastes perceived via the specific taste sensor cells on the tongue. Recent studies suggest the specific glutamate sensors exist in the gastric mucosa and contribute to the regulation of gastrointestinal functions, yet the precise mechanism remains still unknown. We established the method to enrich various cell fractions from the isolated rat gastric mucosa and characterized the expression of putative glutamate sensors using such cell fractions. The gastric mucosal cell fractions such as surface mucous, parietal, chief, and endocrine cells were successfully prepared by mucosal protease digestion, elutriation, and gradient centrifugation. The characteristics of these cells were confirmed by real-time RT-PCR using the respective cell-specific markers. Parietal cell fraction exclusively expressed putative umami receptor molecules such as T1R1 and mGluR1 compared to other fractions, although the degree of expression was low. In contrast, the representative taste cell specific markers such as PLCbeta2 and TRPM5 were specifically expressed in the smaller endocrine cell fraction. Both parietal and smaller endocrine cell fractions also positively expressed some mGluR subtypes. The chief-cell fraction less expressed T1R1 and mGluR1. These results suggest that multiple glutamate sensors, probably different mechanisms from taste buds, contribute to the glutamate sensing in the gastric mucosa.

The calcium-sensing receptor in taste tissue.

Calcium is an essential nutrient that induces a distinctive taste quality, but the sensing mechanism of calcium in the tongue is poorly understood. A recent study linked calcium to T1R3 receptor. Here, we propose another system for calcium taste involving the extracellular calcium-sensing receptor (CaSR). This G protein-coupled receptor that responds to calcium and magnesium cations is involved in calcium homeostasis regulating parathyroid and kidney functions. In this study, CaSR was found in isolated taste buds from rats and mice. It was expressed in a subset of cells in circumvallate and foliate papillae, with fewer cells in the fungiform papillae. This is the first evidence in mammals that locates CaSR in gustatory tissue and provides the basis for better understanding not only calcium taste but also the taste of multiple CaSR agonists.

mGluR1 in the fundic glands of rat stomach.

l-glutamate not only confers cognitive discrimination for umami taste in the oral cavity, but also conveys sensory information to vagal afferent fibers in the gastric mucosa. We used RT-PCR, western blotting, and immunohistochemistry to demonstrate that mGluR1 is located in glandular stomach. Double staining revealed that mGluR1 is found at the apical membrane of chief cells and possibly in parietal cells. Moreover, a diet with 1% l-glutamate induced changes in the expression of pepsinogen C mRNA in stomach mucosa. These data suggest that mGluR1 is involved in the gastric phase regulation of protein digestion.

Free amino acid content in breast milk of adolescent and adult mothers in Ecuador.

Because of increased incidence of teenage births and high prevalence of lactation in Latin America, we determined the patterning of free amino acids (FAAs) in breast milk of 65 primiparous Ecuadorian women of varying ages (14-27 years). An automatic amino acid analyzer quantified levels of FAAs in milk samples obtained at three lactation stages: colostrum, transition, and mature milk. Regardless of mother's age, most FAAs increased with time postpartum, with taurine, glutamic acid, glutamine, and alanine being most abundant in all stages.

Nutritional and regulatory role of branched-chain amino acids in lactation.

Optimal growth and health of suckling neonates critically depend on milk production by their mothers. In both humans and animals, branched-chain amino acids (BCAA) are not only the major components of milk proteins but are also nitrogenous precursors for the synthesis of glutamate, glutamine, alanine, and aspartate in the mammary gland. These synthetic pathways, which are initiated by BCAA transaminase, contribute to the high abundance of free and peptide-bound glutamate, glutamine, aspartate and asparagine in milk. In mammary epithelial cells, the carbon skeletons of BCAA can be partially oxidized via branched-chain alpha-ketoacid dehydrogenase to provide energy for highly active metabolic processes, including nutrient transport, protein turnover, as well as lipid and lactose syntheses. In addition, results of recent studies indicate that BCAA play regulatory roles in mammary metabolism. For example, leucine can activate the mammalian target of rapamycin cell signaling pathway to enhance protein synthesis in mammary epithelial cells. Dietary supplementation with BCAA may have great potential to enhance milk synthesis by the lactating mammary gland, thereby improving neonatal survival, growth and development.

Taste, visceral information and exocrine reflexes with glutamate through umami receptors.

Chemical substances of foods drive the cognitive recognition of taste with the subsequent regulation of digestion in the gastrointestinal (GI) tract. Tastants like glutamate can bind to taste membrane receptors on the tip of specialized taste cells eliciting umami taste. In chemical-sensing cells diffused through the GI tract, glutamate induces functional changes. Most of the taste-like receptor-expressing cells from the stomach and intestine are neuroendocrine cells. The signaling molecules produced by these neuroendocrine cells either activate afferent nerve endings or release peptide hormones that can regulate neighboring cells in a paracrine fashion or travel through blood to their target receptor. Once afferent sensory fibers transfer the chemical information of the GI content to the central nervous system (CNS) facilitating the gut-brain signaling, the CNS regulates the GI through efferent cholinergic and noradrenergic fibers. Thus, this is a two-way extrinsic communication process. Glutamate within the lumen of the stomach stimulates afferent fibers and increases acid and pepsinogen release; whereas on the duodenum, glutamate increases the production of mucous to protect the mucosa against the incoming gastric acid. The effects of glutamate are believed to be mediated by G protein-coupled receptors expressed at the lumen of GI cells. The specific cell-type and molecular function of each of these receptors are not completely known. Here we will examine some of the glutamate receptors and their already understood role on GI function regulation.

Metabotropic glutamate receptor type 1 in taste tissue.

l-Glutamate confers cognitive discrimination for umami taste (delicious or savory) and dietary information to the brain through the activation of G protein-coupled receptors in specialized taste receptor cells of the tongue. The taste heterologous receptor T1R1 plus T1R3 is not sufficient to detect umami taste in mice. The lack of T1R3 diminished but did not abolish nerve and behavioral responses in null mice that still contained umami-sensitive taste receptor cells. The remnant umami responses in T1R3 knockout mice indicate that there are also T1R3 independent receptors. Metabotropic glutamate receptor 1 (mGluR1), which is widely expressed throughout the central nervous system and regulates synaptic signaling, is another l-glutamate receptor candidate. It is found within taste buds, although the amount of l-glutamate in the perisynaptic region is in the order of micromol/L, whereas free dietary l-glutamate is in the mmol/L range. We reexamined the expression of one mGluR1 variant with a lower affinity for l-glutamate that is found in fungiform and circumvallate papillae. This taste mGluR1 receptor responds in vitro to the concentration of l-glutamate usually found in foodstuffs.

Physiological role of dietary free glutamate in the food digestion.

Gustatory and anticipatory cephalic stimuli during a meal yield nutritional information and aid efficient food digestion. Mammals, including humans, can detect the amount of dietary protein and its quality via cephalic relay to initiate proper digestion in the upper gastrointestinal (GI) tract. In addition to gustatory stimuli, visceral sensing by the abdominal vagus conveys primary afferent nutritional information from the digestive system to the brain. Electrophysiological studies indicated that abdominal vagal afferents, which were innervated into the stomach and intestine sending information to the brain, were activated by luminal glutamate. Histochemical analysis also revealed the existence of a glutamate signalling system (metabotrophic glutamate receptors) in the GI tract. Luminal glutamate in the stomach and intestine provides the efferent reflection of the abdominal vagus, supporting the modulation of exocrine and endocrine excretion during digestion. These results strongly indicate that glutamate has regulatory effects on the food digestive processes through the gut nutrient-sensing system. It plays physiological and nutritional roles and initiates digestion in the stomach as well as anticipates subsequent processes in the small intestine and the liver. We reviewed recent studies on glutamate physiology in the gut including our research, and discussed the physiological significance of dietary free glutamate in the regulation of gut function, focusing on the visceral sensation from the stomach.

Luminal amino acid sensing in the rat gastric mucosa.

Recent advancements in molecular biology in the field of taste perception in the oral cavity have raised the possibility for ingested nutrients to be "tasted" in the upper gastrointestinal tract. The purpose of this study was to identify the existence of a nutrient-sensing system by the vagus in the rat stomach. Afferent fibers of the gastric branch increased their firing rate solely with the intragastric application of the amino acid glutamate. Other amino acids failed to have the same effect. This response to glutamate was blocked by the depletion of serotonin (5-HT) and inhibition of serotonin receptor(3) (5-HT(3)) or nitric oxide (NO) synthase enzyme. Luminal perfusion with the local anesthesia lidocaine abolished the glutamate-evoked afferent activation. The afferent response was also mimicked by luminal perfusion with a NO donor, sodium nitroprusside. In addition, the NO donor-induced afferent activation was abolished by 5-HT(3) blockade as well. Altogether, these results strongly suggest the existence of a sensing system for glutamate in the rat gastric mucosa. Thus luminal glutamate would enhance the electrophysiological firing rate of afferent fibers from the vagus nerve of the stomach through the production of mucosal bioactive substances such as NO and 5-HT. Assuming there is a universal coexistence of free glutamate with dietary protein, a glutamate-sensing system in the stomach could contribute to the gastric phase of protein digestion.