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Leptin-inhibited PBN neurons enhance responses to hypoglycemia in negative energy balance

Nature Neuroscience volume 17, pages 1744–1750 (2014)Cite this article

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Abstract

Hypoglycemia initiates the counter-regulatory response (CRR), in which the sympathetic nervous system, glucagon and glucocorticoids restore glucose to appropriate concentrations. During starvation, low leptin levels restrain energy utilization, enhancing long-term survival. To ensure short-term survival during hypoglycemia in fasted animals, the CRR must overcome this energy-sparing program and nutrient depletion. Here we identify in mice a previously unrecognized role for leptin and a population of leptin-regulated neurons that modulate the CRR to meet these challenges. Hypoglycemia activates neurons of the parabrachial nucleus (PBN) that coexpress leptin receptor (LepRb) and cholecystokinin (CCK) (PBN LepRbCCK neurons), which project to the ventromedial hypothalamic nucleus. Leptin inhibits these cells, and Cckcre-mediated ablation of LepRb enhances the CRR. Inhibition of PBN LepRb cells blunts the CRR, whereas their activation mimics the CRR in a CCK-dependent manner. PBN LepRbCCK neurons are a crucial component of the CRR system and may be a therapeutic target in hypoglycemia.

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Figure 1: VMH-projecting PBN LepRb neurons are activated by low glucose levels and are inhibited by leptin.
Figure 2: Deletion of LepRb from PBN CCK cells blunts IIH and augments the CRR to glucoprivation.
Figure 3: Enhanced CRR during hypoglycemic clamp in LepRbCck KO mice.
Figure 4: Inhibition of PBN LepRb neurons blunts the hyperglycemic response to glucoprivation.
Figure 5: Activation of PBN LepRb neurons mimics the CRR to glucoprivation.
Figure 6: CCK dependence of the hyperglycemic responses to activation of PBN LepRb neurons and glucoprivation.

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  • 10 December 2014

    In the version of this article initially published, the anteroposterior and dorsoventral stereotaxic injection coordinates in the Online Methods were transposed. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank AstraZeneca Pharmaceuticals for the generous gift of leptin, B. Roth (University of North Carolina, Chapel Hill) for CNO and members of the labs of M.G.M. and L.K.H. for helpful discussions. Research support was provided by the Animal Phenotyping Core of the Michigan Diabetes Research Center (US National Institutes of Health (NIH) grant P30 DK020572) and the Michigan Nutrition and Obesity Research Center (P30 DK089503), the American Diabetes Association, the American Heart Association, the Marilyn H. Vincent Foundation and the NIH (DK098853) to M.G.M., NIH DK055267 and DK020595 to C.J.R., NIH DK046060 to R.T.K., NIH DK046409 to L.S. and the Wellcome Trust (098012) and Biotechnology and Biological Sciences Research Council (BB/K001418/1) to L.K.H.

Author information

Author notes

  1. Jonathan N Flak, Christa M Patterson and Alastair S Garfield: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan, USA

    Jonathan N Flak, Christa M Patterson, Mark Germani, Justin C Jones, Michael Rajala & Martin G Myers Jr

  2. Center for Integrative Physiology, University of Edinburgh, Edinburgh, UK

    Alastair S Garfield

  3. Rowett Institute of Nutrition and Health, University of Aberdeen, Aberdeen, UK

    Giuseppe D'Agostino & Lora K Heisler

  4. Department of Pharmacology, University of Cambridge, Cambridge, UK

    Giuseppe D'Agostino & Martin G Myers Jr

  5. Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, USA

    Paulette B Goforth & Leslie Satin

  6. Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan, USA

    Amy K Sutton

  7. Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA

    Paige A Malec, Jenny-Marie T Wong & Robert T Kennedy

  8. Kovler Diabetes Center, University of Chicago, Chicago, Illinois, USA

    Christopher J Rhodes

  9. Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan, USA

    David P Olson

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Contributions

J.N.F. and C.M.P. produced the data in Figures 1,2,3,4,5 and Supplementary Figures 1,2,3,4,5,6,7,8,9,10, with the exception of the data in Figure 1e–i, which was produced by P.B.G. and L.S., and the measurements of catecholamines, which were performed by P.A.M., J.-M.T.W. and R.T.K. M.G. and M.R. helped produce Supplementary Figures 2, 5 and 10. J.C.J. aided with Figures 2,3,4 and with animal genotyping and husbandry. G.D., A.S.G. and L.K.H. performed the experiments in Figure 6. Adenoviral tracers were produced by A.K.S. and C.J.R. Experimental design, interpretation and manuscript preparation were led by M.G.M., L.K.H., J.N.F., C.M.P., A.S.G. and D.P.O.

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Correspondence to Lora K Heisler or Martin G Myers Jr.

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Integrated supplementary information

Supplementary Figure 1 Leptin-responsive LepRb neurons in PBN.

LepRbeYFP mice were injected with IP (a) vehicle or (b) leptin (5 mg/kg) 2 hours prior to perfusion and immunostaining for pSTAT3 (purple) and GFP (eYFP; green). Representative of 2 similar experiments, each with n = 3 animals. The PBN of representative mice is shown. Scale bars = 100 μM. Inset- digital zoom of the indicated area. Arrowheads indicate examples of colocalized neurons; not all colocalized neurons are indicated.

Supplementary Figure 2 Activation of PBN LepRb cells by IIH and glucoprivation but not i.c.v. insulin.

(a,b) LepRbeYFP mice were injected with IP (a) vehicle or 2DG (250 mg/kg) or (b) vehicle or insulin (1 U/kg) 2 hours prior to perfusion and immunostaining for cFos (purple) and GFP (eYFP; green) *p<0.001, {t(13) = −4.371}. (c,d) LepRbeYFP mice were injected with ICV (c) *p = 0.008, {t(12) = −3.19} vehicle or 2DG (1µg) *p<0.001, {t(9) = −6.01} or (d) vehicle or insulin (300mU) 2 hours prior to perfusion and immunostaining for cFos (purple) and GFP (eYFP; green). Colocalized cFos+eYFP and total eYFP neurons were quantified; data are presented as %colocalized/total eYFP. All studies: n = 5–8 animals analyzed by one tailed t-test; each panel representative of two separate studies.

Supplementary Figure 3 Similar pattern of cFos IR in PBN LepRb cells in response to i.p. insulin and 2DG.

LepRbeYFP mice were injected with IP (a) vehicle, (b) insulin (1 U/kg), or or 2DG (250 mg/kg) 2 hours prior to perfusion and immunostaining for cFos (purple) and GFP (eYFP; green). The PBN of representative mice is shown. Scale bars= 100 μM. n = 9 animals per group; representative of two separate studies. Inset- digital zoom of the indicated area. Arrowheads indicate examples of colocalized neurons; not all colocalized neurons are indicated.

Supplementary Figure 4 Leptin blunts fasting-induced enhancement of the CRR.

Male C57Bl6/J mice were allowed to feed ad libitum (Control), were fasted for 12 hours (Fast), or were injected with leptin (5 mg/kg, IP) and fasted for 12 hours (Fast+Leptin) before being injected with 2DG (500 mg/kg, IP). (a) Blood glucose was monitored at the indicated times; p = 0.013 (60 min), <0.001 (90 min), 0.008 (120 min) vs control and p = 0.003 (90 min), 0.008 (120 min) vs Fast + Leptin {F(12,26) = 4.61} by two way repeated measures ANOVA with Fisher LSD post hoc test. N = 8–9 animals per condition; data are plotted as mean +/− SEM. Letters indicate significant differences relative to other conditions by ANOVA with Fishers post-hoc test. (b) Area under the curve (AUC) for the data graphed in (a); *p<0.001 vs control and *0.009 vs Fast + Leptin {F(2,26) = 15.15}by one way ANOVA with Fisher LSD post hoc test.

Supplementary Figure 5 Induction of cFos in PBN CCK neurons by glucoprivation.

(a,b) CCKeYFP mice were injected with IP (a) vehicle or (b) 2DG (250 mg/kg, IP) 2 hours prior to perfusion and immunostaining for cFos (purple) and GFP (eYFP; green). The PBN of representative mice is shown. N=11 animals per condition; representative of two similar studies. Scale bars= 100 μM. Inset- digital zoom of the indicated area. Arrowheads indicate examples of colocalized neurons; not all colocalized neurons are indicated. (c) Quantification of the data. Colocalized cFos+eYFP and total eYFP neurons were quantified; data are presented as %colocalized/total eYFP; *p = 0.017, {t(16) = −2.32}. n = 7–11 animals per condition *p<0.05 by one tailed t-test; representative of two separate studies.

Supplementary Figure 6 Reduced leptin-stimulated pSTAT3 IR in PBN and EW nuclei of LepRbCck KO mice.

Quantitation of pSTAT3-IR cells in the indicated hindbrain nuclei of Control and LepRbCckKO mice following treatment with leptin (IP, 5 mg/kg) 2 hours prior to perfusion. N = 3–4 animals per condition; data are plotted as mean +/− SEM. NTS- nucleus tractus solitarius; PBN- parabrachial nucleus; PAG- periaqueductal grey; DR- dorsal raphe; EW- Edinger-Westphal; LR- linear raphe. EW: t(5) = 4.01, *p = 0.05. PBN: t(5) = 8.28, *p<0.001 by one tailed t-test.

Supplementary Figure 7 Energy balance and glucose homeostasis in LepRbCck KO mice.

(a) Weekly body weight for male Control and LepRbCckKO mice. (b) GTT (blood glucose values at the indicated times following an IP injection of 3 g/kg) in 15-week old male Control and LepRbCckKO mice. Hormone levels at 14 weeks of age in male Control and LepRbCckKO mice: (c) leptin, (d) insulin, (e) corticosterone. N = 12–18 animals per condition; p = NS for all comparisons; all data are plotted as mean +/− SEM.

Supplementary Figure 8 Fasting fails to enhance the CRR to glucoprivation in LepRbCck KO mice.

Male Control and LepRbCckKO mice were fasted for 4 (a,b) or 24 (c,d) hours prior to injection with 2DG (250 mg/kg, IP); blood glucose was monitored for 180 minutes afterwards (a,c); *p<0.001 (90 min), <0.001 (12 0min), {F(6,20) = 19.43} by two way repeated measures ANOVA with Fisher LSD post hoc test. (b,d) Quantification of area under the curve (AUC) from the data in (a,c) respectively; *p = 0.012, {t(19) = −2.79} by two tailed t-test. N = 9–13 animals per condition; all data are plotted as mean +/− SEM.

Supplementary Figure 9 Leptin action via PBNCck neurons does not alter glycemic responses to restraint stress.

(a) Leprcre mice were crossed onto the Rosa26-eYFP background to generate LepRbeYFP mice expressing eYFP in LepRb neurons. (b,c) LepRbeYFP mice were handled briefly (b) or subjected to restraint stress in a conical tube for 30 minutes (c) and then were perfused 2 hours prior to immunostaining for cFos (purple) and GFP (eYFP; green) in one experiment. The PBN of representative mice is shown. Inset- digital zoom of the indicated area. Arrowheads indicate examples of colocalized neurons. (d) Cckcre mice were crossed onto the Leprflox background to generate LepRbCckKO mice. (e,f) Control and LepRbCckKOeYFP mice were subjected to restraint stress for 30 minutes and blood glucose was monitored for 120 minutes (e). (f) Area under the curve (AUC) is plotted for the data in (e). N = 2–4 animals per condition; p = NS for all comparisons; all data are plotted as mean +/− SEM.

Supplementary Figure 10 Induction of cFos in PBN and VMH neurons after DREADD-mediated activation of PBN LepRb neurons.

The cre-inducible AAV-hM3Dq was injected bilaterally into the PBN of male Leprcre mice. Animals were injected with Vehicle (a,c) or CNO (b,d; 0.3 mg/kg, IP) and monitored for 120 minutes (see Figure 6). At the end of the experiment, animals were perfused and brain sections were stained for cFos (green; panels a-d) and/or dsRed (red; panels a-b). Representative images are shown for the PBN (a,b) and VMH (c,d) in one experiment. (e) Quantification of cFos in the VMH of Vehicle and CNO treated animals. N = 9–11 animals per condition; *p<0.001, {t(18) = −6.83} by one-tailed t-test; all data are plotted as mean +/− SEM.

Supplementary Figure 11 Working model of PBN LepRb neurons.

Our data suggest that PBN LepRb expressing neurons are excited by hypoglycemia, inhibited by leptin, release CCK in the VMH to drive counterregulatory responses. Our data indicates the importance of this pathway in modulating counter-regulatory responses during fasting conditions to promote survival of the individual.

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Flak, J., Patterson, C., Garfield, A. et al. Leptin-inhibited PBN neurons enhance responses to hypoglycemia in negative energy balance. Nat Neurosci 17, 1744–1750 (2014). https://doi.org/10.1038/nn.3861

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