Anthony, Tracy G.a; Gietzen, Dorothy W.b
In metazoans, a requirement for dietary protein is necessary in large part because of the inability to synthesize de novo a subset of amino acids, called the nutritionally indispensable or essential amino acids (EAAs), and because there is no storage depot for EAA per se since release via protein degradation risks loss of protein function. In the absence of EAA biosynthesis and storage, when an animal is faced with a severely inadequate (i.e. devoid, significantly deficient, or imbalanced) EAA supply, an alternative control strategy is necessary to support homeostasis and survival. In the case of omnivores, an almost-immediate behavior ensues: the foodstuff being consumed is promptly, that is, within 20 min  rejected in favor of foraging for other edible choices. Further, if no adequate foods become available, the animal will continue to self-restrict its intake of the insufficient meal no matter how ample its supply and hunt for alternate choices until death. These behavioral traits were originally reported in rodents nearly 100 years ago in the search to explain why severely protein-deprived diets cause growth failure; subsequently, food rejection was instrumental in helping to identify the EAA . Decades of work since then (reviewed in ) describe how this basic response is conserved across all tested species (recently described in pigs [5▪]), is not dependent on taste, olfactory or vagally mediated cues, and is initiated by nutrient sensing in an easily excitable region of the brain called the anterior piriform cortex (APC) (Fig. 1). Whereas the initial signaling events have been identified [3,4], subsequent propagating events extending to the appetitive motor control centers and hedonic areas that reinforce the food rejection behavior are not fully elucidated.
ESSENTIAL AMINO ACID SENSING IN THE ANTERIOR PIRIFORM CORTEX
Consumption of a meal inadequate in EAA reduces concentrations of the deficient or limiting EAA in plasma within minutes of the onset of feeding [2,5▪]. This internal reflection of the diet also occurs in cerebrospinal fluid  and brain  in a remarkably short period of time since dietary amino acids are efficiently moved across the blood–brain barrier via several transporters with overlapping affinity . As dietary EAA drops, the intracellular availability of the deficient EAA is also reduced, resulting in a lower concentration of the cognate tRNA that is aminoacylated . Proximal to the ribosome, the activity of an enzyme called general control nonderepressible kinase 2 (GCN2) is awakened upon interaction with these deacylated or ‘uncharged’ tRNAs, triggering phosphorylation of its only substrate (apart from itself), the mRNA translation factor called eukaryotic initiation factor 2 (eIF2). Phosphorylation of eIF2 effectively slows the assembly of 80S ribosomes onto mRNA, reducing protein synthesis but favoring the translation of a few mRNAs with special features in their 5′ leader sequence . These unusual mRNAs encode transcription factors (e.g. ATF4) which serve to reconfigure gene expression toward adaptive outcomes . The reduction of protein synthesis via initiation of translation occurs quickly, but gene expression following gene-specific translation takes hours, too long to play a role in the 20-min response of animals in detecting an EAA-depleting diet.
Through a series of ablation, microinjection, and dietary studies in transgenic mice and other animal models, the APC became identified as the brain's EAA chemosensor  (Fig. 1). Indeed, the isolated APC shows independent excitation and sensory function to EAA depletion [11▪▪]. Within this area, GCN2 is identified as the ‘first responder’ in sensing an EAA-insufficient meal via binding uncharged tRNA and phosphorylating eIF2 [3,4]. Studies utilizing pharmacological inhibitors further clarify that other neurochemical signaling events, such as phosphorylation of the mitogen-activated protein kinases ERK1/2, and activation of ribosomal protein S6 kinase 1 (S6K1) via mammalian target of rapamycin (mTOR), do not contribute to the nutrient sensing seen at 20 min in the APC . Rather, phosphorylation of the glutamate receptor, GluR1, and loss of GABAergic/inhibitory control contribute to excitation of the APC's recurrent excitatory circuits, which project glutamatergic output to other brain regions . How exactly glutamatergic signaling is activated in the APC remains undefined, but clearly, rapidly turning-over inhibitory proteins cannot be restored while protein synthesis is compromised via eIF2 phosphorylation; such proteins include the potassium-chloride co-transporter, KCC2, which maintains the chloride levels required for GABAergic functioning as an inhibitor in the APC circuitry, and the GABAA receptor itself . Recently, the circuitry projecting from the APC was described in detail based on tract-tracing studies [14▪]. Brain regions that receive output from the APC include those associated with sensing (e.g. olfactory cortex), feeding/reward/preferences (e.g. hypothalamus, striatum), and locomotion. Furthermore, whereas projections to brain regions are mapped, identification of the precise target cells distal to the APC and how they function in the integrated response to EAA insufficiency remain unknown.
THE HYPOTHALAMUS IN SENSING ESSENTIAL AMINO ACID
Evidence that APC connections with the hypothalamus are relevant during EAA insufficiency is revealed by increased intake of EAA-imbalanced diet in rodents with transections of fibers around (but not cell body lesions of) the dorsomedial hypothalamus [14▪]. In addition, the dorsal region of the lateral hypothalamus receives axons directly from the APC and has been shown to be activated in EAA-deficient rats when they are injected into the APC with the limiting EAA . Furthermore, whereas nutrient sensing in the APC is critical for rejection of a novel EAA-deficient meal, longer-term adaptive changes in appetitive and consummatory behavior (i.e. aversion conditioning and metabolic adaptation to mild imbalances) are regulated elsewhere. This is emphasized by the fact that even GCN2 knockout mice demonstrate reduced intake of a leucine-devoid diet over several days [15,16]. These data point out that chronic aversion to EAA deficiency involves mechanisms apart from tRNA charging and brain regions outside the APC.
In this regard, the hypothalamus deserves special attention because it plays a major role in regulating ingestive behavior. Here, several regions direct feeding behavior, including responses to the presence (versus absence) of EAA: the lateral hypothalamus, the ventromedial hypothalamus, and the mediobasal hypothalamus (MBH), among others (Fig. 1). The lateral hypothalamus responds to repletion of a limiting EAA in deficient animals . The MBH is recognized as the location sensing high doses of leucine by mTOR  in an MBH-brainstem circuit which relates to the satiating effect of higher-protein diets , a topic that expands beyond the scope of this review, but see [19,20▪] for recent critical discussions of this issue.
In the context of EAA deficiency, several regulatory neurocircuits within the hypothalamus play important roles in nutrient signaling  and sensing of EAA repletion. Ventral to the MBH, the arcuate nucleus contains centrally projecting neurons which produce appetite regulators in the form of neuropeptide Y (NPY, orexigenic), agouti-related protein (AGRP, orexigenic), and pro-opiomelanocortin (anorexigenic) [22,23]. These neurons innervate the lateral hypothalamus and paraventricular nucleus, and serve to increase and decrease food intake, respectively. For example, starvation activates NPY neurons via increased circulating levels of the hunger-stimulating hormone, ghrelin. Whereas ghrelin is a potent stimulator of food intake, it also stimulates hedonic food anticipatory behavior by activating NPY neurons in the arcuate nucleus. A mechanism by which an orexigenic or anorexigenic peptide mediates the anorexia caused by EAA insufficiency has recently received some attention. Goto et al. found that 6–12 days exposure to a valine-devoid diet produces a hyperghrelinemia similar to starvation. However, subsequent intracerebroventricular (icv) administration of ghrelin, NPY, or AGRP only slightly improved intake of a valine-devoid diet, suggesting redundancy in the system; that is, more than one regulatory system is involved. Indeed, additional work by this group used microarray analysis after 2–3 days (no effect after 24 h) to identify hypothalamic somatostatin as involved in the food rejection behavior, although the precise site of action within the hypothalamus was not identified [24▪]. In the same study, nine novel gene targets were identified, including three unknown genes. Also in the arcuate nucleus of the hypothalamus, a different research team found expression of a novel gene called fat mass and obesity-related transcript (FTO) to be decreased by 6 h of EAA (but not dispensable amino acid) deficiency and in particular by sulfur EAA deficiency . In total, recent studies are showing that changes in signaling and gene expression are occurring in the hypothalamus following ingestion of EAA-deficient diets. How important these events are to regulating behavioral responses are as yet unclear. Further studies are required to delineate the relationship of somatostatin, FTO, and other novel gene products to the adaptation to EAA insufficiency. Furthermore, because several neurochemical systems in the APC have been associated with responses to EAA deficiency at different times after introduction of the deficient diet , it will be important to correlate the time course of the neurochemistry with the behavioral responses such as initial detection versus adaptation, along with the anatomical substrates involved.
ESSENTIAL AMINO ACID DEFICIENCY AND HYPERPHAGIA
Given a choice, rodents reject a diet very low in protein or devoid in EAA . On the contrary, marginally deficient diets produce the opposite effect; a hyperphagia ensues in an attempt to satisfy the need for EAA . This behavior has recently been proposed as playing an important role in energy intake and the development of obesity in humans [27▪▪]. According to the ‘protein leverage hypothesis’, the appetite for protein, and in particular EAA, supersedes that of energy. As such, habitual intake of protein-insufficient foods drives excess energy intake, resulting in weight gain with unfavorable changes in body composition. An apparent exception to this paradigm exists in the feeding of methionine-restricted diets. Feeding methionine-restricted diets induces hyperphagia similar to low-protein diets, but fat deposition is limited. Rodents fed a methionine-restricted diet become leaner and live longer despite consuming more calories . Further, obese adult humans fed a methionine-restricted diet demonstrate increased fat oxidation and reduced intrahepatic lipid content [29▪▪]. Cysteine supplementation reverses these effects in rats, suggesting a specific effect of sulfur-containing amino acids . Soy-fed rats (soy is imbalanced for, but not deficient in, sulphur-containing amino acids) adapted to the imbalance had increased eIF2 phosphorylation and showed increased levels of transcription factors like ATF4 in their livers after 7 days . Whereas the neurobiology is ill-defined, the appetitive effect of methionine restriction (MR) does not appear to involve GCN2 as both wild-type and GCN2-deleted mice demonstrate similar hyperphagia . Rather, the metabolic adaptations to the diet as well as sympathetic outflow to the periphery appear to be influenced by GCN2. These observations are currently under investigation.
Diets devoid of EAA do not support life, but during the period of survival they cause a loss of body fat due to increased energy expenditure and activation of lipolysis genes . Recent work identifies expression of corticotrophin-releasing hormone (CRH) in the hypothalamus as a key event regulating both processes [34▪]. Stress has long been recognized in the responses to EAA deficiency, as adrenalectomized rats were unable to adapt to a severely imbalanced diet, but neither blockade of glucocorticoid synthesis nor adrenalectomy altered the detection of EAA depletion . In the recent work, mice fed a leucine-devoid diet demonstrate activation of the sympathetic nervous system via increased CRH [34▪]. Interestingly, icv administration of leucine to leucine-deficient mice attenuates expression of lipolytic genes in white adipose and thermogenic genes in brown adipose. The mechanisms underlying central nervous system (CNS) regulation of CRH in leucine deficiency require additional study but appear to be mediated via S6K1 . Follow-up experiments clarifying if and how the GCN2-eIF2 pathway contributes to these metabolic adaptations will be important to conduct. Nevertheless, these data provide evidence that signaling in the hypothalamus is relevant not just for ingestive behavior but also for peripheral metabolism. How CNS sensing of EAA-deficient versus devoid diets control ingestive (hypophagia versus hyperphagia) and food-seeking behaviors, as well as metabolic outcomes, are areas in need of additional research.
Additional research is necessary to determine where and how EAA deficiency is recognized in the brain, not only in the hypothalamus, but also in the hedonic and brain stem regions, and how these findings relate to the regulation of protein intake, metabolism, and other physiological functions, such as activation of peripheral nervous systems. Also, the mechanisms involved in adaptations to varying levels of protein and amino acids over the longer term (hours to days) must be determined. Even though mTOR is not part of the signaling in the APC  the role of mTOR as well as that of the GCN2-eIF2 pathway has yet to be fully explored in the hypothalamus. Clearly, there is much work left to be done in understanding how the central events that sense EAA availability regulate and link appetitive and consummatory behavior to peripheral metabolism.
Increased public interest in energy balance and appetite control continues to provoke scientific inquiry into the mechanism by which EAA insufficiency regulates appetite and influences energy metabolism. Although the initial sensor of EAA insufficiency is known, much work remains. Clarification of the signaling and gene expression events occurring in the different regions of the hypothalamus following consumption of EAA-deficient versus EAA-devoid diets is needed. Integrating these signals with the events identified in the APC as well as projections to other brain regions are gaps that require additional investigation.
T.G.A. has received support from NIH (HD070487, GM49164). D.W.G. has received support from NIH (NS43210) and Ajinomoto Co. USA.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
▪ of special interest
▪▪ of outstanding interest
Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 117).
1. Koehnle TJ, Russell MC, Morin AS, et al. Diets deficient in indispensable amino acids rapidly decrease the concentration of the limiting amino acid in the anterior piriform cortex of rats. J Nutr 2004; 134:2365–2371.
2. Gietzen DW, Hao S, Anthony TG. Mechanisms of food intake repression in indispensable amino acid deficiency. Ann Rev Nutr 2007; 27:63–78.
3. Hao S, Sharp JW, Ross-Inta CM, et al. Uncharged tRNA and sensing of amino acid deficiency in mammalian piriform cortex. Science 2005; 307:1776–1778.
4. Maurin AC, Jousse C, Averous J, et al. The GCN2 kinase biases feeding behavior to maintain amino acid homeostasis in omnivores. Cell Metabol 2005; 1:273–277.
5▪. Gloaguen M, Le Floc’h N, Corrent E, et al. Providing a diet deficient in valine but with excess leucine results in a rapid decrease in feed intake and modifies the postprandial plasma amino acid and alpha-keto acid concentrations in pigs. J Anim Sci 2012; 90:3135–3142.
This study demonstrates that a valine-deficient diet reduces food intake in pigs and that circulating valine declines rapidly following ingestion of a valine-deficient diet.
6. Goto S, Nagao K, Bannai M, et al. Anorexia in rats caused by a valine-deficient diet is not ameliorated by systemic ghrelin treatment. Neurosci 2010; 166:333–340.
7. Hawkins RA, O’Kane RL, Simpson IA, Vina JR. Structure of the blood-brain barrier and its role in the transport of amino acids. J Nutr 2006; 136:218S–226S.
8. Huynh LN, Thangavel M, Chen T, et al. Linking tRNA localization with activation of nutritional stress responses. Cell Cycle 2010; 9:3112–3118.
9. Baird TD, Wek RC. Eukaryotic initiation factor 2 phosphorylation and translational control in metabolism. Adv Nutr 2012; 3:307–321.
10. Kilberg MS, Balasubramanian M, Fu L, Shan J. The transcription factor network associated with the amino acid response in mammalian cells. Adv Nutr 2012; 3:295–306.
11▪▪. Rudell JB, Rechs AJ, Kelman TJ, et al. The anterior piriform cortex is sufficient for detecting depletion of an indispensable amino acid, showing independent cortical sensory function. J Neurosci 2011; 31:1583–1590.
This study shows that direct primary sensing of EAA insufficiency occurs in the APC.
12. Hao S, Ross-Inta CM, Gietzen DW. The sensing of essential amino acid deficiency in the anterior piriform cortex, that requires the uncharged tRNA/GCN2 pathway, is sensitive to wortmannin but not rapamycin. Pharmacol Biochem Behav 2010; 94:333–340.
13. Gietzen D. Sensing amino acid deficiency: mechanism of initial brain activation in the anterior piriform cortex. Annals Nutr Metabol 2009; 55:68–69.
14▪. Gietzen DW, Aja SM. The brain's response to an essential amino acid-deficient diet and the circuitous route to a better meal. Molec Neurobiol 2012 [ePub 07 June].
This study uses tract tracing studies to describe the neural projections of the APC following EAA insufficiency.
15. Anthony TG, McDaniel BJ, Byerley RL, et al. Preservation of liver protein synthesis during dietary leucine deprivation occurs at the expense of skeletal muscle mass in mice deleted for eIF2 kinase GCN2. J Biol Chem 2004; 279:36553–36561.
16. Guo F, Cavener DR. The GCN2 eIF2alpha kinase regulates fatty-acid homeostasis in the liver during deprivation of an essential amino acid. Cell Metabol 2007; 5:103–114.
17. Cota D, Proulx K, Smith KA, et al. Hypothalamic mTOR signaling regulates food intake. Science 2006; 312:927–930.
18. Blouet C, Jo YH, Li X, Schwartz GJ. Mediobasal hypothalamic leucine sensing regulates food intake through activation of a hypothalamus-brainstem circuit. J Neurosci 2009; 29:8302–8311.
19. Journel M, Chaumontet C, Darcel N, et al. Brain responses to high-protein diets. Adv Nutr 2012; 3:322–329.
20▪. Morrison CD, Reed SD, Henagan TM. Homeostatic regulation of protein intake: in search of a mechanism. Am J Physiol 2012; 302:R917–928.
An outstanding perspective on the signaling systems in the central nervous system that play an important role in mediating the homeostatic regulation of protein balance.
21. Morton GJ, Cummings DE, Baskin DG, et al. Central nervous system control of food intake and body weight. Nature 2006; 443:289–295.
22. Aponte Y, Atasoy D, Sternson SM. AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training. Nature Neurosci 2011; 14:351–355.
23. Nguyen AD, Mitchell NF, Lin S, et al. Y1 and Y5 receptors are both required for the regulation of food intake and energy homeostasis in mice. PloS One 2012; 7:e40191.
24▪. Nakahara K, Takata S, Ishii A, et al. Somatostatin is involved in anorexia in mice fed a valine-deficient diet. Amino Acids 2012; 42:1397–1404.
This study identifies several novel genes that may be involved in hypothalamic signaling following chronic ingestion of a valine-devoid diet.
25. Cheung MK, Gulati P, O’Rahilly S, Yeo GS. FTO expression is regulated by availability of essential amino acids. Int J Obes (Lond) 2012 [Epub ahead of print]. doi:10.1038/ijo.2012.77
26. White BD, Du F, Higginbotham DA. Low dietary protein is associated with an increase in food intake and a decrease in the in vitro release of radiolabeled glutamate and GABA from the lateral hypothalamus. Nutr Neurosci 2003; 6:361–367.
27▪▪. Gosby AK, Conigrave AD, Lau NS, et al. Testing protein leverage in lean humans: a randomised controlled experimental study. PloS One 2011; 6:e25929.
This clinical trial tests innovative ideas about how a diet low in protein increases appetite and promotes the development of obesity.
28. Hasek BE, Stewart LK, Henagan TM, et al. Dietary methionine restriction enhances metabolic flexibility and increases uncoupled respiration in both fed and fasted states. Am J Physiol 2010; 299:R728–739.
29▪▪. Plaisance EP, Greenway FL, Boudreau A, et al. Dietary methionine restriction increases fat oxidation in obese adults with metabolic syndrome. J Clin Endocrinol Metabol 2011; 96:E836–840.
This clinical trial assesses dietary MR for the purpose of improving lipid profiles and reducing fat deposition in adults with metabolic syndrome.
30. Elshorbagy AK, Valdivia-Garcia M, Mattocks DA, et al. Cysteine supplementation reverses methionine restriction effects on rat adiposity: significance of stearoyl-coenzyme A desaturase. J Lipid Res 2011; 52:104–112.
31. Sikalidis AK, Stipanuk MH. Growing rats respond to a sulfur amino acid-deficient diet by phosphorylation of the alpha subunit of eukaryotic initiation factor 2 heterotrimeric complex and induction of adaptive components of the integrated stress response. J Nutr 2010; 140:1080–1085.
32. Plaisance EP, Van N, Orgeron M, et al. Role of general control nonderepressible 2 (GCN2) kinase in mediating responses to dietary methionine restriction. FASEB J. Experimental Biology 2012. Abstract 255.251.
33. Cheng Y, Meng Q, Wang C, et al. Leucine deprivation decreases fat mass by stimulation of lipolysis in white adipose tissue and upregulation of uncoupling protein 1 (UCP1) in brown adipose tissue. Diabetes 2010; 59:17–25.
34▪. Cheng Y, Zhang Q, Meng Q, et al. Leucine deprivation stimulates fat loss via increasing CRH expression in the hypothalamus and activating the sympathetic nervous system. Mol Endocrinol 2011; 25:1624–1635.
This study shows that increased abdominal fat loss during dietary leucine deprivation is mediated by increased corticotrophin-releasing hormone in the hypothalamus and activation of the sympathetic nervous system.
35. Xia T, Cheng Y, Zhang Q, et al. S6K1 in the central nervous system regulates energy expenditure via MC4R/corticotropin-releasing hormone pathways in response to deprivation of an essential amino acid. Diabetes 2012; 61:2461–2471.
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