Slit3 secreted from M2-like macrophages increases sympathetic activity and thermogenesis in adipose tissue.

Beiging of white adipose tissue (WAT) is associated with an increase of anti-inflammatory M2-like macrophages in WAT. However, mechanisms through which M2-like macrophages affect beiging are incompletely understood. Here, we show that the macrophage cytokine Slit3 is secreted by adipose tissue macrophages and promotes cold adaptation by stimulating sympathetic innervation and thermogenesis in mice. Analysing the transcriptome of M2-like macrophages in murine inguinal WAT (iWAT) after cold exposure, we identify Slit3 as a secreted cytokine. Slit3 binds to the ROBO1 receptor on sympathetic neurons to stimulate Ca2+/calmodulin-dependent protein kinase II signalling and norepinephrine release, which enhances adipocyte thermogenesis. Adoptive transfer of Slit3-overexpressing M2 macrophages to iWAT promotes beiging and thermogenesis, whereas mice that lack Slit3 in myeloid cells are cold-intolerant and gain more weight. Our findings shed new light on the integral role of M2-like macrophages for adipose tissue homeostasis and uncover the macrophage-Slit3-sympathetic neuron-adipocyte signalling axis as a regulator of long-term cold adaptation.

Paraventricular nucleus is involved in the central pathway of adipose afferent reflex in rats.

Increasing evidence indicates a link between sympathetic nervous system activation and obesity, but the underlying mechanisms remain elusive. The adipose afferent reflex (AAR) is a sympathoexcitatory reflex that is activated by afferent neurotransmission from the white adipose tissue (WAT). This study aimed to investigate whether the hypothalamic paraventricular nucleus (PVH) is an important component of the central neurocircuitry of the AAR. In anesthetized rats, the discharge activity of individual PVH neurons was recorded in vivo. Activation of WAT afferents was initiated by capsaicin injection, and the AAR was evaluated by monitoring renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses. The responses of PVH neurons to activation of WAT afferents were evaluated by c-fos immunoreactivity and the discharge activity of individual PVH neurons, which was recorded using extracellular single-unit recording. After activation of WAT afferents, both individual PVH neuron discharge activity and c-fos immunoreactivity increased. Bilateral selective lesions of the neurons in the PVH with kainic acid abolished the AAR. These results indicate that PVH is an important component of the central neurocircuitry of the AAR.

Fish oil intake induces UCP1 upregulation in brown and white adipose tissue via the sympathetic nervous system.

Brown adipose tissue (BAT) plays a central role in regulating energy homeostasis, and may provide novel strategies for the treatment of human obesity. BAT-mediated thermogenesis is regulated by mitochondrial uncoupling protein 1 (UCP1) in classical brown and ectopic beige adipocytes, and is controlled by sympathetic nervous system (SNS). Previous work indicated that fish oil intake reduces fat accumulation and induces UCP1 expression in BAT; however, the detailed mechanism of this effect remains unclear. In this study, we investigated the effect of fish oil on energy expenditure and the SNS. Fish oil intake increased oxygen consumption and rectal temperature, with concomitant upregulation of UCP1 and the ß3 adrenergic receptor (ß3AR), two markers of beige adipocytes, in the interscapular BAT and inguinal white adipose tissue (WAT). Additionally, fish oil intake increased the elimination of urinary catecholamines and the noradrenaline (NA) turnover rate in interscapular BAT and inguinal WAT. Furthermore, the effects of fish oil on SNS-mediated energy expenditure were abolished in transient receptor potential vanilloid 1 (TRPV1) knockout mice. In conclusion, fish oil intake can induce UCP1 expression in classical brown and beige adipocytes via the SNS, thereby attenuating fat accumulation and ameliorating lipid metabolism.

Sympathetic Nerve Activity Maintains an Anti-Inflammatory State in Adipose Tissue in Male Mice by Inhibiting TNF-α Gene Expression in Macrophages.

Adipose tissue macrophages (ATMs) play an important role in the inflammatory response in obese animals. How ATMs are regulated in lean animals has remained elusive, however. We now show that the sympathetic nervous system (SNS) is necessary to maintain the abundance of the mRNA for the proinflammatory cytokine TNF-α at a low level in ATMs of lean mice. Intracerebroventricular injection of agouti-related neuropeptide increased the amount of TNF-α mRNA in epididymal (epi) white adipose tissue (WAT), but not in interscapular brown adipose tissue (BAT), through inhibition of sympathetic nerve activity in epiWAT. The surgical denervation and ß-adrenergic antagonist propranolol up-regulated TNF-α mRNA in both epiWAT and BAT in vivo. Signaling by the ß2-adrenergic receptor (AR) and protein kinase A down-regulated TNF-α mRNA in epiWAT explants and suppressed lipopolysaccharide-induced up-regulation of TNF-α mRNA in the stromal vascular fraction of this tissue. ß-AR-deficient (ß-less) mice manifested an increased plasma TNF-α concentration and increased TNF-α mRNA abundance in epiWAT and BAT. TNF-α mRNA abundance was greater in ATMs (CD11b(+) cells of the stromal vascular fraction) from epiWAT or BAT of wild-type mice than in corresponding CD11b(-) cells, and ß2-AR mRNA abundance was greater in ATMs than in CD11b(-) cells of epiWAT. Our results show that the SNS and ß2-AR-protein kinase A pathway maintain an anti-inflammatory state in ATMs of lean mice in vivo, and that the brain melanocortin pathway plays a role in maintaining this state in WAT of lean mice via the SNS.

Central PACAP mediates the sympathetic effects of leptin in a tissue-specific manner.

We previously demonstrated that the peptidergic neurotransmitter pituitary adenylate cyclase-activating polypeptide (PACAP) affects the autonomic system and contributes to the control of metabolic and cardiovascular functions. Previous studies have demonstrated the importance of centrally-mediated sympathetic effects of leptin for obesity-related hypertension. Here we tested whether PACAP signaling in the brain is implicated in leptin-induced sympathetic excitation and appetite suppression. In anesthetized mice, intracerebroventricular (ICV) pre-treatment with PACAP6-38, an antagonist of the PACAP receptors (PAC1-R and VPAC2), inhibited the increase in white adipose tissue sympathetic nerve activity (WAT-SNA) produced by ICV leptin (2µg). In contrast, leptin-induced stimulation of renal sympathetic nerve activity (RSNA) was not affected by ICV pre-treatment with PACAP6-38. Moreover, in PACAP-deficient (Adcyap1-/-) mice, ICV leptin-induced WAT-SNA increase was impaired, whereas RSNA response was preserved. The reductions in food intake and body weight evoked by ICV leptin were attenuated in Adcyap1-/- mice. Our data suggest that hypothalamic PACAP signaling plays a key role in the control by leptin of feeding behavior and lipocatabolic sympathetic outflow, but spares the renal sympathetic traffic.

Neurochemical characterization and sexual dimorphism of projections from the brain to abdominal and subcutaneous white adipose tissue in the rat.

Retroperitoneal white adipose tissue (rWAT) and subcutaneous (inguinal) white adipose tissue (iWAT) are both innervated and regulated by sympathetic efferents, but the distribution and identity of the cells in the brain that regulate sympathetic outflow are poorly characterized. Our aim was to use two isogenic strains of a neurotropic virus (pseudorabies, Bartha) tagged with either green or red fluorescent reporters to identify cells in the brain that project to rWAT and/or iWAT. These viruses were injected into separate WAT depots in male and female Sprague Dawley rats. Retrogradely labeled neurons in the CNS were characterized by immunohistochemistry and PCR. For the latter, laser capture of individual virally labeled neurons was used. All virally labeled brain regions contained neurons projecting to either and both WAT depots. Neurons to abdominal fat were the most abundant in males, whereas females contained a greater proportion of neurons to subcutaneous via private lines and collateral branches. Retrogradely labeled neurons directed to WAT expressed estrogen receptor-α (ERα), and fewer neurons to subcutaneous WAT expressed ERα in males. Regardless of sex, projections from the arcuate nucleus were predominantly from pro-opiomelanocortin cells, with a notable lack of projections from agouti-related protein-expressing neurons. Within the lateral hypothalamus, neurons directed to rWAT and iWAT expressed orexin and melanin-concentrating hormone (MCH), but male rats had a predominance of MCH directed to iWAT. In conclusion, the neurochemical substrates that project through polysynaptic pathways to iWAT and rWAT are different in male and female rats, suggesting that metabolic regulation of rWAT and iWAT is sexually dimorphic.

Enhanced adipose afferent reflex contributes to sympathetic activation in diet-induced obesity hypertension.

We recently found that adipose afferent reflex (AAR) induced by chemical stimulation of white adipose tissue (WAT) increased sympathetic outflow and blood pressure in normal rats. The study was designed to test the hypothesis that AAR contributes to sympathetic activation in obesity hypertension. Male rats were fed with a control diet (12% kcal as fat) or high-fat diet (42% kcal as fat) for 12 weeks to induce obesity hypertension. Stimulation of WAT with capsaicin increased renal sympathetic nerve activity and mean arterial pressure. Both AAR and WAT afferent activity were enhanced in obesity hypertension (OH) compared with obesity nonhypertension (ON) and in ON compared with obesity-resistant or control diet rats. WAT sensory denervation induced by resiniferatoxin caused greater decreases in renal sympathetic nerve activity and mean arterial pressure in OH than ON and in ON than obesity-resistant or control. The depressor effect of resiniferatoxin lasted ≥ 3 weeks in OH. Leptin antagonist in WAT reduced renal sympathetic nerve activity and mean arterial pressure in OH. WAT injection of capsaicin increased plasma renin, angiotensin II, and norepinephrine levels in OH and caused more c-fos expression in paraventricular nucleus in OH than ON and in ON than obesity-resistant or control rats. Inhibiting paraventricular nucleus neurons with lidocaine attenuated renal sympathetic nerve activity in OH and ON, decreased mean arterial pressure in OH, and abolished the capsaicin-induced AAR in all groups. The results indicate that enhanced AAR contributes to sympathetic activation in OH, and paraventricular nucleus plays an important role in the enhanced AAR and sympathetic activation in OH.

Sympathetic activation by chemical stimulation of white adipose tissues in rats.

Injection of leptin into white adipose tissue (WAT) increases sympathetic outflow. The present study was designed to determine the effects of capsaicin and other chemicals in WAT on the sympathetic outflow and blood pressure and the roles of WAT afferents and hypothalamic paraventricular nucleus (PVN) in the adipose afferent reflex (AAR). The AAR was induced by injection of capsaicin, bradykinin, adenosine, adenosine triphosphate (ATP), or leptin into inguinal WAT (iWAT) or retroperitoneal WAT (rWAT) in anesthetized rats. The iWAT injection of capsaicin increased the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) but not the heart rate. Bradykinin, adenosine, or leptin but not ATP in the iWAT caused similar effects to capsaicin on the RSNA and MAP. Intravenous, intramuscular, or intradermal injection of capsaicin had no significant effects on the RSNA and MAP. The effects of capsaicin in rWAT were similar to that in iWAT on the RSNA and MAP. Furthermore, injection of capsaicin into the iWAT increased the WAT afferent nerve activities, WAT efferent nerve activity, and brown adipose tissue efferent nerve activity. The iWAT denervation or chemical lesion of the PVN neurons with kainic acid abolished the AAR induced by the iWAT injection of capsaicin. These results indicate that the stimulation of iWAT afferents with capsaicin, bradykinin, adenosine, or leptin reflexly increases the RSNA and blood pressure. The iWAT afferents and the PVN are involved in the AAR induced by capsaicin in the iWAT.

Short photoperiod-induced decrease of histamine H3 receptors facilitates activation of hypothalamic neurons in the Siberian hamster.

Nonhibernating seasonal mammals have adapted to temporal changes in food availability through behavioral and physiological mechanisms to store food and energy during times of predictable plenty and conserve energy during predicted shortage. Little is known, however, of the hypothalamic neuronal events that lead to a change in behavior or physiology. Here we show for the first time that a shift from long summer-like to short winter-like photoperiod, which induces physiological adaptation to winter in the Siberian hamster, including a body weight decrease of up to 30%, increases neuronal activity in the dorsomedial region of the arcuate nucleus (dmpARC) assessed by electrophysiological patch-clamping recording. Increased neuronal activity in short days is dependent on a photoperiod-driven down-regulation of H3 receptor expression and can be mimicked in long-day dmpARC neurons by the application of the H3 receptor antagonist, clobenproprit. Short-day activation of dmpARC neurons results in increased c-Fos expression. Tract tracing with the trans-synaptic retrograde tracer, pseudorabies virus, delivered into adipose tissue reveals a multisynaptic neuronal sympathetic outflow from dmpARC to white adipose tissue. These data strongly suggest that increased activity of dmpARC neurons, as a consequence of down-regulation of the histamine H3 receptor, contributes to the physiological adaptation of body weight regulation in seasonal photoperiod.

White adipose tissue lacks significant vagal innervation and immunohistochemical evidence of parasympathetic innervation.

Converging evidence indicates that white adipose tissue (WAT) is innervated by the sympathetic nervous system (SNS) based on immunohistochemical labeling of a SNS marker (tyrosine hydroxylase [TH]), tract tracing of WAT sympathetic postganglionic innervation, pseudorabies virus (PRV) transneuronal labeling of WAT SNS outflow neurons, and functional evidence from denervation studies. Recently, WAT para-SNS (PSNS) innervation was suggested because local surgical WAT sympathectomy (sparing hypothesized parasympathetic innervation) followed by PRV injection yielded infected cells in the vagal dorsomotor nucleus (DMV), a traditionally-recognized PSNS brain stem site. In addition, local surgical PSNS WAT denervation triggered WAT catabolic responses. We tested histologically whether WAT was parasympathetically innervated by searching for PSNS markers in rat, and normal (C57BL) and obese (ob/ob) mouse WAT. Vesicular acetylcholine transporter, vasoactive intestinal peptide and neuronal nitric oxide synthase immunoreactivities were absent in WAT pads (retroperitoneal, epididymal, inguinal subcutaneous) from all animals. Nearly all nerves innervating WAT vasculature and parenchyma that were labeled with protein gene product 9.5 (PGP9.5; pan-nerve marker) also contained TH, attesting to pervasive SNS innervation. When Siberian hamster inguinal WAT was sympathetically denervated via local injections of catecholaminergic toxin 6-hydroxydopamine (sparing putative parasympathetic nerves), subsequent PRV injection resulted in no central nervous system (CNS) or sympathetic chain infections suggesting no PSNS innervation. By contrast, vehicle-injected WAT subsequently inoculated with PRV had typical CNS/sympathetic chain viral infection patterns. Collectively, these data indicate no parasympathetic nerve markers in WAT of several species, with sparse DMV innervation and question the claim of PSNS WAT innervation as well as its functional significance.