AgRP neurons are not indispensable for body weight maintenance in adult mice

•AgRP neurons were selectively ablated in adult mice with DTX (i.c.v.) or AAV caspase-3•Ablation of AgRP neurons in adult mice does not affect normal feeding and body weight•Ablation of AgRP neurons blunts fasting refeeding In addition to their role in promoting feeding and obesity development, hypothalamic arcuate agouti-related protein/neuropeptide Y (AgRP/NPY) neurons are widely perceived to be indispensable for maintaining normal feeding and body weight in adults, and consistently, acute inhibition of AgRP neurons is known to reduce short-term food intake. Here, we adopted complementary methods to achieve nearly complete ablation of arcuate AgRP/NPY neurons in adult mice and report that lesioning arcuate AgRP/NPY neurons in adult mice causes no apparent alterations in ad libitum feeding or body weight. Consistent with previous studies, loss of AgRP/NPY neurons blunts fasting refeeding. Thus, our studies show that AgRP/NPY neurons are not required for maintaining ad libitum feeding or body weight homeostasis in adult mice. In addition to their role in promoting feeding and obesity development, hypothalamic arcuate agouti-related protein/neuropeptide Y (AgRP/NPY) neurons are widely perceived to be indispensable for maintaining normal feeding and body weight in adults, and consistently, acute inhibition of AgRP neurons is known to reduce short-term food intake. Here, we adopted complementary methods to achieve nearly complete ablation of arcuate AgRP/NPY neurons in adult mice and report that lesioning arcuate AgRP/NPY neurons in adult mice causes no apparent alterations in ad libitum feeding or body weight. Consistent with previous studies, loss of AgRP/NPY neurons blunts fasting refeeding. Thus, our studies show that AgRP/NPY neurons are not required for maintaining ad libitum feeding or body weight homeostasis in adult mice. Studies from recent decades have established the importance of the melanocortin pathway in body weight regulation in both rodents and humans. Arcuate agouti-related protein/neuropeptide Y (AgRP/NPY) neurons in the melanocortin pathway have emerged as a key regulator in feeding and related behaviors pertinent to environmental adaption.1Deem J.D. Faber C.L. Morton G.J. AgRP neurons: Regulators of feeding, energy expenditure, and behavior.FEBS J. 2022; 289: 2362-2381https://doi.org/10.1111/febs.16176Google Scholar Recent optogenetic and chemogenetic studies have demonstrated a compelling role for AgRP neurons in promoting feeding and positive energy balance.2Aponte Y. Atasoy D. Sternson S.M. AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training.Nat. Neurosci. 2011; 14: 351-355https://doi.org/10.1038/nn.2739Google Scholar,3Krashes M.J. Koda S. Ye C. Rogan S.C. Adams A.C. Cusher D.S. Maratos-Flier E. Roth B.L. Lowell B.B. Rapid, reversible activation of AgRP neurons drives feeding behavior in mice.J. Clin. Invest. 2011; 121: 1424-1428https://doi.org/10.1172/JCI46229Google Scholar Consistently, chronic activation of AgRP neurons through targeted deletion of leptin receptors or expression of neuron-activating channels leads to massive obesity comparable to the body weight increase caused by leptin deficiency.4Xu J. Bartolome C.L. Low C.S. Yi X. Chien C.H. Wang P. Kong D. Genetic identification of leptin neural circuits in energy and glucose homeostases.Nature. 2018; 556: 505-509https://doi.org/10.1038/s41586-018-0049-7Google Scholar,5Zhu C. Jiang Z. Xu Y. Cai Z.L. Jiang Q. Xu Y. Xue M. Arenkiel B.R. Wu Q. Shu G. Tong Q. Profound and redundant functions of arcuate neurons in obesity development.Nat. Metab. 2020; 2: 763-774https://doi.org/10.1038/s42255-020-0229-2Google Scholar These results collectively show that AgRP neurons are sufficient to drive feeding and obesity development. In addition to their role in promoting feeding, AgRP neurons have also been widely perceived to be indispensable for maintaining normal feeding and body weight.6Bewick G.A. Gardiner J.V. Dhillo W.S. Kent A.S. White N.E. Webster Z. Ghatei M.A. Bloom S.R. Post-embryonic ablation of AgRP neurons in mice leads to a lean, hypophagic phenotype.Faseb. J. 2005; 19: 1680-1682https://doi.org/10.1096/fj.04-3434fjeGoogle Scholar,7Gropp E. Shanabrough M. Borok E. Xu A.W. Janoschek R. Buch T. Plum L. Balthasar N. Hampel B. Waisman A. et al.Agouti-related peptide-expressing neurons are mandatory for feeding.Nat. Neurosci. 2005; 8: 1289-1291https://doi.org/10.1038/nn1548Google Scholar,8Luquet S. Perez F.A. Hnasko T.S. Palmiter R.D. NPY/AgRP neurons are essential for feeding in adult mice but can be ablated in neonates.Science. 2005; 310: 683-685https://doi.org/10.1126/science.1115524Google Scholar Acute inhibition of AgRP neurons reduces short-term feeding in mice.3Krashes M.J. Koda S. Ye C. Rogan S.C. Adams A.C. Cusher D.S. Maratos-Flier E. Roth B.L. Lowell B.B. Rapid, reversible activation of AgRP neurons drives feeding behavior in mice.J. Clin. Invest. 2011; 121: 1424-1428https://doi.org/10.1172/JCI46229Google Scholar,9Betley J.N. Xu S. Cao Z.F.H. Gong R. Magnus C.J. Yu Y. Sternson S.M. Neurons for hunger and thirst transmit a negative-valence teaching signal.Nature. 2015; 521: 180-185https://doi.org/10.1038/nature14416Google Scholar,10Denis R.G.P. Joly-Amado A. Webber E. Langlet F. Schaeffer M. Padilla S.L. Cansell C. Dehouck B. Castel J. Delbès A.S. et al.Palatability Can Drive Feeding Independent of AgRP Neurons.Cell Metab. 2017; 25: 975https://doi.org/10.1016/j.cmet.2017.03.001Google Scholar Based on a mouse model with selective expression of diphtheria toxin receptors (DTRs) in AgRP neurons, whereas the lesion of AgRP neurons by systemic delivery of diphtheria toxin (DTX) produced a little effect in neonates, it caused a rapid arrest in feeding and lethal weight loss in adult mice, which could last up to 2 weeks in surviving obese leptin-deficient mice.8Luquet S. Perez F.A. Hnasko T.S. Palmiter R.D. NPY/AgRP neurons are essential for feeding in adult mice but can be ablated in neonates.Science. 2005; 310: 683-685https://doi.org/10.1126/science.1115524Google Scholar,11Wu Q. Whiddon B.B. Palmiter R.D. Ablation of neurons expressing agouti-related protein, but not melanin concentrating hormone, in leptin-deficient mice restores metabolic functions and fertility.Proc. Natl. Acad. Sci. USA. 2012; 109: 3155-3160https://doi.org/10.1073/pnas.1120501109Google Scholar Other studies showed that postembryonic partial loss of AgRP neurons led to a lean and hypophagic phenotype.6Bewick G.A. Gardiner J.V. Dhillo W.S. Kent A.S. White N.E. Webster Z. Ghatei M.A. Bloom S.R. Post-embryonic ablation of AgRP neurons in mice leads to a lean, hypophagic phenotype.Faseb. J. 2005; 19: 1680-1682https://doi.org/10.1096/fj.04-3434fjeGoogle Scholar,7Gropp E. Shanabrough M. Borok E. Xu A.W. Janoschek R. Buch T. Plum L. Balthasar N. Hampel B. Waisman A. et al.Agouti-related peptide-expressing neurons are mandatory for feeding.Nat. Neurosci. 2005; 8: 1289-1291https://doi.org/10.1038/nn1548Google Scholar However, recent studies with chronic or chemogenetic inhibition of AgRP neurons showed no or minimal impact on body weight,5Zhu C. Jiang Z. Xu Y. Cai Z.L. Jiang Q. Xu Y. Xue M. Arenkiel B.R. Wu Q. Shu G. Tong Q. Profound and redundant functions of arcuate neurons in obesity development.Nat. Metab. 2020; 2: 763-774https://doi.org/10.1038/s42255-020-0229-2Google Scholar,12Üner A.G. Keçik O. Quaresma P.G.F. De Araujo T.M. Lee H. Li W. Kim H.J. Chung M. Bjørbæk C. Kim Y.B. Role of POMC and AgRP neuronal activities on glycaemia in mice.Sci. Rep. 2019; 913068https://doi.org/10.1038/s41598-019-49295-7Google Scholar raising the question of whether AgRP neurons are required for feeding and body weight regulation. To address this issue, we first employed the DTX-induced AgRP neuron lesion approach in adult mice by using the previously described adult AgRP-DTR mouse strain.7Gropp E. Shanabrough M. Borok E. Xu A.W. Janoschek R. Buch T. Plum L. Balthasar N. Hampel B. Waisman A. et al.Agouti-related peptide-expressing neurons are mandatory for feeding.Nat. Neurosci. 2005; 8: 1289-1291https://doi.org/10.1038/nn1548Google Scholar,8Luquet S. Perez F.A. Hnasko T.S. Palmiter R.D. NPY/AgRP neurons are essential for feeding in adult mice but can be ablated in neonates.Science. 2005; 310: 683-685https://doi.org/10.1126/science.1115524Google Scholar Since AgRP and NPY are nearly completely colocalized in the hypothalamic arcuate (ARC),13Hahn T.M. Breininger J.F. Baskin D.G. Schwartz M.W. Coexpression of Agrp and NPY in fasting-activated hypothalamic neurons.Nat. Neurosci. 1998; 1: 271-272https://doi.org/10.1038/1082Google Scholar we visualized AgRP neurons with GFP expression in AgRP-DTR::NPY-GFP mice. We utilized intracerebroventricular (i.c.v.) delivery of DTX to limit the effects of ablating peripheral AgRP-expressing cells14Gupta R. Ma Y. Wang M. Whim M.D. AgRP-Expressing Adrenal Chromaffin Cells Are Involved in the Sympathetic Response to Fasting.Endocrinology. 2017; 158: 2572-2584https://doi.org/10.1210/en.2016-1268Google Scholar (Figure 1A). Due to the potentially toxic effects of DTX,15Goldwich A. Steinkasserer A. Gessner A. Amann K. Impairment of podocyte function by diphtheria toxin--a new reversible proteinuria model in mice.Lab. Invest. 2012; 92: 1674-1685https://doi.org/10.1038/labinvest.2012.133Google Scholar,16Chapman T.J. Georas S.N. Adjuvant effect of diphtheria toxin after mucosal administration in both wild type and diphtheria toxin receptor engineered mouse strains.J. Immunol. Methods. 2013; 400-401: 122-126https://doi.org/10.1016/j.jim.2013.10.010Google Scholar,17Cerpa V. Gonzalez A. Richerson G.B. Diphtheria toxin treatment of Pet-1-Cre floxed diphtheria toxin receptor mice disrupts thermoregulation without affecting respiratory chemoreception.Neuroscience. 2014; 279: 65-76https://doi.org/10.1016/j.neuroscience.2014.08.018Google Scholar,18Valek L. Tegeder I. Failure of Diphtheria Toxin Model to Induce Parkinson-Like Behavior in Mice.Int. J. Mol. Sci. 2021; 229496https://doi.org/10.3390/ijms22179496Google Scholar we performed studies with the administration of DTX at the dose of 5 ng since it could cause complete lesions of brain neurons with DTR expression.19Xi D. Gandhi N. Lai M. Kublaoui B.M. Ablation of Sim1 neurons causes obesity through hyperphagia and reduced energy expenditure.PLoS One. 2012; 7e36453https://doi.org/10.1371/journal.pone.0036453Google Scholar Indeed, i.c.v. injections of 5 ng DTX in 8- to 10-week-old mice ablated almost all NPY-GFP neurons and AgRP-immunoreactive (AgRP-ir) signals in the paraventricular nucleus (PVN) (Figures 1B, 1C, S1, and S2). The ablation is almost complete since the same i.c.v. injection with a double amount of toxin (10 ng) failed to increase the ablation efficiency (Figure S3). A few neurons retained GFP expression (Figures 1B, 1C, and S1), which is presumably due to NPY expression in a minor number of non-AgRP neurons within the ARC.20Campbell J.N. Macosko E.Z. Fenselau H. Pers T.H. Lyubetskaya A. Tenen D. Goldman M. Verstegen A.M.J. Resch J.M. McCarroll S.A. et al.A molecular census of arcuate hypothalamus and median eminence cell types.Nat. Neurosci. 2017; 20: 484-496https://doi.org/10.1038/nn.4495Google Scholar,21Chen R. Wu X. Jiang L. Zhang Y. Single-Cell RNA-Seq Reveals Hypothalamic Cell Diversity.Cell Rep. 2017; 18: 3227-3241https://doi.org/10.1016/j.celrep.2017.03.004Google Scholar We also measured individual AgRP transcripts using RNAscope probes against AgRP, which showed a virtual absence of the transcripts, confirming a complete ablation of AgRP neurons (Figures 1D and S4). Compared to NPY-GFP littermate controls, AgRP-DTR::NPY-GFP mice showed no changes in daily body weight during the first 2 weeks (Figure 1E) or weekly body weight within 8 weeks (Figure 1F) or in daily ad libitum food intake (Figure 1G) post 5 ng DTX delivery, suggesting that AgRP neurons are not required for ad libitum feeding or energy balance. Surprisingly, i.c.v. injection of 40 ng DTX, which was used in a previous study for the lesion of AgRP neurons,8Luquet S. Perez F.A. Hnasko T.S. Palmiter R.D. NPY/AgRP neurons are essential for feeding in adult mice but can be ablated in neonates.Science. 2005; 310: 683-685https://doi.org/10.1126/science.1115524Google Scholar led to rapid lethal phenotypes with a feeding arrest within the first 10 days after injections in both NPY-GFP and AgRP-DTR::NPY-GFP mice (Figures 1E–1H), suggesting a nonspecific toxic effect. It is important to note that at the time point when feeding was arrested in mice, a significant number of GFP neurons remained in the ARC (Figure S3), suggesting that the observed lethality was not due to AgRP neuron lesions. In fact, it was not until 12 days post injection that DTX induced a nearly complete lesion (Figure S3). However, 5 ng DTX had no impact on survival in either group (Figure 1H). Of note, we also observed no impact of AgRP neuron lesions on ad libitum feeding or body weight in female AgRP-DTR mice (Figure 2). In line with previous observations,22Tan K. Knight Z.A. Friedman J.M. Ablation of AgRP neurons impairs adaption to restricted feeding.Mol. Metab. 2014; 3: 694-704https://doi.org/10.1016/j.molmet.2014.07.002Google Scholar AgRP lesions caused reduced fasting refeeding (Figure 1I). To avoid toxicity issues associated with using DTX, we adopted another approach by expressing caspase-3, which is known to induce targeted cell apoptosis.23Yang C.F. Chiang M.C. Gray D.C. Prabhakaran M. Alvarado M. Juntti S.A. Unger E.K. Wells J.A. Shah N.M. Sexually dimorphic neurons in the ventromedial hypothalamus govern mating in both sexes and aggression in males.Cell. 2013; 153: 896-909https://doi.org/10.1016/j.cell.2013.04.017Google Scholar We bred AgRP-Cre mice with Ai14 reporter mice to track and visualize AgRP neurons (referred to as AgRP-Cre::Ai14 mice). The AAV-flex-taCasp3-TEVp viral vectors were bilaterally injected into the ARC of 8- to 10-week-old AgRP-Cre::Ai14 mice to ablate AgRP neurons (Figure 3A). Sham-injected littermates were used as controls. Expression of caspase-3 effectively ablated ARC AgRP neurons approximately 2 weeks after viral injection (Figures 3B and S5A). We injected AAV-flex-taCasp3-TEVp into 31 mice of both sexes, among which 19 mice exhibited nearly complete loss of AgRP neurons, with only 26–245 neurons remaining (Figures 3C and S5B), as well as less than 1% AgRP expression in downstream areas such as the PVN, paraventricular thalamic nucleus, dorsomedial hypothalamic nucleus, and lateral hypothalamus compared to control mice (Figures 3B, 3D, and 3E). There were only approximately 100 or fewer remaining AgRP neurons in the ARC of 10 mice that were screened. Correlation analysis showed that the body weight changes at weeks 4 and 8 after viral injection were unrelated to the number of remaining AgRP neurons (Figures 3F and 4A ). We did not observe any starvation or moribund phenotype in all mice.Figure 4AgRP neuron lesion did not impair body weight gain and ad libitum feeding in female adult miceShow full caption(A) The correlation between the numbers of remaining AgRP neurons and body weight gain at week 8 after viral injection. Each symbol represents an individual mouse. n = 19 mice of both sexes.(B and C) Comparison in weekly absolute body weight (B) and normalized body weight change (C) between control and AAV-flex-taCasp3-TEVp injection groups after viral delivery. Two-way ANOVA followed by Bonferroni’s multiple comparisons, p = 0.3388 in (B), p = 0.9669 in (C). ns, not significant.(D and E) Daily food intake between the control and AAV-flex-taCasp3-TEVp injection groups over 3–5 consecutive days at weeks 5 (D) and 9 (E) after viral delivery. Two-way ANOVA followed by Bonferroni’s multiple comparisons, p = 0.7977 in (D), p = 0.8118 in (E). All data represent the mean ± SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) The correlation between the numbers of remaining AgRP neurons and body weight gain at week 8 after viral injection. Each symbol represents an individual mouse. n = 19 mice of both sexes. (B and C) Comparison in weekly absolute body weight (B) and normalized body weight change (C) between control and AAV-flex-taCasp3-TEVp injection groups after viral delivery. Two-way ANOVA followed by Bonferroni’s multiple comparisons, p = 0.3388 in (B), p = 0.9669 in (C). ns, not significant. (D and E) Daily food intake between the control and AAV-flex-taCasp3-TEVp injection groups over 3–5 consecutive days at weeks 5 (D) and 9 (E) after viral delivery. Two-way ANOVA followed by Bonferroni’s multiple comparisons, p = 0.7977 in (D), p = 0.8118 in (E). All data represent the mean ± SEM. We compared the body weight change and food intake of 19 completely ablated mice with controls. In corroboration with the results from DTX-induced ablation, caspase-3-mediated ablations showed no effect on body weight over 8 weeks in either male or female adult mice (Figures 3G, 3H, 4B, and 4C). We did not observe any significant difference in daily ad libitum food intake at weeks 5 and 9 after viral injections between controls and AgRP-ablated mice (Figures 3I, 3J, 4D, and 4E). Consistent with the DTX-induced ablations, caspase-3-mediated ablations significantly impaired refeeding after a 24-h fast (Figure 3K). Thus, based on two compelling animal models with AgRP neuron lesions associated with no alterations in ad libitum feeding or body weight, we conclude that AgRP neurons are not indispensable for ad libitum feeding or body weight regulation. It is unlikely that the maintenance of body weight and ad libitum food intake were due to incomplete ablation. Prior optogenetic work has demonstrated that increased food intake was observed only in mice with more than 300 activated AgRP neurons, whereas there was no feeding stimulation effect in those containing fewer than 100 activated AgRP neurons.2Aponte Y. Atasoy D. Sternson S.M. AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training.Nat. Neurosci. 2011; 14: 351-355https://doi.org/10.1038/nn.2739Google Scholar In our caspase-3-mediated ablation experiments, the number of remaining AgRP neurons in 19 completely ablated mice was less than 300. Even when there were approximately 100 or fewer AgRP neurons left, the mice still exhibited normal body weight gain (Figures 3F and 4A). It is currently unknown why previous studies with an intraperitoneal (i.p.) or intramuscular (i.m.) injection dose of 40 or 50 μg/kg caused differential effects between the AgRP-DTR and control groups.7Gropp E. Shanabrough M. Borok E. Xu A.W. Janoschek R. Buch T. Plum L. Balthasar N. Hampel B. Waisman A. et al.Agouti-related peptide-expressing neurons are mandatory for feeding.Nat. Neurosci. 2005; 8: 1289-1291https://doi.org/10.1038/nn1548Google Scholar,8Luquet S. Perez F.A. Hnasko T.S. Palmiter R.D. NPY/AgRP neurons are essential for feeding in adult mice but can be ablated in neonates.Science. 2005; 310: 683-685https://doi.org/10.1126/science.1115524Google Scholar However, a previous study on AgRP-DTR mice also reported mice death in the control group treated with DTX at a dose of 50 μg/kg.8Luquet S. Perez F.A. Hnasko T.S. Palmiter R.D. NPY/AgRP neurons are essential for feeding in adult mice but can be ablated in neonates.Science. 2005; 310: 683-685https://doi.org/10.1126/science.1115524Google Scholar Other studies reported i.p. administration of DTX at 40 μg/kg or higher doses were highly toxic even in wild-type mice.17Cerpa V. Gonzalez A. Richerson G.B. Diphtheria toxin treatment of Pet-1-Cre floxed diphtheria toxin receptor mice disrupts thermoregulation without affecting respiratory chemoreception.Neuroscience. 2014; 279: 65-76https://doi.org/10.1016/j.neuroscience.2014.08.018Google Scholar,18Valek L. Tegeder I. Failure of Diphtheria Toxin Model to Induce Parkinson-Like Behavior in Mice.Int. J. Mol. Sci. 2021; 229496https://doi.org/10.3390/ijms22179496Google Scholar We found that both AgRP-DTR and control mice injected with 40 ng DTX (i.c.v.) exhibited lethargy and movement difficulty during the first few days after toxin injection (Videos S1 and S2), which may cause difficulty in reaching food provided. Using the same strategy as previously employed to administer DTX peripherally (50 μg/kg, i.m.) twice with 2 days apart, we observed no difference in body weight or food intake between AgRP-DTR mice and control mice (Figures S6A, S6E, and S6F). Of note, three out of nine AgRP-DTR mice showed a lethal phenotype (Figure S6G), yet these animals displayed an incomplete ablation of AgRP neurons in the ARC (Figures S6B–S6D), suggesting that the lethal phenotype is not correlated with the extent of AgRP neurons. Thus, it is conceivable that diet conditions, aging, obese states, strain differences, and loss of peripheral AgRP function, as well as defective fast-refeeding by AgRP lesions, may cause differential responses to DTX.8Luquet S. Perez F.A. Hnasko T.S. Palmiter R.D. NPY/AgRP neurons are essential for feeding in adult mice but can be ablated in neonates.Science. 2005; 310: 683-685https://doi.org/10.1126/science.1115524Google Scholar,11Wu Q. Whiddon B.B. Palmiter R.D. Ablation of neurons expressing agouti-related protein, but not melanin concentrating hormone, in leptin-deficient mice restores metabolic functions and fertility.Proc. Natl. Acad. Sci. USA. 2012; 109: 3155-3160https://doi.org/10.1073/pnas.1120501109Google Scholar,15Goldwich A. Steinkasserer A. Gessner A. Amann K. Impairment of podocyte function by diphtheria toxin--a new reversible proteinuria model in mice.Lab. Invest. 2012; 92: 1674-1685https://doi.org/10.1038/labinvest.2012.133Google Scholar https://www.cell.com/cms/asset/26205d57-333e-4747-8c9a-7af406719147/mmc2.mp4Loading ... Download .mp4 (2.33 MB) Help with .mp4 files Video S1. 40 ng DTX caused movement difficulty in an AgRP-DTR mouse https://www.cell.com/cms/asset/08c8bbf7-a578-42eb-abbe-ef722a702fce/mmc3.mp4Loading ... Download .mp4 (0.98 MB) Help with .mp4 files Video S2. 40 ng DTX caused movement difficulty in a control mouse Our results establish a concept that AgRP neurons are sufficient but not required for ad libitum feeding or body weight maintenance in adult mice. This contrasts with the widely accepted notion that AgRP neurons are mandatory for feeding and body weight maintenance.7Gropp E. Shanabrough M. Borok E. Xu A.W. Janoschek R. Buch T. Plum L. Balthasar N. Hampel B. Waisman A. et al.Agouti-related peptide-expressing neurons are mandatory for feeding.Nat. Neurosci. 2005; 8: 1289-1291https://doi.org/10.1038/nn1548Google Scholar,8Luquet S. Perez F.A. Hnasko T.S. Palmiter R.D. NPY/AgRP neurons are essential for feeding in adult mice but can be ablated in neonates.Science. 2005; 310: 683-685https://doi.org/10.1126/science.1115524Google Scholar,24Dietrich M.O. Horvath T.L. GABA keeps up an appetite for life.Cell. 2009; 137: 1177-1179https://doi.org/10.1016/j.cell.2009.06.002Google Scholar Given the widely perceived critical role of AgRP neurons in feeding and survival, our findings suggest a necessary revision of the neural circuit mechanism, in which AgRP neurons are permissive for feeding and body weight regulation during ad libitum feeding conditions. This notion is consistent with no or little body weight phenotypes from loss of function in AgRP and NPY in neonatal8Luquet S. Perez F.A. Hnasko T.S. Palmiter R.D. NPY/AgRP neurons are essential for feeding in adult mice but can be ablated in neonates.Science. 2005; 310: 683-685https://doi.org/10.1126/science.1115524Google Scholar,25Könner A.C. Janoschek R. Plum L. Jordan S.D. Rother E. Ma X. Xu C. Enriori P. Hampel B. Barsh G.S. et al.Insulin action in AgRP-expressing neurons is required for suppression of hepatic glucose production.Cell Metab. 2007; 5: 438-449https://doi.org/10.1016/j.cmet.2007.05.004Google Scholar,26Krashes M.J. Shah B.P. Koda S. Lowell B.B. Rapid versus delayed stimulation of feeding by the endogenously released AgRP neuron mediators GABA, NPY, and AgRP.Cell Metab. 2013; 18: 588-595https://doi.org/10.1016/j.cmet.2013.09.009Google Scholar,27Ste Marie L. Luquet S. Cole T.B. Palmiter R.D. Modulation of neuropeptide Y expression in adult mice does not affect feeding.Proc. Natl. Acad. Sci. USA. 2005; 102: 18632-18637https://doi.org/10.1073/pnas.0509240102Google Scholar or adult mice,26Krashes M.J. Shah B.P. Koda S. Lowell B.B. Rapid versus delayed stimulation of feeding by the endogenously released AgRP neuron mediators GABA, NPY, and AgRP.Cell Metab. 2013; 18: 588-595https://doi.org/10.1016/j.cmet.2013.09.009Google Scholar,27Ste Marie L. Luquet S. Cole T.B. Palmiter R.D. Modulation of neuropeptide Y expression in adult mice does not affect feeding.Proc. Natl. Acad. Sci. USA. 2005; 102: 18632-18637https://doi.org/10.1073/pnas.0509240102Google Scholar blocking GABA release from AgRP neurons in neonatal28Tong Q. Ye C.P. Jones J.E. Elmquist J.K. Lowell B.B. Synaptic release of GABA by AgRP neurons is required for normal regulation of energy balance.Nat. Neurosci. 2008; 11: 998-1000https://doi.org/10.1038/nn.2167Google Scholar or adult mice,29Meng F. Han Y. Srisai D. Belakhov V. Farias M. Xu Y. Palmiter R.D. Baasov T. Wu Q. New inducible genetic method reveals critical roles of GABA in the control of feeding and metabolism.Proc. Natl. Acad. Sci. USA. 2016; 113: 3645-3650https://doi.org/10.1073/pnas.1602049113Google Scholar or chronic or acute inhibition of AgRP neurons in adult mice.5Zhu C. Jiang Z. Xu Y. Cai Z.L. Jiang Q. Xu Y. Xue M. Arenkiel B.R. Wu Q. Shu G. Tong Q. Profound and redundant functions of arcuate neurons in obesity development.Nat. Metab. 2020; 2: 763-774https://doi.org/10.1038/s42255-020-0229-2Google Scholar,12Üner A.G. Keçik O. Quaresma P.G.F. De Araujo T.M. Lee H. Li W. Kim H.J. Chung M. Bjørbæk C. Kim Y.B. Role of POMC and AgRP neuronal activities on glycaemia in mice.Sci. Rep. 2019; 913068https://doi.org/10.1038/s41598-019-49295-7Google Scholar In addition, the previously described lethal starvation phenotype due to AgRP neuronal lesions can be rescued by obesity,11Wu Q. Whiddon B.B. Palmiter R.D. Ablation of neurons expressing agouti-related protein, but not melanin concentrating hormone, in leptin-deficient mice restores metabolic functions and fertility.Proc. Natl. Acad. Sci. USA. 2012; 109: 3155-3160https://doi.org/10.1073/pnas.1120501109Google Scholar suggesting that the lethal starvation was not due to a specific inhibition mechanism on feeding behaviors, which otherwise should have caused starvation independent of body weight. Given previous results on acute inhibition of AgRP neurons causing a short-term reduction in feeding,3Krashes M.J. Koda S. Ye C. Rogan S.C. Adams A.C. Cusher D.S. Maratos-Flier E. Roth B.L. Lowell B.B. Rapid, reversible activation of AgRP neurons drives feeding behavior in mice.J. Clin. Invest. 2011; 121: 1424-1428https://doi.org/10.1172/JCI46229Google Scholar,9Betley J.N. Xu S. Cao Z.F.H. Gong R. Magnus C.J. Yu Y. Sternson S.M. Neurons for hunger and thirst transmit a negative-valence teaching signal.Nature. 2015; 521: 180-185https://doi.org/10.1038/nature14416Google Scholar,10Denis R.G.P. Joly-Amado A. Webber E. Langlet F. Schaeffer M. Padilla S.L. Cansell C. Dehouck B. Castel J. Delbès A.S. et al.Palatability Can Drive Feeding Independent of AgRP Neurons.Cell Metab. 2017; 25: 975https://doi.org/10.1016/j.cmet.2017.03.001Google Scholar combined with our current results, acute effects on feeding may not be translated to chronic body weight changes. Our results also argue against a widely perceived role for developmental compensation underlying the normal body weight observed in mice with lesions of AgRP neurons from neonatal stages. The concept that AgRP neurons are sufficient but not mandatory for body weight regulation supports our previous observations that ARC GABAergic neurons promote feeding and drive obesity in a redundant mechanism.5Zhu C. Jiang Z. Xu Y. Cai Z.L. Jiang Q. Xu Y. Xue M. Arenkiel B.R. Wu Q. Shu G. Tong Q. Profound and redundant functions of arcuate neurons in obesity development.Nat. Metab. 2020; 2: 763-774https://doi.org/10.1038/s42255-020-0229-2Google Scholar This redundant mechanism predicts that while activation of random subsets of ARC GABAergic neurons is sufficient to promote feeding and obesity, inhibition or loss of function of single subsets has no impact on feeding or body weight. Interestingly, both current and previous studies show that AgRP neurons are necessary to mount appropriate feeding responses to fasting.3Krashes M.J. Koda S. Ye C. Rogan S.C. Adams A.C. Cusher D.S. Maratos-Flier E. Roth B.L. Lowell B.B. Rapid, reversible activation of AgRP neurons drives feeding behavior in mice.J. Clin. Invest. 2011; 121: 1424-1428https://doi.org/10.1172/JCI46229Google Scholar,5Zhu C. Jiang Z. Xu Y. Cai Z.L. Jiang Q. Xu Y. Xue M. Arenkiel B.R. Wu Q. Shu G. Tong Q. Profound and redundant functions of arcuate neurons in obesity development.Nat. Metab. 2020; 2: 763-774https://doi.org/10.1038/s42255-020-0229-2Google Scholar,30Chen Y. Lin Y.C. Zimmerman C.A. Essner R.A. Knight Z.A. Hunger neurons drive feeding through a sustained, positive reinforcement signal.Elife. 2016; 5e18640https://doi.org/10.7554/eLife.18640Google Scholar Thus, although AgRP neurons are not required for normal feeding or energy balance in a laboratory setting, these neurons appear to be implicated in stressful conditions such as fasting and may be needed for survival in more challenging natural environment