Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Genetic identification of leptin neural circuits in energy and glucose homeostases

Nature volume 556, pages 505–509 (2018)Cite this article

Subjects

Abstract

Leptin, a hormone produced in white adipose tissue, acts in the brain to communicate fuel status, suppress appetite following a meal, promote energy expenditure and maintain blood glucose stability1,2. Dysregulation of leptin or its receptors (LEPR) results in severe obesity and diabetes3,4,5. Although intensive studies on leptin have transformed obesity and diabetes research2,6, clinical applications of the molecule are still limited7, at least in part owing to the complexity and our incomplete understanding of the underlying neural circuits. The hypothalamic neurons that express agouti-related peptide (AGRP) and pro-opiomelanocortin (POMC) have been hypothesized to be the main first-order, leptin-responsive neurons. Selective deletion of LEPR in these neurons with the Cre–loxP system, however, has previously failed to recapitulate, or only marginally recapitulated, the obesity and diabetes that are seen in LEPR-deficient Leprdb/db mice, suggesting that AGRP or POMC neurons are not directly required for the effects of leptin in vivo8,9,10. The primary neural targets of leptin are therefore still unclear. Here we conduct a systematic, unbiased survey of leptin-responsive neurons in streptozotocin-induced diabetic mice and exploit CRISPR–Cas9-mediated genetic ablation of LEPR in vivo. Unexpectedly, we find that AGRP neurons but not POMC neurons are required for the primary action of leptin to regulate both energy balance and glucose homeostasis. Leptin deficiency disinhibits AGRP neurons, and chemogenetic inhibition of these neurons reverses both diabetic hyperphagia and hyperglycaemia. In sharp contrast to previous studies, we show that CRISPR-mediated deletion of LEPR in AGRP neurons causes severe obesity and diabetes, faithfully replicating the phenotype of Leprdb/db mice. We also uncover divergent mechanisms of acute and chronic inhibition of AGRP neurons by leptin (presynaptic potentiation of GABA (γ-aminobutyric acid) neurotransmission and postsynaptic activation of ATP-sensitive potassium channels, respectively). Our findings identify the underlying basis of the neurobiological effects of leptin and associated metabolic disorders.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Overactivation of AGRP neurons induces diabetic hyperphagia and hyperglycaemia.
Fig. 2: CRISPR-mediated deletion of leptin receptors in AGRP neurons results in severe obesity and diabetes.
Fig. 3: KATP channels in AGRP neurons are required for leptin-mediated regulation of body weight and blood glucose.
Fig. 4: Presynaptic GABAergic afferents to AGRP neurons mediate acute leptin inhibition of feeding.

Similar content being viewed by others

References

  1. Friedman, J. 20 years of leptin: leptin at 20: an overview. J. Endocrinol. 223, T1–T8 (2014).

    Article  CAS  Google Scholar 

  2. Flak, J. N. & Myers, M. G. Jr. Minireview: CNS mechanisms of leptin action. Mol. Endocrinol. 30, 3–12 (2016).

    Article  CAS  Google Scholar 

  3. Chen, H. et al. Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell 84, 491–495 (1996).

    Article  MathSciNet  CAS  Google Scholar 

  4. Zhang, Y. et al. Positional cloning of the mouse obese gene and its human homologue. Nature 372, 425–432 (1994).

    Article  ADS  CAS  Google Scholar 

  5. Farooqi, I. S. et al. Beneficial effects of leptin on obesity, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction of human congenital leptin deficiency. J. Clin. Invest. 110, 1093–1103 (2002).

    Article  CAS  Google Scholar 

  6. Morton, G. J. & Schwartz, M. W. Leptin and the central nervous system control of glucose metabolism. Physiol. Rev. 91, 389–411 (2011).

    Article  CAS  Google Scholar 

  7. Kelesidis, T., Kelesidis, I., Chou, S. & Mantzoros, C. S. Narrative review: the role of leptin in human physiology: emerging clinical applications. Ann. Intern. Med. 152, 93–100 (2010).

    Article  Google Scholar 

  8. Balthasar, N. et al. Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis. Neuron 42, 983–991 (2004).

    Article  CAS  Google Scholar 

  9. van de Wall, E. et al. Collective and individual functions of leptin receptor modulated neurons controlling metabolism and ingestion. Endocrinology 149, 1773–1785 (2008).

    Article  Google Scholar 

  10. Vong, L. et al. Leptin action on GABAergic neurons prevents obesity and reduces inhibitory tone to POMC neurons. Neuron 71, 142–154 (2011).

    Article  CAS  Google Scholar 

  11. Fujikawa, T., Chuang, J.-C., Sakata, I., Ramadori, G. & Coppari, R. Leptin therapy improves insulin-deficient type 1 diabetes by CNS-dependent mechanisms in mice. Proc. Natl Acad. Sci. USA 107, 17391–17396 (2010).

    Article  ADS  CAS  Google Scholar 

  12. Perry, R. J. et al. Leptin reverses diabetes by suppression of the hypothalamic-pituitary-adrenal axis. Nat. Med. 20, 759–763 (2014).

    Article  CAS  Google Scholar 

  13. Knight, Z. A. et al. Molecular profiling of activated neurons by phosphorylated ribosome capture. Cell 151, 1126–1137 (2012).

    Article  CAS  Google Scholar 

  14. Delovitch, T. L. & Singh, B. The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD. Immunity 7, 727–738 (1997).

    Article  CAS  Google Scholar 

  15. Könner, A. C. et al. Insulin action in AGRP-expressing neurons is required for suppression of hepatic glucose production. Cell Metab. 5, 438–449 (2007).

    Article  Google Scholar 

  16. van den Top, M., Lee, K., Whyment, A. D., Blanks, A. M. & Spanswick, D. Orexigen-sensitive NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus. Nat. Neurosci. 7, 493–494 (2004).

    Article  Google Scholar 

  17. Cowley, M. A. et al. Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature 411, 480–484 (2001).

    Article  ADS  CAS  Google Scholar 

  18. Aponte, Y., Atasoy, D. & Sternson, S. M. AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training. Nat. Neurosci. 14, 351–355 (2011).

    Article  CAS  Google Scholar 

  19. Krashes, M. J. et al. Rapid, reversible activation of AgRP neurons drives feeding behavior in mice. J. Clin. Invest. 121, 1424–1428 (2011).

    Article  CAS  Google Scholar 

  20. van den Pol, A. N. et al. Neuromedin B and gastrin-releasing peptide excite arcuate nucleus neuropeptide Y neurons in a novel transgenic mouse expressing strong Renilla green fluorescent protein in NPY neurons. J. Neurosci. 29, 4622–4639 (2009).

    Article  Google Scholar 

  21. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

    Article  ADS  CAS  Google Scholar 

  22. Luquet, 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 310, 683–685 (2005).

    Article  ADS  CAS  Google Scholar 

  23. Wu, Q., Clark, M. S. & Palmiter, R. D. Deciphering a neuronal circuit that mediates appetite. Nature 483, 594–597 (2012).

    Article  ADS  CAS  Google Scholar 

  24. Platt, R. J. et al. CRISPR–Cas9 knockin mice for genome editing and cancer modeling. Cell 159, 440–455 (2014).

    Article  CAS  Google Scholar 

  25. Spanswick, D., Smith, M. A., Groppi, V. E., Logan, S. D. & Ashford, M. L. Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels. Nature 390, 521–525 (1997).

    Article  ADS  CAS  Google Scholar 

  26. Seghers, V., Nakazaki, M., DeMayo, F., Aguilar-Bryan, L. & Bryan, J. Sur1 knockout mice. A model for KATP channel-independent regulation of insulin secretion. J. Biol. Chem. 275, 9270–9277 (2000).

    Article  CAS  Google Scholar 

  27. Miki, T. et al. Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice. Proc. Natl Acad. Sci. USA 95, 10402–10406 (1998).

    Article  ADS  CAS  Google Scholar 

  28. Pinto, S. et al. Rapid rewiring of arcuate nucleus feeding circuits by leptin. Science 304, 110–115 (2004).

    Article  ADS  CAS  Google Scholar 

  29. Rudolph, U. & Möhler, H. Analysis of GABAA receptor function and dissection of the pharmacology of benzodiazepines and general anesthetics through mouse genetics. Annu. Rev. Pharmacol. Toxicol. 44, 475–498 (2004).

    Article  CAS  Google Scholar 

  30. Garfield, A. S. et al. Dynamic GABAergic afferent modulation of AgRP neurons. Nat. Neurosci. 19, 1628–1635 (2016).

    Article  CAS  Google Scholar 

  31. Tong, 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. 11, 998–1000 (2008).

    Article  CAS  Google Scholar 

  32. Parton, L. E. et al. Glucose sensing by POMC neurons regulates glucose homeostasis and is impaired in obesity. Nature 449, 228–232 (2007).

    Article  ADS  CAS  Google Scholar 

  33. Leiter, E. H. The NOD mouse: a model for analyzing the interplay between heredity and environment in development of autoimmune disease. ILAR J. 35, 4–14 (1993).

    Article  Google Scholar 

  34. Liu, T. et al. Fasting activation of AgRP neurons requires NMDA receptors and involves spinogenesis and increased excitatory tone. Neuron 73, 511–522 (2012).

    Article  Google Scholar 

  35. Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  Google Scholar 

  36. Montague, T. G., Cruz, J. M., Gagnon, J. A., Church, G. M. & Valen, E. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42, W401–W407 (2014).

    Article  CAS  Google Scholar 

  37. Henry, F. E., Sugino, K., Tozer, A., Branco, T. & Sternson, S. M. Cell type-specific transcriptomics of hypothalamic energy-sensing neuron responses to weight-loss. eLife 4, e09800 (2015).

    Article  Google Scholar 

  38. Opland, D. et al. Loss of neurotensin receptor-1 disrupts the control of the mesolimbic dopamine system by leptin and promotes hedonic feeding and obesity. Mol. Metab. 2, 423–434 (2013).

    Article  CAS  Google Scholar 

  39. Kong, D. et al. A postsynaptic AMPK→p21-activated kinase pathway drives fasting-induced synaptic plasticity in AGRP neurons. Neuron 91, 25–33 (2016).

    Article  CAS  Google Scholar 

  40. Kong, D. et al. GABAergic RIP–Cre neurons in the arcuate nucleus selectively regulate energy expenditure. Cell 151, 645–657 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank all members of the Kong laboratory for helpful discussions and comments on the manuscript; F. Zhang for providing pX330 plasmid and Rosa26-LSL-Cas9-GFP mice; Tufts CNR for confocal imaging (supported by NIH/NINDS P30 NS047243); Boston Children’s Hospital Viral Core for AAV virus packaging (supported by NIH/NEI P30 EY012196-17); the Adipose Tissue Biology and Nutrient Metabolism Core and A. Greenberg for help with body mass and oxygen consumption measurement (supported by NIH/NIDDK P30 DK046200-26); BIDMC-FNL and G. Blackburn for equipment support; and P. Haydon and M. Rios for reading the manuscript. This research is supported by the following grants: to C.L.B., NINDS T32NS061764-09; to C.-H.C., AHA-Postdoctoral Fellowship 17POST33661185; to D.K., NIH/NIDDK K01 DK094943, R01 DK108797, NINDS R21 NS097922, BNORC Transgenic core, BNORC P&F grant, BNORC small grant program (NIDDK P30 DK046200) and Charles Hood Foundation Award.

Reviewer information

Nature thanks R. Palmiter and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

  1. These authors contributed equally: Jie Xu, Christopher L. Bartolome.

Authors and Affiliations

  1. Department of Neuroscience, Tufts University School of Medicine, Boston, MA, USA

    Jie Xu, Christopher L. Bartolome, Cho Shing Low, Xinchi Yi, Cheng-Hao Chien, Peng Wang & Dong Kong

  2. Program of Neuroscience, Tufts University Sackler School of Graduate Biomedical Sciences, Boston, MA, USA

    Christopher L. Bartolome & Dong Kong

  3. Program of Cellular, Molecular, and Developmental Biology, Tufts University Sackler School of Graduate Biomedical Sciences, Boston, MA, USA

    Cho Shing Low & Dong Kong

Authors
  1. Jie Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christopher L. Bartolome
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cho Shing Low
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xinchi Yi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Cheng-Hao Chien
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Peng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Dong Kong
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.X., C.L.B. and D.K. designed the experiments, analysed data and wrote the manuscript. J.X. and C.L.B. performed the experiments with the help of C.S.L., X.Y., C.-H.C. and P.W. J.X. constructed AAV vectors. J.X. and C.-H.C. performed electrophysiology. C.L.B. and C.S.L. performed STZ-related studies. J.X., C.L.B. and X.Y. performed surgery. C.L.B. and P.W. performed the ciLepr re-expression study. D.K. conceived and supervised the project.

Corresponding author

Correspondence to Dong Kong.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Characterization of STZ-induced diabetic mice.

Additional analysis of alterations in neuronal activity in STZ-treated animals and following leptin administration; similar activation of neurons in the ARC was also observed in NOD diabetic mice. a, Development of hyperglycaemia in C57BL/6 mice after one-time intraperitoneal administration of STZ at different doses from 75 mg kg−1 to 150 mg kg−1 (n = 4 mice per group). STZ treatments in subsequent experiments used a dose of 125 mg kg−1. b, Experimental protocol. c–g, Serum insulin levels (c), body weight (d), daily water intake (e), representative cages in which a saline or STZ-treated mouse were housed (f) and serum leptin levels (g) after one week post-STZ-injection (n = 14 mice per group), suggesting that treatment with 125 mg kg−1 STZ effectively induces insulin deficiency and diabetes, as well as emaciation, polydipsia, polyuria and leptin deficiency. h, Representative sections of FOS immunostaining in the hypothalamus of saline- or STZ-treated mice. i, j, Representative sections and quantification of pS6 (Ser235,236) immunostaining in the mediobasal hypothalamus of saline- or STZ-treated mice 3 h after the administration of saline or leptin (n = 3 mice per group). k, l, Comparison of FOS expression in the mediobasal hypothalamus of STZ-treated mice following 3-h or 24-h leptin treatment (n = 3 per group). m, Schematic diagram of the development of the non-obese diabetic (NOD) mouse model. n–r, Body weight (n), daily food intake (o), ad libitum-fed blood glucose levels (p), and representative sections (q) and quantification (r) of FOS immunostaining in the ARC of non-diabetic or diabetic NOD littermates (n = 4 mice per group). Data are mean ± s.e.m. and representative of three independent experiments; *P < 0.05, **P < 0.01, ****P < 0.0001; Student’s two-tailed, unpaired t-test (c–e, g, j, n–p, r) or two-way ANOVA (l).

Extended Data Fig. 2 Characterization of feeding behaviours in STZ-treated diabetic mice.

Additional analyses of ectopic activation of AGRP neurons and its pathologic contributions to STZ-induced hyperphagia and hyperglycaemia. a, Quantitative PCR results showing that expression of Agrp and Npy is significantly upregulated in the mediobasal hypothalamus of STZ-treated animals, which is consistent with increased AGRP neuronal activity following STZ injection (n = 5 mice per group). b–f, Food intake during light cycle (07:00 to 19:00) (b) and dark cycle (19:00 to 07:00) (c), representative 1-h heat map (10:00 to 11:00) showing per cent occupancy time in food zone (upper right corner) and nesting zone (lower right corner) (d), feeding duration (e), and 1-h food intake (f) in saline- or STZ-treated C57BL/6 mice (n = 8 mice per group). g, h, Representative sections and quantification of hrGFP immunostaining in the ARC of saline- or STZ-treated Npy-hrGFP transgenic mice, suggesting that STZ treatment does not induce obvious loss of AGRP neurons (n = 3 mice per group). i, Representative sections and quantification of hrGFP and pS6 co-immunostaining in the ARC of saline- or STZ-treated Npy-hrGFP transgenic mice (n = 4 mice per group). j, Schematic of chemogenetic inhibition of AGRP neurons in virus-transduced Agrp-IRES-cre mice. k, Four-hour food-intake measurement during dark cycle (20:00 to 00:00) following the administration of saline or CNO (n = 6 mice per group). l, m, Four-hour food-intake assay (10:00 to 14:00) (l) and blood glucose measurement (m, without food in the cage) in saline- or CNO-treated female Agrp-IRES-cre littermates that had been injected with AAV pSyn-FLEX-hM4Di-mCherry virus into the ARC, (n = 8 mice per group). n–q, Schematic of experiments to assess effects of CNO on Agrp-IRES-cre mice injected with Cre-dependent AAV-FLEX-mCherry virus in the ARC (n). Representative brain sections (o) and quantification (p, n = 3 mice per group) of mCherry and FOS co-immunostaining, and food intake assay (q; 10:00 to 14:00, n = 8 mice per group) in STZ-treated mice following intraperitoneal injection of saline or CNO, demonstrating that CNO administration without hM4Di expression in AGRP neurons has no effect and the changes observed in Fig. 1g–i are caused by the chemogenetic inhibition of AGRP neurons. Data are mean ± s.e.m. and representative of three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Student’s two-tailed, unpaired t-test (a–c, e, f, h, p), paired t-test (m) or two-way ANOVA analysis (l, q) with Šidák post hoc test (k).

Extended Data Fig. 3 Additional information and analyses for CRISPR-mediated disruption of LEPRs in AGRP neurons.

a, sgRNA design for targeting the mouse Lepr genomic locus. b–h, AAV pU6-sgRNALepr::pEF1α-FLEX-mCherry (AAV-sgLepr) was injected unilaterally into the ARC of Agrp-IRES-cre::LSL-Cas9-GFP mice (b). Representative images (c) and quantification (d) of mCherry and leptin-induced pSTAT3 (Tyr705) immunostaining in the hypothalamus. Cell counting of GFP+ cells from the ARC suggests that deletion of Lepr in AGRP neurons does not induce cell death (e). Representative images of mCherry immunostaining (left) and ISH against Lepr mRNA (right) indicating efficient deletion of Lepr (f). Representative images (g) and quantification (h) of mCherry and FOS co-immunostaining in the ARC of ad libitum-fed mice, indicating disinhibition of AGRP neurons (n = 3 mice per group). i, j, AAV pU6-sgRNALepr::pEF1α-FLEX-mCherry (AAV-sgLepr) was injected bilaterally into the ARC of Agrp-IRES-cre::LSL-Cas9-GFP or Agrp-IRES-cre mice (i); reverse transcription with qPCR showing Lepr, Agrp, Npy and Pomc mRNA expression in the ventromedial hypothalamus of fed mice (j, n = 4 mice per group). k, Serum insulin levels in ad libitum-fed Agrp-IRES-cre (Cas9−) and Agrp-IRES-cre::LSL-Cas9-GFP mice (Cas9+) bilaterally injected with AAV-sgLepr virus (n = 4 mice per group). l, CRISPR-mediated deletion of Lepr in AGRP neurons also induces severe obesity in female mice (n = 6 mice per group). m, Representative near-infrared thermal images and quantification of iBAT temperature in virus-transduced, ad libitum-fed Agrp-IRES-cre (Cas9−) and Agrp-IRES-cre::LSL-Cas9-GFP (Cas9+) male littermates. n, Assay design to compare mice with global Lepr mutations and mice with specific deletion of Lepr in AGRP neurons. o, p, Weekly blood glucose measurement and daily food intake at eight weeks of age of ad libitum-fed, virus-transduced Leprdb/+, Leprdb/db, Agrp-IRES-cre (Cas9−), and Agrp-IRES-cre::LSL-Cas9-GFP (Cas9+) mice (n = 9 mice per group). q–s, Experimental design (q), changes in body weight (r) and daily food intake (s) five days post pump surgery in STZ-treated, virus-transduced Agrp-IRES-cre (Cas9−) and Agrp-IRES-cre::LSL-Cas9-GFP (Cas9+) mice following chronic administration of saline or leptin with osmotic pump (n = 6 mice per group). t–v, Experimental design (t), changes of body weight (u) and daily food intake (v) in non-STZ treated, virus-transduced Agrp-IRES-cre (Cas9−) and Agrp-IRES-cre::LSL-Cas9-GFP (Cas9+) mice following three-day intraperitoneal injection of leptin (n = 7 mice per group). Data are mean ± s.e.m. and representative of three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Student’s two-tailed, unpaired t-test (d, e, h, j, k, m, o, s) or two-way ANOVA (l, p, u, v) with Šidák post hoc test (r).

Extended Data Fig. 4 Analysis of off-target effects of CRISPR-mediated genome editing.

Expression of CRISPR-insensitive leptin receptors in AGRP neurons occludes alterations in body weight, food intake or blood glucose levels caused by CRISPR-mediated disruption of Lepr. a, Predicted off-target site of sgLepr on the first exon of Gpr108 locus. b, Plasmid for co-expression of sgLepr and Cas9-GFP proteins (top left); GFP immunofluorescence following transfection of the plasmid into mouse N2a cells (bottom left); on-target and off-target indel detection, demonstrating that sgLepr effectively induces mutations in the Lepr locus but not in the Gpr108 locus (right). c, sgRNA targeting the mouse Lepr locus (upper) and CRISPR-insensitive Lepr cDNA encoding the long-form leptin receptors (ciLepr, lower) with indicated silent mutations to prevent binding of sgRNA. d, Cre-dependent AAV pEF1α-FLEX-ciLepr (AAV-FLEX-ciLepr) injected unilaterally into the ARC of Agrp-IRES-cre::Leprdb/db mice (left) and representative images of leptin-induced pSTAT3 (Tyr705) immunostaining (right), indicating that ciLepr is fully responsive to leptin stimulation. e–g, Body weight (e), food intake (f) and blood glucose (g) of non-cre and Agrp-IRES-cre littermates following bilateral injection of AAV-FLEX-ciLepr into the ARC (n = 8 mice per group), suggesting that AAV-mediated expression of ciLepr in AGRP neurons produces no obvious effects on energy or glucose balance. h–k, Experimental protocol (h), body weight (i), daily food intake (j) and blood glucose measurements (k) in virus-transduced ad libitum-fed littermates (n = 8 mice per group). Since expression of ciLepr in AGRP neurons prevented body weight gain, increased food intake and hyperglycaemia induced by CRISPR-mediated deletion, potential contributions from off-site mutagenesis are excluded. Data are mean ± s.e.m. and representative of three independent experiments; **P < 0.01, ***P < 0.001; Student’s two-tailed, unpaired t-test (f, j) or two-way ANOVA (e, g, i, k).

Extended Data Fig. 5 CRISPR-mediated disruption of leptin receptors in hypothalamic POMC neurons does not alter energy or blood glucose balance.

a, b, Representative sections and quantification of hrGFP and FOS co-immunostaining in the ARC of saline- or STZ-treated Pomc-hrGFP transgenic mice (n = 3 mice per group). Reduced FOS expression in POMC neurons was observed following STZ treatment. c, Results of reverse transcription with qPCR, showing that Pomc mRNA levels are significantly reduced in the mediobasal hypothalamus of STZ-treated animals, which is consistent with inhibited POMC neuronal activity following STZ injection (n = 5 mice per group). d, Schematics, representative sections and quantification of tdTomato and hrGFP co-immunostaining in the ARC of Pomc-cre::LSL-tdTomato::Npy-hrGFP mice (top) and mCherry and hrGFP co-immunostaining in the ARC of Pomc-cre::Npy-hrGFP mice with Cre-dependent AAV pEF1α-FLEX-mCherry injected into the ARC (bottom). Co-expression was observed in Pomc-cre::LSL-tdTomato::Npy-hrGFP mice but not in virus-transduced Pomc-cre::Npy-hrGFP mice, suggesting that Pomc-cre ectopically induces Cre activity in AGRP neurons at an early developmental stage, consistent with previous findings. These data also demonstrate that Cre-dependent AAV injected into the ARC of Pomc-cre transgenic mice provides an efficient approach to specifically express genes of interest in POMC ARC neurons, without perturbing intermingled AGRP neurons (n = 3 mice per group). e, Two approaches to achieve CRISPR-mediated deletion of Lepr in POMC ARC neurons. Left, AAV-sgLepr virus was bilaterally injected into the ARC of Pomc-cre::LSL-Cas9-GFP mice. Right, a viral mix of AAV-sgLepr and Cre-dependent AAV pMeCP2-FLEX-spCas9 (AAV-FLEX-spCas9, see Methods) was bilaterally injected into the ARC of Pomc-cre mice. f–j, Body weight (f), accumulated weekly food intake (g), ad libitum-fed-state blood glucose levels (h), glucose-tolerance test (i) and insulin-tolerance test (j) in the virus-transduced animals (n = 6 mice per group). Notably, single virus-transduced Pomc-Cre::LSL-Cas9-GFP mice, but not dual virus-transduced Pomc-Cre mice, exhibited mildly increased body weight and food intake, and slightly impaired glucose tolerance and insulin sensitivity. Since Cas9 protein would be expected to be expressed in some AGRP neurons in Pomc-cre::LSL-Cas9-GFP mice, the difference observed between the two approaches is likely to be explained by the ectopic Cre activity of Pomc-cre in AGRP neurons. The phenotypes observed in Pomc-cre::LSL-Cas9-GFP mice mimic those following genetic ablation of Lepr with a conventional Cre–loxP system. Inactivation of LEPR in POMC neurons of adult mice does not appear to affect energy balance or glucose homeostasis under the assayed conditions. Data are mean ± s.e.m. and representative of three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001; Student’s two-tailed, unpaired t-test (b, c) or two-way ANOVA with Šidák post hoc test (f–j).

Extended Data Fig. 6 Additional information on experimental design related to CRISPR-mediated deletion of Kcnj11 in AGRP neurons.

Expression of a CRISPR-insensitive Kir6.2 in AGRP neurons prevents alterations in body weight, food intake or blood glucose following CRISPR-mediated deletion. a, Representative images and quantification of mCherry and pSTAT3 co-immunostaining (n = 4 mice per group) following CRISPR-mediated disruption of Kcnj11 in AGRP neurons. b, Experimental protocol to test chronic effects of leptin on regulation of body weight and food intake. c, sgRNA design targeting the mouse Kcnj11 locus and CRISPR-insensitive Kcnj11 cDNA encoding the mouse KATP channel subunit Kir6.2 (ciKcnj11) with indicated silent mutations to prevent binding of sgRNA. d, Schematic of Cre-dependent AAV pEF1α-FLEX-ciKcnj11-IRES-mCherry (AAV-FLEX-ciKcnj11) injected unilaterally into the ARC of Agrp-IRES-cre::Leprdb/db mice and representative image of mCherry immunostaining. e–h, Experimental protocol (e), body weight (f), daily food intake (g) and blood glucose measurements (h) in virus-transduced ad libitum-fed littermates (n = 8 mice per group). Expression of ciKcnj11 in AGRP neurons completely prevented body weight gain, increased food intake and hyperglycaemia induced by CRISPR-mediated deletion, suggesting that contributions from off-site mutagenesis are excluded. i–k, Body weight (i), food intake (j), and blood glucose (k) of non-cre and Agrp-IRES-cre littermates following bilateral injection of AAV-FLEX-ciKcnj11 into the ARC (n = 8 mice per group), suggesting that AAV-mediated expression of ciKcnj11 in AGRP neurons produces no obvious effects on energy or glucose balances. Data are mean ± s.e.m. and representative of three independent experiments; ***P < 0.001, ****P < 0.0001; Student’s two-tailed, unpaired t-test (a, g, h, j) or two-way ANOVA (f, i, k).

Extended Data Fig. 7 Additional characterization of inhibition of fasting-induced hunger by leptin.

Fasting- and leptin-induced alterations in GABAergic neurotransmission on AGRP neurons. a, Assays to test acute effects of leptin in suppressing 24-h fasting-induced hunger in Agrp-IRES-cre (Cas9−) and Agrp-IRES-cre::LSL-Cas9-GFP (Cas9+) mice, following bilateral injection of AAV-sgKATP into the ARC. These data indicate that KATP channels in AGRP neurons are not required for acute inhibition of hunger by leptin following fasting. n = 6 mice per group. b, Representative traces of spontaneous inhibitory postsynaptic currents (sIPSCs) on AGRP neurons and quantification of their frequency and amplitude in ad libitum-fed or 24-h-fasted Npy-hrGFP transgenic mice. No difference was observed in sIPSC amplitude. n = 10 cells from three mice per group. c, Representative traces and quantification of sIPSCs on AGRP neurons before or 15 min post-incubation with leptin in ad libitum-fed Npy-hrGFP transgenic mice, suggesting that leptin does not further regulate GABAergic neurotransmission on AGRP neurons in fed animals. n = 10 cells from three mice per group. d, Schematic of the mouse GABAA receptor complex and the genes encoding its subunits. e, Genomic structures and design of sgRNAs targeting the mouse Gabrb1–Gabrb3 loci encoding GABAA receptor subunits β1 to β3, respectively. Data are mean ± s.e.m. and representative of three independent experiments; **P < 0.01, ***P < 0.001, ****P < 0.0001; Student’s two-tailed, unpaired t-test (a, b).

Extended Data Fig. 8 Dynamic changes of neuronal activity and synaptic neurotransmission following CRISPR-mediated deletion of GABAA receptors in AGRP neurons.

a, b, Body weight (a) and daily food intake (b) of Agrp-IRES-cre (Cas9−) and Agrp-IRES-cre::LSL-Cas9-GFP (Cas9+) mice following bilateral injection of AAV-sgGABAA-R into the ARC (n = 7 mice per group). c–f, Representative sections and quantification of mCherry and FOS co-immunostaining (c, n = 3 mice per group), representative traces and quantification of spontaneous excitatory postsynaptic currents (sEPSCs) (d, n = 15 neurons from three mice; e, n = 8 neurons from two mice) on AGRP neurons of Agrp-IRES-cre::LSL-Cas9-GFP mice after one-week or four-week unilateral injection of AAV-sgGABAA-R into the ARC. Reduced frequency and amplitude of sEPSCs were observed in AGRP neurons at a later stage following GABAA receptor deletion, together with concurrent elimination of the observed disinhibition of these neurons (c), body weight gain (a) and hyperphagia (b), suggesting that excitatory and inhibitory afferents cooperate dynamically to modulate AGRP neuronal activities. In addition, the compensatory reduction in sEPSCs in virus-transduced neurons but not in the neurons from the contralateral side also indicates a cell-autonomous mechanism. Data are mean ± s.e.m. and representative of three independent experiments; **P < 0.01, ****P < 0.0001; Student’s two-tailed, unpaired t-test (b, d, e) or two-way ANOVA with Šidák post hoc test (a).

Extended Data Fig. 9 Additional characterization of leptin regulation of energy balance and GABAergic neurotransmission on AGRP neurons following CRISPR-mediated deletion or expression of CRISPR-insensitive GABAA receptors.

a, b, Body weight and daily food intake changes during three-consecutive-day treatment with saline or leptin in Agrp-IRES-cre (Cas9−) and Agrp-IRES-cre::LSL-Cas9-GFP (Cas9+) mice following bilateral injection of AAV-sgGABAA-R into the ARC (n = 7 mice per group), suggesting that GABAergic neurotransmission on AGRP neurons is not required for chronic effects of leptin on regulation of body weight or food intake. c, sgRNA design targeting the mouse Gabrb3 locus and CRISPR-insensitive Gabrb3 cDNA encoding the mouse β3 subunit of GABAA receptors (ciGabrb3) with indicated silent mutations to prevent binding of sgRNA. d, Schematic of Cre-dependent AAV pEF1α-FLEX-ciGabrb3-GFP (AAV-FLEX-ciGabrb3) and representative image of GFP immunostaining following bilateral injection of the virus into the ARC of Agrp-IRES-cre mice. e–g, Body weight (e), food intake (f) and blood glucose (g) of non-cre and Agrp-IRES-cre littermates following bilateral injection of AAV-FLEX-ciGabrb3 into the ARC (n = 8 mice per group), suggesting that AAV-mediated expression of ciGabrb3 in AGRP neurons produces no obvious effects on energy or glucose balances. h–j, Representative traces and quantification of sIPSCs and sEPSCs of AGRP neurons in Agrp-IRES-cre::LSL-Cas9-GFP mice, following bilateral injection of AAV-sgGABAA-R (red) or a mix of AAV-sgGABAA-R and AAV-FLEX-ciGabrb3 (purple) into the ARC (n = 8 neurons from three mice per group). k, Assays to test acute effects of leptin in suppressing 24-h-fasting-induced hunger in Agrp-IRES-cre (Cas9−) and Agrp-IRES-cre::LSL-Cas9-GFP (Cas9+) mice, following bilateral injection of a mix of AAV-sgGABAA-R and AAV-FLEX-ciGabrb3 into the ARC. These data indicate that expression of CRISPR-insensitive β3 subunit of GABAA receptor in AGRP neurons following deletion of GABAA receptor subunits β1–β3 restores acute inhibition of hunger by leptin following fasting (n = 7 mice per group). Functional contributions from CRISPR-mediated off-site mutagenesis of the triple deletion can therefore be excluded. l, Representative traces (left) and quantification (right) of electrically evoked IPSC (eIPSC) paired-pulse ratios in AGRP neurons of ad libitum-fed Npy-hrGFP mice before or after leptin incubation (n = 8 neurons from three mice per group). m, Representative anterograde tracing image (image from Allen Institute, experiment #113314337-DMH) showing intensive GFP-labelled projections in the ARC following unilateral viral injections in DMH of Lepr-IRES-cre mice. n, Schematic of unilateral injection of Cre-dependent AAV pEF1α-FLEX-synaptophysin-mCherry into the ventral DMH (vDMH) of Vgat-IRES-cre::Npy-hrGFP mice (left) and mCherry and hrGFP co-immunostaining (middle) to enable anterograde tracing of the projections of vDMH GABAergic neurons (vGATvDMH neurons). vGATvDMH neurons predominantly project to the ARC and their axon terminal puncta are intensively stained on the soma of AGRP neurons (right). o, Schematic summarizing leptin action on AGRP neurons and on GABAergic neurons in the DMH. Data are mean ± s.e.m. and representative of three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Student’s two-tailed, unpaired t-test (g, j, k, l) or two-way ANOVA (a, b, e, f).

Extended Data Table 1 Summary of FOS expression in saline- or STZ-treated C57BL/6 mice
Full size table

Supplementary information

Supplementary Tables 1-4

Supplementary Table 1 contains a detailed description of all statistical analyses performed for Figs. 1–4, including a brief description for each figure, sample size, statistical tests performed, P values, post hoc test, and post hoc test P values. Supplementary Table 2 contains a detailed description of all statistical analyses performed for Extended Data Figures 1–9, including a brief description for each figure, sample size, statistical tests performed, P values, post hoc test, and post hoc test P values. Supplementary Table 3 contains a detailed description of the raw data in Figs. 1–4, in which individual data points were not displayed. Supplementary Table 4 contains a detailed description of the raw data in Extended Data Figures 1–9, in which individual data points were not displayed.

Reporting Summary

Video 1: Hyperphagia developed in Streptozotocin-treated diabetic mice

A video of Saline- and STZ-treated littermates during light cycle (10:00 a.m.-11:00 a.m.), showing that STZ-treated mice display voracious feeding behavior during a time when mice are normally at rest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xu, J., Bartolome, C.L., Low, C.S. et al. Genetic identification of leptin neural circuits in energy and glucose homeostases. Nature 556, 505–509 (2018). https://doi.org/10.1038/s41586-018-0049-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-018-0049-7

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing