Melanocortin 4 receptors in autonomic neurons regulate thermogenesis and glycemia

SUMMARY Melanocortin 4 receptors (Mc4rs) are expressed by extra-hypothalamic neurons including cholinergic autonomic pre-ganglionic neurons. However, whether Mc4rs in these neurons are required to control energy and glucose homeostasis is unclear. Here we report that Mc4rs in sympathetic, but not parasympathetic, pre-ganglionic neurons are required to regulate energy expenditure and body weight including brown and white adipose tissue thermogenic responses to diet and cold exposure. In addition, deletion of Mc4rs in both sympathetic and parasympathetic cholinergic neurons impairs glucose homeostasis.

diet and cold exposure. In addition, deletion of Mc4rs in both sympathetic and parasympathetic cholinergic neurons impairs glucose homeostasis.
Distinct sub-sets of melanocortin 4 receptors (MC4Rs) in the CNS are established to exert segregated effects on tissues and processes underlying energy balance, glucose homeostasis, and cardiovascular function [1][2][3][4][5] . Notably, MC4Rs expressed outside the hypothalamus in preganglionic cholinergic neurons of the sympathetic and parasympathetic nervous system have emerged as potential regulatory sites [3][4][5] , thus raising the possibility that these subpopulations are crucial to control energy and glucose homeostasis. Extension of these and other data [6][7][8][9] further suggests that MC4Rs in sympathetic pre-ganglionic neurons may be required to stimulate thermogenesis in brown and white adipose tissue depots (BAT and WAT, respectively). This is noteworthy since thermogenic properties of BAT and WAT have recently become avid areas of interest to treat obesity and diabetes. However, the central pathway(s) that govern these processes are not fully understood.
To study requirement(s) for MC4Rs in the autonomic nervous system (ANS) to modulate energy and glucose homeostasis including role(s) of BAT and WAT depots, we generated genetically modified mice selectively lacking Mc4rs in pre-ganglionic autonomic neurons. Since MC4R loss-of-function causes obesity in humans 1, 11, 12 and animals 1, 12 , our first goal was to assess body weight. Chow-fed Mc4r flox/flox x Chat-cre mice initially exhibited normal body weight, but diverged at eight weeks-of-age and were ~ 40% heavier at 20 weeks-of-age ( Figure 1A). This obesity was intermediate versus Mc4r-nulls and due to higher lean and fat mass ( Figures 1A and 1B). It was also noted that body length was normal in Mc4r flox/flox x Chat-cre mice whereas Mc4r-null snout-to-anus distance was longer (data not shown). This is, to our knowledge, the first MC4R mutation causing obesity that is not confounded by altered growth. Metabolic cages were then used in young mice [to minimize differences in body weight ( Figure 1A)] to isolate that Mc4r flox/flox x Chat-cre mice were obese due to low EE, not hyperphagia ( Figures 1C-1D). As expected, global Mc4r-null mice were hypometabolic and hyperphagic ( Figures 1C-1D). In contrast, chow-fed Mc4r flox/flox x Phox2B-cre revealed no differences in body weight or body composition ( Supplementary  Figures 2A-2B). Collectively, these results indicate that Mc4r signaling in cholinergic neurons, including sympathetic pre-ganglionic neurons, are required to maintain normal EE and thus body weight. Mc4rs in Phox2b neurons including those in the DMV, however, are dispensable for normal body weight homeostasis.
In addition to established roles of Mc4rs to control energy balance, growing evidence identifies role(s) to directly regulate glycemia. In agreement with this literature, we found that circulating glucose, insulin, and glucagon were elevated in young, body weight-  Figure 2G). In contrast, impaired insulin-stimulated suppression of endoR a is the sole contributor to reduced GIR in Mc4r flox/flox x Phox2B-cre mice ( Figure 2H). These data demonstrate that Mc4r flox/flox x Chat-cre exhibit gluco-regulatory defects including hyperglycemia, hyperinsulinemia, hepatic insulin resistance, and hyperglucagonemia that in many respects are similar to Mc4r-null mice. Mc4r flox/flox x Phox2B-cre mice exhibit modest insulin resistance, but not overt hyperglycemia likely due to hyperinsulinemia. Collectively, these results demonstrate that Mc4r signaling in autonomic pre-ganglionic neurons are required to regulate glucose metabolism and insulin sensitivity.
We next challenged energy homeostasis in both Mc4r flox/flox x Chat-cre and Mc4r flox/flox x Phox2B-cre cohorts using high-fat/high-sucrose (HFHS) diet-induced obesity. Prior work has shown that whole-body Mc4r-deficient mice are more sensitive to diet-induced obesity 6,7,13 . Time-course analyses in Mc4r flox/flox x Chat-cre mice show that HFHS diet accelerated weight-gain resulting in obesity nearly comparable to Mc4r-null littermates ( Figure 3A). Results from a metabolic cage paradigm similar to prior work 6 where HFHS diet is introduced to naïve, young (to minimize differences in body weight) chow-fed adult mice pinpoints that both hyperphagia and failure to fully increase EE likely underlies obesity in Several studies have also suggested that Mc4r expressing neurons regulate intrascapular BAT (iBAT), which is potently regulated by the sympathetic nervous system and a key component of adaptive thermogenesis [6][7][8][9] . We found that transcripts associated with heat production, fatty acid oxidation, and mitochondrial functions were reduced in chow-fed Mc4r flox/flox x Chat-cre iBAT (Supplementary Figure 4D). This was not the case in chowfed Mc4r flox/flox x Phox2B-cre mice (Supplementary Figure 4E). Remote biotelemetry probes were next used to assess body temperature at 23°C and in response to cold (5 days at 6°C). These results show that basal body temperature was not reduced in Mc4r flox/flox x Chat-cre ( Figure 3D). However, Mc4r flox/flox x Chat-cre mice were unable to engage adaptive thermogenesis and body temperature in cold-exposed Mc4r flox/flox x Chat-cre mice declined rapidly to Mc4r-null values within 4 h ( Figure 3D). Analyses of iBAT histology and uncoupling protein 1 (UCP1) protein content after 120 h at 6°C further confirmed impaired thermogenic responses in Mc4r flox/flox x Chat-cre mice on par with defects in Mc4r-null littermates ( Figure 3E and 3F). In addition, inguinal WAT (iWAT) in coldexposed Mc4r flox/flox x Chat-cre mice failed to show evidence of "browning" based on gene expression (Supplementary Figure 4F) or histology for UCP1 ( Figure 3G). Together, these data suggest that Mc4rs in sympathetic pre-ganglionic neurons are essential for adaptive thermogenesis including recruitment of iBAT and browning of iWAT. Notably, Mc4rs directly in these neurons are also required to maintain body temperature following cold exposure.
These current studies extend and confirm conclusions that MC4Rs in both parasympathetic and sympathetic pre-ganglionic neurons are sufficient and required to maintain aspects of normal energy and glucose homeostasis 3 . Specifically, selective loss of Mc4r in the sympathetic pre-ganglionic neurons deleteriously and independently impacts both processes while deletion in the vagal motor neurons results in hyperinsulinemia and modest insulin resistance. Our evidence also demonstrates that Mc4rs in sympathetic pre-ganglionic neurons are required for diet-and cold-induced thermogenesis in iBAT and "browning" of iWAT. These are topical findings because existence of functional depots of iBAT in adult humans [14][15][16][17][18] and the ability of WAT 19,20 to exhibit thermogenic properties characteristic of BAT are recently described findings. However, it is important to clarify that our data cannot firmly establish that defects in iBAT and/or ability to "brown" iWAT contribute to low EE and/or hyperglycemia in mice lacking Mc4rs in sympathetic pre-ganglionic neurons. Nonetheless, synthesis of these data with the existing literature connecting specific subpopulations of Mc4rs to specific tissues and processes shows that SNS-mediated mechanisms regulate an array of tissues including the pancreas, liver, BAT, and WAT. In contrast, Mc4r-mediated control via the DMV appears restricted to pancreatic beta cells and circulating insulin. Outlining these functional connections is potentially important to develop more selective strategies to target Mc4rs or related pathways. Caution is warranted, however, as targeting Mc4rs to stimulate iBAT, browning of iWAT, or other EE mechanisms to exert beneficial effects in contexts of obesity and/or diabetes may cause sympathetic nervous system side effects. We propose that these extra-hypothalamic Mc4rs underlie key defects associated with melanocortin 4 receptor mutations in humans and that these sites are critical components involved in coordinated control of energy and glucose homeostasis.

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Animal Care
Mc4r flox/flox , Chat-cre, and Phox2B-cre mice on a C57BL/6J background have been previously described 3,4 . Mice were group housed (1-5 mice per cage) in a barrier facility at 23°C unless otherwise noted. Mice were provided Harlan Teklad 2016 chow diet and water ad libitum unless otherwise noted. High-fat/high-sucrose (HFHS; Research Diets D12331) diet, if applicable, was removed and refilled weekly. Mice losing >10% of body weight during the acclimation period for metabolic cage studies (see below) or post-surgery prior to clamp studies (see below) were not studied. Body composition was measured using NMR (Bruker Mini-Spec).

Hormone Measurements
All measurements were done in duplicate. Insulin was measured using a commercial ELISA kit (Crystal Chem.). Glucagon was measured via RIA at the Vanderbilt University MMPC.

Metabolic Cages
Experiments were performed in a temperature-controlled room containing 36 TSE metabolic cages maintained by UTSW Animal Resources personnel. One week prior to study, mice were singly housed to acclimate to new housing. Three days prior to study, mice were transported to room containing metabolic cages to acclimate to a new environment. HFHS diet, if applicable, was also introduced at the beginning of this acclimation period. After 3 d acclimation, cages were connected to TSE system for a total of 5 d. Days 2-4 were used for data analyses.

Cold Exposure
Body temperature was assessed using biotelemetry probes (IPTT-300, BioMedic Data Systems) injected under anesthesia one-week prior. Mice were housed in cold chambers maintained at 6°C by UTSW Animal Resources personnel.

Clamp Studies
Experiments were done with the assistance of the UTSW Mouse Phenotyping Core.

Protein Extraction and Western Blot Analysis
Protein was extracted from BAT by using RIPA buffer (Boston BioProducts) supplemented with complete protease inhibitor cocktail (Roche). For western blot analyses, 60 mg protein was subjected to SDS-PAGE under reducing conditions, transferred, and blotted with the anti-UCP1 antibody (Abcam, ab10983).

Histology
Tissues were dissected and fixed in formalin for 48 h at 4°C followed by 50% ethanol. BAT histology was performed with assistance from the UTSW Histology Core. Inguinal white adipose tissue was performed with assistance from the Harvard Histology Core.

Analysis of Gene Expression by Quantitative PCR
Total RNA was extracted from tissues with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. 1 μg total RNA was converted into first-strand cDNA with oligo(dT) primers as described by the manufacturer (Clontech). PCR was performed in an Mx3000P Q-PCR system (Stratagene) with specific primers and SYBR Green PCR Master Mix (Stratagene). The relative abundance of mRNAs was standardized with 36B4 mRNA as the invariant control.

Statistics
Sample sizes were determined using prior experience and power calculations designed to detect p<0.05 with a 15% variance. There were no methods to randomize mice to experimental groups. There were also no methods to blind investigators to genotype during experiments. Pre-established criteria for excluding data points were data two standard deviations outside the mean or any data obtained from mice that died or lost > 10% of body weight due to metabolic cage acclimation or clamp studies.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.  x Chat-cre mice using two-tailed unpaired Student's t-tests. ϕ indicates p<0.05 comparing basal versus clamp within genotype using two-tailed paired Student's t-tests. All mice were male and results are means ± SEM.