Although the short-term effects of fasting or energy deficit on hypothalamic neuropeptide circuitries are now better understood, the effects of long-term energy deficit and refeeding remain to be elucidated. We showed that after a long-term energy deficit, mice exhibited persistent hypoleptinemia following the refeeding period despite restoration of fat mass, ovarian activity, and feeding behavior. We aimed to examine the hypothalamic adaptations after 10 weeks of energy deficit and after 10 further weeks of nutritional recovery. To do so, we assessed the mRNA levels of the leptin receptor and the main orexigenic and anorexigenic peptides, and their receptors regulated by leptin. Markers of hypothalamic inflammation were assessed as leptin can also participate in this phenomenon. Long-term time-restricted feeding and separation induced significant increase in mRNA levels of hypothalamic orexigenic peptides, while both Y1 and Y5 receptor mRNAs were downregulated. No changes occurred in the mRNA levels of orexin (OX), melanin-concentrating hormone, pro-opiomelanocortin, 26RFa (26-amino acid RF-amide peptide), and their receptors despite an increase in the expression of melanocortin receptors (MC3-R and MC4-R) and OXR1 (OX receptor 1). The refeeding period induced an overexpression of leptin receptor mRNA in the hypothalamus. The other assessed mRNA levels were normalized except for Y2, Y5, MC3-R, and MC4-R, which remained upregulated. No convincing changes were observed in neuroinflammatory markers, even if interleukin-1β mRNA levels were increased in parallel with those of Iba1 (ionized calcium-binding adaptor molecule 1), a marker of microglial activation. Normalization of leptin-regulated functions and hypothalamic gene expressions in refed mice with low plasma leptin levels could be sustained by recalibration of hypothalamic sensitivity to leptin.

Keywords:
Animal model, Refeeding, Long-term energy deficit, Hypothalamus, Leptin, Anorexigenic peptides, Orexigenic peptides, Anorexia nervosa
1.
Zgheib S, Méquinion M, Lucas S, Leterme D, Ghali O, Tolle V, et al: Long-term physiological alterations and recovery in a mouse model of separation associated with time-restricted feeding: a tool to study anorexia nervosa related consequences. PLoS One 2014;9: e103775.
2.
van Leeuwen SD, Bonne OB, Avraham Y, Berry EM: Separation as a new animal model for self-induced weight loss. Physiol Behav 1997;62:77-81.
3.
Carrera O, Fraga Á, Pellón R, Gutiérrez E: Rodent model of activity-based anorexia. Curr Protoc Neurosci 2014;67:9.47.1-11.
4.
Chowdhury TG, Chen Y-W, Aoki C: Using the activity-based anorexia rodent model to study the neurobiological basis of anorexia nervosa. J Vis Exp JoVE 2015;e52927.
5.
Balland E, Dam J, Langlet F, Caron E, Steculorum S, Messina A, et al: Hypothalamic tanycytes are an ERK-gated conduit for leptin into the brain. Cell Metab 2014;19:293-301.
6.
Cowley MA: Hypothalamic melanocortin neurons integrate signals of energy state. Eur J Pharmacol 2003;480:3-11.
7.
Marks DL, Hruby V, Brookhart G, Cone RD: The regulation of food intake by selective stimulation of the type 3 melanocortin receptor (MC3R). Peptides 2006;27:259-264.
8.
Gelez H, Poirier S, Facchinetti P, Allers KA, Wayman C, Bernabé J, et al: Neuroanatomical distribution of the melanocortin-4 receptors in male and female rodent brain. J Chem Neuroanat 2010;40:310-324.
9.
Xu T-R, Yang Y, Ward R, Gao L, Liu Y: Orexin receptors: multi-functional therapeutic targets for sleeping disorders, eating disorders, drug addiction, cancers and other physiological disorders. Cell Signal 2013;25:2413-2423.
10.
Qu D, Ludwig DS, Gammeltoft S, Piper M, Pelleymounter MA, Cullen MJ, et al: A role for melanin-concentrating hormone in the central regulation of feeding behaviour. Nature 1996;380:243-247.
11.
Chartrel N, Dujardin C, Anouar Y, Leprince J, Decker A, Clerens S, et al: Identification of 26RFa, a hypothalamic neuropeptide of the RFamide peptide family with orexigenic activity. Proc Natl Acad Sci USA 2003;100:15247-15252.
12.
Paz-Filho G, Mastronardi C, Franco CB, Wang KB, Wong M-L, Licinio J: Leptin: molecular mechanisms, systemic pro-inflammatory effects, and clinical implications. Arq Bras Endocrinol Metabol 2012;56:597-607.
13.
Tang C-H, Lu D-Y, Yang R-S, Tsai H-Y, Kao M-C, Fu W-M, et al: Leptin-induced IL-6 production is mediated by leptin receptor, insulin receptor substrate-1, phosphatidylinositol 3- kinase, Akt, NF-kappaB, and p300 pathway in microglia. J Immunol 2007;179:1292-1302.
14.
Wisse BE, Ogimoto K, Morton GJ, Wilkinson CW, Frayo RS, Cummings DE, et al: Physiological regulation of hypothalamic IL-1beta gene expression by leptin and glucocorticoids: implications for energy homeostasis. Am J Physiol Endocrinol Metab 2004;287: E1107-E1113.
15.
Lafrance V, Inoue W, Kan B, Luheshi GN: Leptin modulates cell morphology and cytokine release in microglia. Brain Behav Immun 2010;24:358-365.
16.
Gao Y, Ottaway N, Schriever SC, Legutko B, García-Cáceres C, de la Fuente E, et al: Hormones and diet, but not body weight, control hypothalamic microglial activity. Glia 2014;62:17-25.
17.
de Git KCG, Adan RAH: Leptin resistance in diet-induced obesity: the role of hypothalamic inflammation: leptin resistance and inflammation. Obes Rev 2015;16:207-224.
18.
Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-159.
19.
Radler ME, Hale MW, Kent S: Calorie restriction attenuates lipopolysaccharide (LPS)-induced microglial activation in discrete regions of the hypothalamus and the subfornical organ. Brain Behav Immun 2014;38:13-24.
20.
Govic A, Levay EA, Hazi A, Penman J, Kent S, Paolini AG: Alterations in male sexual behaviour, attractiveness and testosterone levels induced by an adult-onset calorie restriction regimen. Behav Brain Res 2008;190:140-146.
21.
Sonti G, Ilyin SE, Plata-Salamán CR: Neuropeptide Y blocks and reverses interleukin-1 beta-induced anorexia in rats. Peptides 1996;17:517-520.
22.
Ferreira R, Xapelli S, Santos T, Silva AP, Cristóvão A, Cortes L, et al: Neuropeptide Y modulation of interleukin-1β (IL-1β)-induced nitric oxide production in microglia. J Biol Chem 2010;285:41921-41934.
23.
Felies M, von Hörsten S, Pabst R, Nave H: Neuropeptide Y stabilizes body temperature and prevents hypotension in endotoxaemic rats. J Physiol 2004;561:245-252.
24.
Stephens TW, Basinski M, Bristow PK, Bue-Valleskey JM, Burgett SG, Craft L, et al: The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature 1995;377:530-532.
25.
de Rijke CE, Hillebrand JJG, Verhagen LA, Roeling TA, Adan RA: Hypothalamic neuropeptide expression following chronic food restriction in sedentary and wheel-running rats. J Mol Endocrinol 2005;35:381-390.
26.
Mercer RE, Chee MJS, Colmers WF: The role of NPY in hypothalamic mediated food intake. Front Neuroendocrinol 2011;32:398-415.
27.
Acuna-Goycolea C, Tamamaki N, Yanagawa Y, Obata K, van den Pol AN: Mechanisms of neuropeptide Y, peptide YY, and pancreatic polypeptide inhibition of identified green fluorescent protein-expressing GABA neurons in the hypothalamic neuroendocrine arcuate nucleus. J Neurosci Off J Soc Neurosci 2005;25:7406-419.
28.
Galas L, Tonon M-C, Beaujean D, Fredriksson R, Larhammar D, Lihrmann I, et al: Neuropeptide Y inhibits spontaneous alpha-melanocyte-stimulating hormone (alpha-MSH) release via a Y(5) receptor and suppresses thyrotropin-releasing hormone-induced alpha-MSH secretion via a Y(1) receptor in frog melanotrope cells. Endocrinology 2002;143:1686-1694.
29.
King PJ, Widdowson PS, Doods HN, Williams G: Regulation of neuropeptide Y release by neuropeptide Y receptor ligands and calcium channel antagonists in hypothalamic slices. J Neurochem 1999;73:641-646.
30.
King PJ, Williams G, Doods H, Widdowson PS: Effect of a selective neuropeptide Y Y(2) receptor antagonist, BIIE0246 on neuropeptide Y release. Eur J Pharmacol 2000;396:R1-R3.
31.
Tolle V, Low MJ: In vivo evidence for inverse agonism of Agouti-related peptide in the central nervous system of proopiomelanocortin-deficient mice. Diabetes 2008;57:86-94.
32.
Harrold JA, Widdowson PS, Williams G: Altered energy balance causes selective changes in melanocortin-4 (MC4-R), but not melanocortin-3 (MC3-R), receptors in specific hypothalamic regions: further evidence that activation of MC4-R is a physiological inhibitor of feeding. Diabetes 1999;48:267-271.
33.
Gelegen C, Collier DA, Campbell IC, Oppelaar H, Kas MJH: Behavioral, physiological, and molecular differences in response to dietary restriction in three inbred mouse strains. Am J Physiol Endocrinol Metab 2006;291: E574-E581.
34.
Sakurai T: Orexins and orexin receptors: implication in feeding behavior. Regul Pept 1999;85:25-30.
35.
López M, Seoane L, García MC, Lago F, Casanueva FF, Señarís R, et al: Leptin regulation of prepro-orexin and orexin receptor mRNA levels in the hypothalamus. Biochem Biophys Res Commun 2000;269:41-45.
36.
Girault EM, Yi C-X, Fliers E, Kalsbeek A: Orexins, feeding, and energy balance. Prog Brain Res 2012;198:47-64.
37.
Forray C: The MCH receptor family: feeding brain disorders? Curr Opin Pharmacol 2003;3:85-89.
38.
Shimada M, Tritos NA, Lowell BB, Flier JS, Maratos-Flier E: Mice lacking melanin-concentrating hormone are hypophagic and lean. Nature 1998;396:670-674.
39.
Marsh DJ, Weingarth DT, Novi DE, Chen HY, Trumbauer ME, Chen AS, et al: Melanin-concentrating hormone 1 receptor-deficient mice are lean, hyperactive, and hyperphagic and have altered metabolism. Proc Natl Acad Sci USA 2002;99:3240-3245.
40.
Takayasu S, Sakurai T, Iwasaki S, Teranishi H, Yamanaka A, Williams SC, et al: A neuropeptide ligand of the G protein-coupled receptor GPR103 regulates feeding, behavioral arousal, and blood pressure in mice. Proc Natl Acad Sci USA 2006;103:7438-7443.
41.
Moriya R, Sano H, Umeda T, Ito M, Takahashi Y, Matsuda M, et al: RFamide peptide QRFP43 causes obesity with hyperphagia and reduced thermogenesis in mice. Endocrinology 2006;147:2916-2922.
42.
Lectez B, Jeandel L, El-Yamani F-Z, Arthaud S, Alexandre D, Mardargent A, et al: The orexigenic activity of the hypothalamic neuropeptide 26RFa is mediated by the neuropeptide Y and proopiomelanocortin neurons of the arcuate nucleus. Endocrinology 2009;150:2342-2350.
43.
Primeaux SD: QRFP in female rats: effects on high fat food intake and hypothalamic gene expression across the estrous cycle. Peptides 2011;32:1270-1275.
44.
Galusca B, Jeandel L, Germain N, Alexandre D, Leprince J, Anouar Y, et al: Orexigenic neuropeptide 26RFa: new evidence for an adaptive profile of appetite regulation in anorexia nervosa. J Clin Endocrinol Metab 2012;97:2012-2018.
45.
Swart I, Jahng JW, Overton JM, Houpt TA: Hypothalamic NPY, AGRP, and POMC mRNA responses to leptin and refeeding in mice. Am J Physiol Regul Integr Comp Physiol 2002;283:R1020-R1026.
46.
Becskei C, Lutz TA, Riediger T: Blunted fasting-induced hypothalamic activation and refeeding hyperphagia in late-onset obesity. Neuroendocrinology 2009;90:371-382.
47.
Wu Q, Lemus MB, Stark R, Bayliss JA, Reichenbach A, Lockie SH, et al: The temporal pattern of cfos activation in hypothalamic, cortical, and brainstem nuclei in response to fasting and refeeding in male mice. Endocrinology 2014;155:840-853.
48.
Sucajtys-Szulc E, Goyke E, Korczynska J, Stelmanska E, Rutkowski B, Swierczynski J: Refeeding after prolonged food restriction differentially affects hypothalamic and adipose tissue leptin gene expression. Neuropeptides 2009;43:321-325.
49.
Modan-Moses D, Stein D, Pariente C, Yaroslavsky A, Ram A, Faigin M, et al: Modulation of adiponectin and leptin during refeeding of female anorexia nervosa patients. J Clin Endocrinol Metab 2007;92:1843-1847.
50.
Seitz J, Bühren K, Biemann R, Timmesfeld N, Dempfle A, Winter SM, et al: Leptin levels in patients with anorexia nervosa following day/inpatient treatment do not predict weight 1 year post-referral. Eur Child Adolesc Psychiatry 2016;25:1019-1025.
51.
Luheshi GN, Gardner JD, Rushforth DA, Loudon AS, Rothwell NJ: Leptin actions on food intake and body temperature are mediated by IL-1. Proc Natl Acad Sci USA 1999;96:7047-7052.
52.
Otero M, Lago R, Gomez R, Dieguez C, Lago F, Gómez-Reino J, et al: Towards a pro-inflammatory and immunomodulatory emerging role of leptin. Rheumatol Oxf Engl 2006;45:944-950.
53.
Lago R, Gómez R, Lago F, Gómez-Reino J, Gualillo O: Leptin beyond body weight regulation - current concepts concerning its role in immune function and inflammation. Cell Immunol 2008;252:139-145.
54.
Pinteaux E, Inoue W, Schmidt L, Molina-Holgado F, Rothwell NJ, Luheshi GN: Leptin induces interleukin-1beta release from rat microglial cells through a caspase 1 independent mechanism. J Neurochem 2007;102:826-833.
55.
Imai Y, Kohsaka S: Intracellular signaling in M-CSF-induced microglia activation: role of Iba1. Glia 2002;40:164-174.
56.
Aguilar-Valles A, Aguliar-Valles A, Kim J, Jung S, Woodside B, Luheshi GN: Role of brain transmigrating neutrophils in depression-like behavior during systemic infection. Mol Psychiatry 2014;19:599-606.
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2016
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