Adipocytes store excess energy in the form of triglycerides and signal the levels of stored energy to the brain. Here we show that adipocyte-specific deletion of Arntl (also known as Bmal1), a gene encoding a core molecular clock component, results in obesity in mice with a shift in the diurnal rhythm of food intake, a result that is not seen when the gene is disrupted in hepatocytes or pancreatic islets. Changes in the expression of hypothalamic neuropeptides that regulate appetite are consistent with feedback from the adipocyte to the central nervous system to time feeding behavior. Ablation of the adipocyte clock is associated with a reduced number of polyunsaturated fatty acids in adipocyte triglycerides. This difference between mutant and wild-type mice is reflected in the circulating concentrations of polyunsaturated fatty acids and nonesterified polyunsaturated fatty acids in hypothalamic neurons that regulate food intake. Thus, this study reveals a role for the adipocyte clock in the temporal organization of energy regulation, highlights timing as a modulator of the adipocyte-hypothalamic axis and shows the impact of timing of food intake on body weight.

The innervation of brown adipose tissue (BAT) by the sympathetic nervous system (SNS) is incontrovertible and, with its activation, functions as the principal, if not exclusive, stimulator of BAT thermogenesis. The parasympathetic innervation of BAT only appears in two minor BAT depots, but not in the major interscapular BAT (IBAT) depot. BAT thermogenesis is triggered by the release of norepinephrine from its sympathetic nerve terminals, stimulating β3-adrenoceptors that turns on a cascade of intracellular events ending in activation of uncoupling protein-1 (UCP-1). BAT also has sensory innervation that may function to monitor BAT lipolysis, a response necessary for activation of UCP-1 by fatty acids, or perhaps responding in a feedback manner to BAT temperature changes. The central sympathetic outflow circuits ultimately terminating in BAT have been revealed by injecting the retrograde viral transneuronal tract tracer, pseudorabies virus, into the tissue; moreover, there is a high degree of colocalization of melanocortin 4-receptor mRNA on these neurons across the neural axis. The necessary and sufficient central BAT SNS outflow sites that are activated by various thermogenic stimuli are not precisely known. In a chronic decerebration procedure, IBAT UCP-1 gene expression can be triggered by fourth ventricular injections of melanotan II, the melanocortin 3/4 receptor agonist, suggesting that there is sufficient hindbrain neural circuitry to generate thermogenic responses with this stimulation. The recent recognition of BAT in normal adult humans suggests a potential target for stimulation of energy expenditure by BAT to help mitigate increased body fat storage.

Summary Circulating factors are typically invoked to explain bidirectional communication between the CNS and white adipose tissue (WAT). Thus, initiation of lipolysis has been relegated primarily to adrenal medullary secreted catecholamines and the inhibition of lipolysis primarily to pancreatic insulin, whereas signals of body fat levels to the brain have been ascribed to adipokines such as leptin. By contrast, evidence is given for bidirectional communication between brain and WAT occurring via the sympathetic nervous system (SNS) and sensory innervation of this tissue. Using retrograde transneuronal viral tract tracers, the SNS outflow from brain to WAT has been defined. Functionally, sympathetic denervation of WAT blocks lipolysis to a variety of lipolytic stimuli. Using anterograde transneuronal viral tract tracers, the sensory input from WAT to brain has been defined. Functionally, these WAT sensory nerves respond electrophysiologically to increases in WAT SNS drive suggesting a possible neural negative feedback loop to regulate lipolysis.

Song CK, Vaughan CH, Keen-Rhinehart E, Harris RB, Richard D, Bartness TJ. Melanocortin-4 receptor mRNA expressed in sympathetic outflow neurons to brown adipose tissue: neuroanatomical and functional evidence. Am J Physiol Regul Integr Comp Physiol 295: R417-R428, 2008. First published June 11, 2008 doi:10.1152/ajpregu.00174.2008.-A precise understanding of neural circuits controlling lipid mobilization and thermogenesis remains to be determined. We have been studying the sympathetic nervous system (SNS) contributions to white adipose tissue (WAT) lipolysis largely in Siberian hamsters. Central melanocortins are implicated in the control of the sympathetic outflow to WAT, and, moreover, the melanocortin 4 receptors (MC4-R) appear to be principally involved. We previously found that acute third ventricular melanotan II (MTII; an MC3/4-R agonist) injections increase sympathetic drive (norepinephrine turnover) to interscapular brown adipose tissue (IBAT) and IBAT temperature. Here we tested whether MC4-R mRNA is expressed in IBAT SNS outflow neurons using in situ hybridization for the former and injections of the transneuronal viral retrograde tract tracer, pseudorabies virus (PRV) into IBAT, for the latter. Significant numbers of double-labeled cells for PRV and MC4-R mRNA were found across the neuroaxis (mean of all brain sites ϳ60%), including the hypothalamic paraventricular nucleus (PVH; ϳ80%). Acute parenchymal MTII microinjections into the PVH of awake, freely-moving hamsters, using doses below those able to increase IBAT temperature when injected into the third ventricle, increased IBAT temperature for as long as 4 h, as measured by temperature transponders implanted below the tissue. Collectively, these data add significant support to the view that central melanocortins are important in controlling IBAT thermogenesis via the SNS innervation of this tissue, likely through the MC4-Rs.Siberian hamsters; in situ hybridization; pseudorabies virus; tract tracing; melanocortins A COMPLETE UNDERSTANDING OF the neural circuits controlling the sympathetic nervous system (SNS) mobilization of lipid from white adipose tissue (WAT) and those triggering thermogenesis in brown adipose tissue (BAT) remain to be determined, although our knowledge has increased markedly in the past decade (for review, see Refs. 6 and 16). We have been studying the SNS contributions to the reversal of the naturally-occurring seasonal obesity of Siberian hamsters (Phodopus sungorus; for a review, see Ref. 5) in WAT, and to a lesser extent BAT (4,11,12,18,49). Of the many remaining unknown details of the SNS outflow circuitry to WAT and BAT is the identification of their neurochemical phenotype, although some progress has been made for both tissues (e.g., 8,39,48,51,53,56). One of the neurochemicals strongly implicated in the sympathetic control of these adipose tissues is the melanocortins (for review, see Refs. 6 and 45). The most important members of the melanocortin family for the control of energy balance is ␣-melanocyte stimulating hor...

Oxytocin (OT) administration elicits weight loss in diet-induced obese (DIO) rodents, nonhuman primates, and humans by reducing energy intake and increasing energy expenditure. Although the neurocircuitry underlying these effects remains uncertain, OT neurons in the paraventricular nucleus are positioned to control both energy intake and sympathetic nervous system outflow to interscapular brown adipose tissue (BAT) through projections to the hindbrain nucleus of the solitary tract and spinal cord. The current work was undertaken to examine whether central OT increases BAT thermogenesis, whether this effect involves hindbrain OT receptors (OTRs), and whether such effects are associated with sustained weight loss following chronic administration. To assess OT-elicited changes in BAT thermogenesis, we measured the effects of intracerebroventricular administration of OT on interscapular BAT temperature in rats and mice. Because fourth ventricular (4V) infusion targets hindbrain OTRs, whereas third ventricular (3V) administration targets both forebrain and hindbrain OTRs, we compared responses to OT following chronic 3V infusion in DIO rats and mice and chronic 4V infusion in DIO rats. We report that chronic 4V infusion of OT into two distinct rat models recapitulates the effects of 3V OT to ameliorate DIO by reducing fat mass. While reduced food intake contributes to this effect, our finding that 4V OT also increases BAT thermogenesis suggests that increased energy expenditure may contribute as well. Collectively, these findings support the hypothesis that, in DIO rats, OT action in the hindbrain evokes sustained weight loss by reducing energy intake and increasing BAT thermogenesis.

Here, we provide a detailed account of how to denervate white and brown adipose tissue (WAT and BAT) and how to measure sympathetic nervous system (SNS) activity to these and other tissues neurochemically. The brain controls many of the functions of WAT and BAT via the SNS innervation of the tissues, especially lipolysis and thermogenesis, respectively. There is no clearly demonstrated parasympathetic innervation of WAT or the major interscapular BAT (IBAT) depot. WAT and BAT communicate with the brain neurally via sensory nerves. We detail the surgical denervation (eliminating both innervations) of several WAT pads and IBAT. We also detail more selective chemical denervation of the SNS innervation via intra-WAT/IBAT 6-hydroxy-dopamine (a catecholaminergic neurotoxin) injections and selective chemical sensory denervation via intra-WAT/IBAT capsaicin (a sensory nerve neurotoxin) injections. Verifications of the denervations are provided (HPLC-EC detection for SNS, ELIA for calcitonin gene-related peptide (proven sensory nerve marker)). Finally, assessment of the SNS drive to WAT/BAT or other tissues is described using the alpha-methyl-para-tyrosine method combined with HPLC-EC, a direct neurochemical measure of SNS activity. These methods have proven useful for us and for other investigators interested in innervation of adipose tissues. The chemical denervation approach has been extended to nonadipose tissues as well.

Brown adipose tissue (BAT) is an important source of thermogenesis which is nearly exclusively dependent on its sympathetic nervous system (SNS) innervation. We previously demonstrated the SNS outflow from brain to BAT using the retrograde SNS-specific transneuronal viral tract tracer, pseudorabies virus (PRV152) and demonstrated the sensory system (SS) inflow from BAT to brain using the anterograde SS-specific transneuronal viral tract tracer, H129 strain of herpes simplex virus-1. Several brain areas were part of both the SNS outflow to, and receive SS inflow from, interscapular BAT (IBAT) in these separate studies suggesting SNS-SS feedback loops. Therefore, we tested whether individual neurons participated in SNS-SS crosstalk by injecting both PRV152 and H129 into IBAT of Siberian hamsters. To define which dorsal root ganglia (DRG) are activated by BAT SNS stimulation, indicated by c-Fos immunoreactivity (IR), we prelabeled IBAT DRG innervating neurons by injecting the retrograde tracer Fast Blue (FB) followed 1 week later by intra-BAT injections of the specific ␤ 3 -adrenoceptor agonist CL316,243 in one pad and the vehicle in the contralateral pad. There were PRV152ϩH129 dually infected neurons across the neuroaxis with highest densities in the raphe pallidus nucleus, nucleus of the solitary tract, periaqueductal gray, hypothalamic paraventricular nucleus, and medial preoptic area, sites strongly implicated in the control of BAT thermogenesis. CL316,243 significantly increased IBAT temperature, afferent nerve activity, and c-Fos-IR in C2-C4 DRG neurons ipsilateral to the CL316,243 injections versus the contralateral side. The neuroanatomical reality of the SNS-SS feedback loops suggests coordinated and/or multiple redundant control of BAT thermogenesis.

Vaughan CH, Bartness TJ. Anterograde transneuronal viral tract tracing reveals central sensory circuits from brown fat and sensory denervation alters its thermogenic responses. Am J Physiol Regul Integr Comp Physiol 302: R1049 -R1058, 2012. First published February 29, 2012 doi:10.1152/ajpregu.00640.2011.-Brown adipose tissue (BAT) thermogenic activity and growth are controlled by its sympathetic nervous system (SNS) innervation, but nerve fibers containing sensory-associated neuropeptides [substance P, calcitonin gene-related peptide (CGRP)] also suggest sensory innervation. The central nervous system (CNS) projections of BAT afferents are unknown. Therefore, we used the H129 strain of the herpes simplex virus-1 (HSV-1), an anterograde transneuronal viral tract tracer used to delineate sensory nerve circuits, to define these projections. HSV-1 was injected into interscapular BAT (IBAT) of Siberian hamsters and HSV-1 immunoreactivity (ir) was assessed 24, 48, 72, 96, and 114 h postinjection. The 96-and 114-h groups had the most HSV-1-ir neurons with marked infections in the hypothalamic paraventricular nucleus, periaqueductal gray, olivary areas, parabrachial nuclei, raphe nuclei, and reticular areas. These sites also are involved in sympathetic outflow to BAT suggesting possible BAT sensory-SNS thermogenesis feedback circuits. We tested the functional contribution of IBAT sensory innervation on thermogenic responses to an acute (24 h) cold exposure test by injecting the specific sensory nerve toxin capsaicin directly into IBAT pads and then measuring core (Tc) and IBAT (TIBAT) temperature responses. CGRP content was significantly decreased in capsaicin-treated IBAT demonstrating successful sensory nerve destruction. TIBAT and Tc were significantly decreased in capsaicin-treated hamsters compared with the saline controls at 2 h of cold exposure. Thus the central sensory circuits from IBAT have been delineated for the first time, and impairment of sensory feedback from BAT appears necessary for the appropriate, initial thermogenic response to acute cold exposure.herpes simplex virus-1; capsaicin; sensory afferents; iButton; body temperature; interscapular brown adipose tissue temperature; calcitonin gene-related peptide RECENT REPORTS of functional brown adipose tissue (BAT) in humans has sparked more interest in mapping the connections between BAT and the brain in the hope of increasing thermogenesis thereby enhancing energy expenditure and reversing or preventing obesity (18,59,61). BAT is the major effector tissue in rodent thermogenesis (for review see Ref. 10) controlled nearly exclusively by the sympathetic nervous system (SNS) drive to the tissue (for review see Ref. 5). Because of reports of sensory nerve-associated neuropeptides at the level of the BAT pad (23,24,43), the potential for reciprocal connections between BAT and the brain would thus create the possibility of SNS-sensory feedback loops to control the thermogenic activity of the tissue in response to varying environmental and physiological cha...