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Melanocortin 4 receptors in autonomic neurons regulate thermogenesis and glycemia

Abstract

Whether melanocortin 4 receptors (MC4Rs) in extra-hypothalamic neurons, including cholinergic autonomic pre-ganglionic neurons, are required to control energy and glucose homeostasis is unclear. We found that MC4Rs in sympathetic, but not parasympathetic, pre-ganglionic neurons were required to regulate energy expenditure and body weight, including thermogenic responses to diet and cold exposure and 'beiging' of white adipose tissue. Deletion of Mc4r genes in both sympathetic and parasympathetic cholinergic neurons impaired glucose homeostasis.

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Figure 1: Deleting Mc4r in pre-ganglionic cholinergic neurons in the sympathetic nervous system causes obesity as a result of reduced EE.
Figure 2: Selective deletion of Mc4r in pre-ganglionic cholinergic neurons in the sympathetic nervous system results in gluco-regulatory abnormalities.
Figure 3: Deletion Mc4r in pre-ganglionic cholinergic neurons promotes obesity and impairs cold tolerance.

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Acknowledgements

We thank E. Rosen (Beth Israel Deaconess Medical Center and Harvard Medical School) for assistance; personnel in the University of Texas Southwestern Mouse Phenotyping and Histology Cores; and the US National Institutes of Health Vanderbilt University Mouse Metabolic Phenotyping Core Hormone Assay and Analytical Core, supported by DK 059637. This work was supported by grants from the US National Institutes of Health (F32 DK092083 and K01 DK098317 to E.D.B., R01 DK55758 and R01 DK099110 to P.E.S., R01 DK088761 to P.E.S. and J.K.E., K01 DK087780 to K.W.W., R01 DK089044, R01 DK071051, R01 DK075632 and R37 DK053477 to B.B.L., and R01 DK088423 and R37 DK053301 to J.K.E.), a mentor-based postdoctoral fellowship from the American Diabetes Association (7-11-MN-16 to T.L.), a post-doctoral fellowship from American Heart Association (13POST16710016to X.K.), a postdoctoral fellowship from the American Heart Association (12POST8860007 to J.-W.S.), and BNORC and BADERC Transgenic Core grants P30DK046200 and P30DK057521 to B.B.L.

Author information

Authors and Affiliations

Authors

Contributions

E.D.B. designed and performed all experiments except for gene expression and immunoblotting, analyzed the data, and wrote the manuscript. T.L. designed and performed all experiments except gene expression and immunoblotting, analyzed the data, and edited the manuscript. X.K. analyzed gene expression in adipose depots, performed immunoblotting in BAT, analyzed the data and reviewed the manuscript. L.V. and D.P.O. designed and developed the Mc4rloxP/loxP mice. D.P.O. designed and developed the Chat-cre mouse. K.W.W. and J.-W.S. performed electrophysiological experiments. Z.D., S.L. and C.E.L. assisted with experiments. P.E.S. designed experiments and edited the manuscript. B.B.L. and J.K.E. supervised development of the mouse models, designed experiments and edited the manuscript.

Corresponding authors

Correspondence to Bradford B Lowell or Joel K Elmquist.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Validation of hindbrain neurons lacking melanocortin 4 receptors (Mc4r).

(a-b) Representative in situ hybridization images of intact Mc4r expression in Mc4rflox/flox mice versus absence in Mc4rflox/flox x Phox2B-cre littermates (n = 3 each genotype). Insets depict higher magnification of outlined region. (c) Representative trace shows a characteristic hyperpolarization of Phox2B-positive dorsal motor nucleus of the vagus (DMV) neurons in response to the Mc4r agonist THIQ. (c-d) Administration of tolbutamide and corresponding changes in current versus voltage (I-V) relationship suggests this effect is KATP channel-mediated. (e) Representative trace denotes lack of a response following administration of THIQ to cells targeted from the DMV of Mc4rflox/flox x Phox2B-cre mice. (f) Histogram summarizes THIQ induced responses of DMV neurons from Phox2B-cre and Mc4rflox/flox x Phox2B-cre littermate mice (n = 4 and 15, respectively).

Supplementary Figure 2 Deleting melanocortin 4 receptor (Mc4r) in pre-ganglionic cholinergic neurons in the sympathetic, but not parasympathetic nervous system (SNS and PNS), impacts body weight or composition.

(a) Body composition in chow-fed 20-week-old Mc4rflox/flox, Mc4rflox/flox x Chat-cre, and Mc4r-null littermate mice (n = 8, 8,and 7, respectively). (b) Food intake in 7-8 week-old chow-fed Mc4rflox/flox, Mc4rflox/flox x Chat-cre, and Mc4r-null littermate mice (n = 8, 8, and 7, respectively). (c) Body weight in chow-fed Mc4rflox/flox mice and littermates with selective deletion of Mc4rs in Phox2B-cre positive neurons (Mc4rflox/flox x Phox2B-cre; n = 7 and 8, respectively) in the PNS. (d) Body composition in 6 week-old Mc4rflox/flox and Mc4rflox/flox x Phox2B-cre littermate using NMR.

Supplementary Figure 3 Selective deletion of melanocortin 4 receptors (Mc4r) in pre-ganglionic cholinergic neurons in the parasympathetic nervous system (PNS) causes gluco-regulatory abnormalities.

(a) 5 h fasted blood glucose, (b) plasma insulin, and (c) 5 h fasted plasma glucagon in chow-fed mice with intact Mc4r signaling (Mc4rflox/flox) and selective deletion in the PNS (n = 8 per genotype). * indicates p<0.05 versus Mc4rflox/flox mice using two-tailed unpaired Student's t-tests. 18 h-fasted (t = 0 min) and glucose-stimulated changes in (d) blood glucose and (e) plasma insulin during an IPGTT (1.5 mg/kg) in Mc4rflox/flox, Mc4rflox/flox x Chat-cre, and Mc4r-null mice (n = 8, 8, and 7, respectively). * and ** indicate p<0.05 or 0.01 versus Mc4rflox/flox and † indicates p<0.05 versus Mc4rflox/flox x Chat-cre using one-way ANOVA followed by Tukey's post-hoc analyses. Similar values are shown in (f) and (g) for Mc4rflox/flox and Mc4rflox/flox x Phox2B-cre mice (n = 7 and 8, respectively). * indicates p<0.05 versus Mc4rflox/flox using two-tailed paired Student's t-tests. θ indicates p<0.05 comparing basal versus clamp within genotype using two-tailed paired Student's t-tests. 5 h-fasted (t = 0 min) and insulin-stimulated (h) blood glucose and (i) plasma insulin during a hyperinsulinemic-euglycemic clamps in Mc4rflox/flox, Mc4rflox/flox x Chat-cre, and Mc4r-null mice (n = 8, 8, and 7, respectively). * and ** indicate p<0.05 or 0.01 versus Mc4rflox/flox and † indicates p<0.05 versus Mc4rflox/flox x Chat-cre using one-way ANOVA followed by Tukey's post-hoc analyses. θ indicates p<0.05 comparing basal versus clamp within genotype using two-tailed paired Student's t-tests. Similar values are shown in (j) and (k) for Mc4rflox/flox and Mc4rflox/flox x Phox2B-cre mice (n = 7 and 6,respectively).

Supplementary Figure 4 Deleting melanocortin 4 receptor (Mc4r) in pre-ganglionic neurons of the parasypathetic nervous system (PNS) does not promote obesity, while loss in the sympathetic nervous system (SNS) impairs iBAT responses to diet.

(a) Body weight in mice with intact Mc4r signaling (Mc4rflox/flox) and selective deletion in the PNS (Mc4rflox/flox x Phox2B-cre) fed a high-fat/high-sucrose diet at 8 weeks-of-age (n = 8 and 7, respectively). (b) Energy expenditure (EE) and (c) food intake in chow-fed 7- to 8-week-old Mc4rflox/flox and Mc4rflox/flox x Phox2B-cre mice introduced to HFHS diet 3 d prior to metabolic cage assessment (n = 8 and 6, respectively). EE in panel b was not normalized for body weight differences since none exist. There were no statistical differences in panels a-c using two tailed unpaired Student's t-tests. iBAT gene expression in (d) Mc4rflox/flox, Mc4rflox/flox x Chat-cre, and Mc4r-null mice as well as (e) Mc4rflox/flox and Mc4rflox/flox x Phox2B-cre mice fed HFHS diet for 12 weeks (8-20 weeks-of-age). * and † indicate p<0.05 versus Mc4rflox/flox or Mc4rflox/flox x Chat-cre one-way ANOVA followed by Tukey's post-hoc analyses or two tailed unpaired Student's t-tests. All mice were male and results are shown as means ± SEM.

Supplementary Figure 5 White and brown adipose tissues do not respond to cold in mice selectively lacking melanocortin 4 receptors (Mc4r) in pre-ganglionic cholinergic neurons of the sympathetic nervous system.

(a) Inguinal white adipose tissue (iWAT) gene expression in Mc4rflox/flox, Mc4rflox/flox x Chat-cre, and Mc4r-null mice housed in 6° C for 5 days. (b-c) Representative images of intrascapular brown adipose tissue (iBAT; b) histology and (c) UCP1 content. Unaltered picture of this blot is shown in Supplementary Figure 6. (d) Representative iWAT UCP1 staining (brown) in chow-fed in chow-fed Mc4rflox/flox or Mc4rflox/flox x Chat-cre littermate mice after 5d cold exposure (n = 6 per genotype; inset in upper right shows higher magnification of area outlined by dashed lines). * and † indicate p<0.05 versus Mc4rflox/flox or Mc4rflox/flox x Chat-cre one-way ANOVA followed by Tukey's post-hoc analyses or two-tailed unpaired Student's t-tests. All mice were male and results are shown as means ± SEM.

Supplementary Figure 6 Unaltered full-length Western blots of UCP1 and β-actin.

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Berglund, E., Liu, T., Kong, X. et al. Melanocortin 4 receptors in autonomic neurons regulate thermogenesis and glycemia. Nat Neurosci 17, 911–913 (2014). https://doi.org/10.1038/nn.3737

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