The anterior piriform cortex is sufficient for detecting depletion of an indispensable amino acid, showing independent cortical sensory function.

Protein synthesis requires a continuous supply of all of the indispensable (essential) amino acids (IAAs). If any IAA is deficient, animals must obtain the limiting amino acid by diet selection. Sensing of IAA deficiency requires an intact anterior piriform cortex (APC), but does it act alone? Shortly after rats begin eating an IAA-deficient diet, the meal ends and EPSPs are activated in the APC; from there, neurons project to feeding circuits; the meal ends within 20 min. Within the APC in vivo, uncharged tRNA activates the general amino acid control non-derepressing 2 (GCN2) enzyme system increasing phosphorylation of eukaryotic initiation factor (P-eIF2α), which blocks general protein synthesis. If this paleocortex is sufficient for sensing IAA depletion, both neuronal activation and P-eIF2α should occur in an isolated APC slice. We used standard techniques for electrophysiology and immunohistochemistry. After rats ate IAA-devoid or -imbalanced diets, their depleted slices responded to different stimuli with increased EPSP amplitudes. Slices from rats fed a control diet were bathed in artificial CSF replete with all amino acids with or without the IAA, threonine, or a tRNA synthetase blocker, l-threoninol, or its inactive isomer, d-threoninol. Thr depletion in vitro increased both EPSP amplitudes and P-eIF2α. l (but not d)-threoninol also increased EPSP amplitudes relative to control. Thus, we show independent excitation of the APC with responses parallel to those known in vivo. These data suggest a novel idea: in addition to classical processing of peripheral sensory input, direct primary sensing may occur in mammalian cortex.

Protein hydrolysate-induced cholecystokinin secretion from enteroendocrine cells is indirectly mediated by the intestinal oligopeptide transporter PepT1.

Dietary protein is a major stimulant for cholecystokinin (CCK) secretion by the intestinal I cell, however, the mechanism by which protein is detected is unknown. Indirect functional evidence suggests that PepT1 may play a role in CCK-mediated changes in gastric motor function. However, it is unclear whether this oligopeptide transporter directly or indirectly activates the I cell. Using both the CCK-expressing enteroendocrine STC-1 cell and acutely isolated native I cells from CCK-enhanced green fluorescent protein (eGFP) mice, we aimed to determine whether PepT1 directly activates the enteroendocrine cell to elicit CCK secretion in response to oligopeptides. Both STC-1 cells and isolated CCK-eGFP cells expressed PepT1 transcripts. STC-1 cells were activated, as measured by ERK(1/2) phosphorylation, by both peptone and the PepT1 substrate Cefaclor; however, the PepT1 inhibitor 4-aminomethyl benzoic acid (AMBA) had no effect on STC-1 cell activity. The PepT1-transportable substrate glycyl-sarcosine dose-dependently decreased gastric motility in anesthetized rats but had no affect on activation of STC-1 cells or on CCK secretion by CCK-eGFP cells. CCK secretion was significantly increased in response to peptone but not to Cefaclor, cephalexin, or Phe-Ala in CCK-eGFP cells. Taken together, the data suggest that PepT1 does not directly mediate CCK secretion in response to PepT1 specific substrates. PepT1, instead, may have an indirect role in protein sensing in the intestine.

Co-localization of phosphorylated extracellular signal-regulated protein kinases 1/2 (ERK1/2) and phosphorylated eukaryotic initiation factor 2alpha (eIF2alpha) in response to a threonine-devoid diet.

The anterior piriform cortex (APC) has been shown to be an essential brain structure for the detection of dietary indispensable amino acid (IAA) deficiency, but little has been known about possible molecular detection mechanisms. Increased phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2alpha (eIF2alpha) has been directly linked to amino acid deficiency in yeast. Recently, we have shown increased phosphorylation of eIF2alpha (p-eIF2alpha) in the rat APC 20 minutes after ingestion of an IAA-deficient meal. We suggest that if phosphorylation of eIF2alpha is an important mechanism in detection of IAA deficiency, then APC neurons that show p-eIF2alpha should also show molecular evidence of potentiation. The present research demonstrates increased expression and co-localization of p-eIF2alpha and phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) in APC neurons, but not in the primary motor or agranular insular cortices in response to an IAA-deficient diet. ERK1/2 is an element of the mitogen-activated protein kinase cascade, an intraneuronal signaling mechanism associated with neuronal activation. The region of the APC that responds to IAA deficiency with increased p-eIF2alpha and p-ERK1/2 labeling ranges from 3.1 to 2.5 mm rostral of bregma. Within this region, only a few neurons respond to IAA deficiency with co-localization of abundant p-eIF2alpha and p-ERK1/2. These chemosensory neurons probably detect IAA deficiency and generate neuronal signaling to other portions of the brain, changing feeding behavior.

Animal models in the investigation of anorexia.

Anorexia nervosa (AN) is an eating disorder of unknown origin that most commonly occurs in women and usually has its onset in adolescence. Patients with AN invariably have a disturbed body image and an intense fear of weight gain. There is currently no definitive treatment for this disease, which carries a 20% mortality over 20 years. Development of an appropriate animal model of AN has been difficult, as the etiology of this eating disorder likely involves a complex interaction between genetic, environmental, social, and cultural factors. In this review, we focus on several possible rodent models of AN. In our laboratory, we have developed and studied three different mouse models of AN based on clinical profiles of the disease; separation stress, activity, and diet restriction (DR). In addition, we discuss the spontaneous mouse mutation anx/anx and several mouse gene knockout models, which have resulted in an anorexic phenotype. We highlight what has been learned from each of these models and possibilities for future models. It is hoped that a combination of the study of such models, together with genetic and clinical studies in patients, will lead to more rational and successful prevention/treatment of this tragic, and often fatal, disease.

Central Fos expression and conditioned flavor avoidance in rats following intragastric administration of bitter taste receptor ligands.

G protein-coupled receptors that signal bitter taste (T2Rs) are expressed in the mucosal lining of the oral cavity and gastrointestinal (GI) tract. In mice, intragastric infusion of T2R ligands activates Fos expression within the caudal viscerosensory portion of the nucleus of the solitary tract (NTS) through a vagal pathway (Hao S, Sternini C, Raybould HE. Am J Physiol Regul Integr Comp Physiol 294: R33-R38, 2008). The present study was performed in rats to further characterize the distribution and chemical phenotypes of brain stem and forebrain neurons activated to express Fos after intragastric gavage of T2R ligands, and to determine a potential behavioral correlate of this central neural activation. Compared with relatively low brain stem and forebrain Fos expression in control rats gavaged intragastrically with water, rats gavaged intragastrically with T2R ligands displayed significantly increased activation of neurons within the caudal medial (visceral) NTS and caudal ventrolateral medulla, including noradrenergic neurons, and within the lateral parabrachial nucleus, central nucleus of the amygdala, and paraventricular nucleus of the hypothalamus. A behavioral correlate of this Fos activation was evidenced when rats avoided consuming flavors that previously were paired with intragastric gavage of T2R ligands. While unconditioned aversive responses to bitter tastants in the oral cavity are often sufficient to inhibit further consumption, a second line of defense may be provided postingestively by ligand-induced signaling at GI T2Rs that signal the brain via vagal sensory inputs to the caudal medulla.

Role of CCK1 and Y2 receptors in activation of hindbrain neurons induced by intragastric administration of bitter taste receptor ligands.

G-protein-coupled receptors signaling bitter taste (T2Rs) in the oral gustatory system and the alpha-subunit of the taste-specific G-protein gustducin are expressed in the gastrointestinal (GI) tract. alpha-Subunit of the taste-specific G-protein gustducin colocalizes with markers of enteroendocrine cells in human and mouse GI mucosa, including peptide YY. Activation of T2Rs increases cholecystokinin (CCK) release from the enteroendocrine cell line, STC-1. The aim of this study was to determine whether T2R agonists in the GI tract activate neurons in the nucleus of the solitary tract (NTS) and whether this activation is mediated by CCK and peptide YY acting at CCK(1) and Y(2) receptors. Immunocytochemistry for the protooncogene c-Fos protein, a marker for neuronal activation, was used to determine activation of neurons in the midregion of the NTS, the region where vagal afferents from the GI tract terminate. Intragastric administration of the T2R agonist denatonium benzoate (DB), or phenylthiocarbamide (PTC), or a combination of T2R agonists significantly increased the number of Fos-positive neurons in the mid-NTS; subdiaphragmatic vagotomy abolished the NTS response to the mixture of T2R agonists. Deletion of CCK(1) receptor gene or blockade of CCK(1) receptors with devazepide abolishes the activation of NTS neurons in response to DB, but had no effect on the response to PTC. Administration of the Y(2) receptor antagonist BIIE0246 blocks the activation of NTS neurons to DB, but not PTC. These findings suggest that activation of neurons in the NTS following administration of T2R agonists to the GI tract involves CCK(1) and Y(2) receptors located on vagal afferent terminals in the gut wall. T2Rs may regulate GI function via release of regulatory peptides and activation of the vagal reflex pathway.

Mechanisms of food intake repression in indispensable amino acid deficiency.

Animals reject diets that lead to indispensable amino acid (IAA) depletion or deficiency. This behavior is adaptive, as continued IAA depletion is incompatible with maintenance of protein synthesis and survival. Following rejection of the diet, animals begin foraging for a better IAA source and develop conditioned aversions to cues associated with the deficient diet. These responses require a sensory system to detect the IAA depletion and alert the appropriate neural circuitry for the behavior. The chemosensor for IAA deprivation is in the highly excitable anterior piriform cortex (APC) of the brain. Recently, the well-conserved general AA control non-derepressing system of yeast was discovered to be activated by IAA deprivation via uncharged tRNA in mammalian APC. This system provides the sensory limb of the mechanism for recognition of IAA depletion that leads to activation of the APC, diet rejection, and subsequent adaptive strategies.

Uncharged tRNA and sensing of amino acid deficiency in mammalian piriform cortex.

Recognizing a deficiency of indispensable amino acids (IAAs) for protein synthesis is vital for dietary selection in metazoans, including humans. Cells in the brain's anterior piriform cortex (APC) are sensitive to IAA deficiency, signaling diet rejection and foraging for complementary IAA sources, but the mechanism is unknown. Here we report that the mechanism for recognizing IAA-deficient foods follows the conserved general control (GC) system, wherein uncharged transfer RNA induces phosphorylation of eukaryotic initiation factor 2 (eIF2) via the GC nonderepressing 2 (GCN2) kinase. Thus, a basic mechanism of nutritional stress management functions in mammalian brain to guide food selection for survival.

Hypothalamic-pituitary-adrenal responses to weight loss in mice following diet restriction, activity or separation stress: effects of tyrosine.

We have studied three different types of weight-loss stress caused by Diet restriction, Activity or Separation, for their effects on the hypothalamic-pituitary axis in young female mice and their responses to tyrosine 100 mg/kg/day. Plasma was assayed for ACTH and glucocorticoid determinations, and brain catecholamine concentrations were measured by HPLC/ECD. A similar weight loss of 24-28% was observed in the models despite significant differences in food intake. Diet restriction to 60% and Separation models produced a significant increase in hypothalamic noradrenaline (p < 0.01), while there was a significant decrease (p < 0.05) in the Diet restriction to 40% that was restored after tyrosine. After Activity, noradrenaline levels did not change. ACTH concentrations decreased following Diet restriction (p < 0.05) but were unaffected by Separation or Activity. The peripheral glucocorticoid response increased significantly after Activity and Diet restriction (p < 0.001), but decreased significantly after Separation (p < 0.001). Tyrosine increased glucocorticoid concentrations in the Activity and Separation models (p < 0.05), but not after Diet restriction. Despite similar weight loss in the three models there were no predictable associations between hypothalamic noradrenaline metabolism and plasma ACTH or glucocorticoid concentrations. Tyrosine might alleviate some of the different pathophysiological problems associated with the stress of weight loss.

Phosphorylation of eIF2alpha is involved in the signaling of indispensable amino acid deficiency in the anterior piriform cortex of the brain in rats.

Sensing of indispensable amino acid (IAA) deficiency, an acute challenge to protein homeostasis, is demonstrated by rats as rejection of IAA-deficient diets within 20 min. The anterior piriform cortex (APC) of the brain in rats and birds is essential for this nutrient sensing, and is activated by IAA deficiency. Yet the mechanisms that sense and transduce IAA reduction to signaling in the APC, or indeed in any animal cells, are unknown. Because rejection of a deficient diet within 20 min is too rapid to be explained by transcription-derived signals, brain tissue was taken from rats after 20 min access to either a threonine-basal, -devoid, or -corrected diet and examined for proteins associated with early signaling of IAA deficiency in the yeast model. Western blots and immunohistochemistry showed that the phosphorylation of eukaryotic initiation factor 2-alpha (p-eIF2alpha[Ser51]) and translation of its downstream product, c-Jun, were increased (47%, P < 0.005, and 55%, P < 0.025, respectively) in APC from rats offered devoid, but not corrected diets, compared with those offered basal diets. This was not seen in other brain areas. In cells intensely labeled for cytoplasmic p-eIF2alpha, there was intense fluorescence for c-Jun in the nucleus. Thus, p-eIF2alpha, which is pivotal in the initiation of global protein translation, and its downstream product, the leucine zipper protein, c-Jun, are increased in the mammalian APC within the time frame necessary for the behavioral response. We suggest that p-eIF2alpha and c-Jun participate in signaling nutrient deficiency in the IAA-sensitive neurons of the APC.