Noradrenergic neurons of the area postrema mediate amylin's hypophagic action.

Circulating amylin inhibits food intake via activation of the area postrema (AP). The aim of this study was to identify the neurochemical phenotype of the neurons mediating amylin's hypophagic action by immunohistochemical and feeding studies in rats. Expression of c-Fos protein was used as a marker for neuronal activation and dopamine-beta-hydroxylase (DBH), the enzyme-catalyzing noradrenaline synthesis, as a marker for noradrenergic neurons. We found that approximately 50% of amylin-activated AP neurons are noradrenergic. To clarify the functional role of these neurons in amylin's effect on eating, noradrenaline-containing neurons in the AP were lesioned using a saporin conjugated to an antibody against DBH. Amylin (5 or 20 microg/kg s.c.)-induced anorexia was observed in sham-lesioned rats with both amylin doses. Rats with a lesion of > 50% of the noradrenaline neurons were unresponsive to the low dose of amylin (5 microg/kg) and only displayed a reduction in food intake 60 min after injection of the high amylin dose (20 microg/kg). In a terminal experiment, the same rats received amylin (20 microg/kg) or saline. The AP and nucleus of the solitary tract (NTS) were stained for DBH to assess noradrenaline lesion success and for c-Fos expression to evaluate amylin-induced neuronal activation. In contrast to sham-lesioned animals, noradrenaline-lesioned rats did not show a significant increase in amylin-induced c-Fos expression in the AP and NTS. We conclude that the noradrenergic neurons in the AP mediate at least part of amylin's hypophagic effect.

Nonthermal activation of transient receptor potential vanilloid-1 channels in abdominal viscera tonically inhibits autonomic cold-defense effectors.

An involvement of the transient receptor potential vanilloid (TRPV) 1 channel in the regulation of body temperature (T(b)) has not been established decisively. To provide decisive evidence for such an involvement and determine its mechanisms were the aims of the present study. We synthesized a new TRPV1 antagonist, AMG0347 [(E)-N-(7-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl)-3-(2-(piperidin-1-yl)-6-(trifluoromethyl)pyridin-3-yl)acrylamide], and characterized it in vitro. We then found that this drug is the most potent TRPV1 antagonist known to increase T(b) of rats and mice and showed (by using knock-out mice) that the entire hyperthermic effect of AMG0347 is TRPV1 dependent. AMG0347-induced hyperthermia was brought about by one or both of the two major autonomic cold-defense effector mechanisms (tail-skin vasoconstriction and/or thermogenesis), but it did not involve warmth-seeking behavior. The magnitude of the hyperthermic response depended on neither T(b) nor tail-skin temperature at the time of AMG0347 administration, thus indicating that AMG0347-induced hyperthermia results from blockade of tonic TRPV1 activation by nonthermal factors. AMG0347 was no more effective in causing hyperthermia when administered into the brain (intracerebroventricularly) or spinal cord (intrathecally) than when given systemically (intravenously), which indicates a peripheral site of action. We then established that localized intra-abdominal desensitization of TRPV1 channels with intraperitoneal resiniferatoxin blocks the T(b) response to systemic AMG0347; the extent of desensitization was determined by using a comprehensive battery of functional tests. We conclude that tonic activation of TRPV1 channels in the abdominal viscera by yet unidentified nonthermal factors inhibits skin vasoconstriction and thermogenesis, thus having a suppressive effect on T(b).

Fever and hypothermia in systemic inflammation: recent discoveries and revisions.

Systemic inflammation is accompanied by changes in body temperature, either fever or hypothermia. Over the past decade, the rat and mouse have become the predominant animal models, and new species-specific tools (recombinant antibodies and other proteins) and genetic manipulations have been applied to study fever and hypothermia. Remarkable progress has been achieved. It has been established that the same inflammatory agent can induce either fever or hypothermia, depending on several factors. It has also been established that experimental fevers are generally polyphasic, and that different mechanisms underlie different febrile phases. Signaling mechanisms of the most common pyrogen used, bacterial lipopolysaccharide (LPS), have been found to involve the Toll-like receptor 4. The roles of cytokines (such as interleukins-1beta and 6 and tumor necrosis factor-alpha) have been further detailed, and new early mediators (e.g., complement factor 5a and platelet-activating factor) have been proposed. Our understanding of how peripheral inflammatory messengers cross the blood-brain barrier (BBB) has changed. The view that the organum vasculosum of the lamina terminalis is the major port of entry for pyrogenic cytokines has lost its dominant position. The vagal theory has emerged and then fallen. Consensus has been reached that the BBB is not a divider preventing signal transduction, but rather the transducer itself. In the endothelial and perivascular cells of the BBB, upstream signaling molecules (e.g., pro-inflammatory cytokines) are switched to a downstream mediator, prostaglandin (PG) E2. An indispensable role of PGE2 in the febrile response to LPS has been demonstrated in studies with targeted disruption of genes encoding either PGE2-synthesizing enzymes or PGE2 receptors. The PGE2-synthesizing enzymes include numerous phospholipases (PL) A2, cyclooxygenases (COX)-1 and 2, and several newly discovered terminal PGE synthases (PGES). It has been realized that the "physiological," low-scale production of PGE2 and the accelerated synthesis of PGE2 in inflammation are catalyzed by different sets of these enzymes. The "inflammatory" set includes several isoforms of PLA2 and inducible isoforms of COX (COX-2) and microsomal (m) PGES (mPGES-1). The PGE2 receptors are multiple; one of them, EP3 is likely to be a primary "fever receptor." The effector pathways of fever start from EP3-bearing preoptic neurons. These neurons have been found to project to the raphe pallidus, where premotor sympathetic neurons driving thermogenesis in the brown fat and skin vaso-constriction are located. The rapid progress in our understanding of how thermoeffectors are controlled has revealed the inadequacy of set point-based definitions of thermoregulatory responses. New definitions (offered in this review) are based on the idea of balance of active and passive processes and use the term balance point. Inflammatory signaling and thermoeffector pathways involved in fever and hypothermia are modulated by neuropeptides and peptide hormones. Roles for several "new" peptides (e.g., leptin and orexins) have been proposed. Roles for several "old" peptides (e.g., arginine vasopressin, angiotensin II, and cholecystokinin) have been detailed or revised. New pharmacological tools to treat fevers (i.e., selective inhibitors of COX-2) have been rapidly introduced into clinical practice, but have not become magic bullets and appeared to have severe side effects. Several new targets for antipyretic therapy, including mPGES-1, have been identified.

Expression of c-Fos in the mouse Edinger-Westphal nucleus following ethanol administration is not secondary to hypothermia or stress.

Restraint stress, lipopolysaccharide (LPS), and ethanol (EtOH) administration have all been found to induce c-Fos in the brain, and to cause hypothermia. The present study was designed to assess whether the c-Fos expression that occurs in the Edinger-Westphal nucleus (EW) after EtOH administration is independent of the hypothermia or any stress effects that occur. To test this, we used restraint stress and LPS in addition to EtOH, and also examined two control areas, the dorsal raphe nucleus (DRN) and the periaqueductal gray (PAG), in addition to EW. Male C57BL6/J mice were used. Groups of mice received intraperitoneal (IP) injections of EtOH (2 g/kg), LPS (600 microg/kg or 50 microg/kg), or saline. A separate group of mice received no injection, but were placed in plastic restrainers for the entirety of the experiment. For all groups, core temperatures were monitored rectally every 30 min for 3 h postinjection, after which, the animals were sacrificed. Then, the number of Fos-positive cells in the brain regions of the EW, DRN, and PAG was quantified. Both EtOH and restraint stress induced a transient hypothermia, where core temperature (Tc) declined immediately and then rose again. Both doses of LPS induced a slower developing, longer lasting hypothermia, while saline had no effect on Tc. Only EtOH induced a significant amount of c-Fos in EW, while both doses of LPS and restraint stress induced c-Fos in DRN, and only restraint stress caused induction in PAG. These data demonstrate that activation of EW after EtOH is unrelated to hypothermia or stress.

The behavioral thermoregulatory response of febrile female rats is not attenuated by vagotomy.

An oft-overlooked consequence of fever is the occurrence of thermoregulatory heat-seeking/producing behaviors. Subdiaphragmatic vagotomy attenuates fever resulting from low dose, peripherally administered pyrogens, suggesting that the vagus is involved in generating the pathogen-induced rise in core body temperature (T(c)). This study was designed to confirm that rats utilize behavioral thermoregulation to augment fever following systemic administration of lipopolysaccharide (LPS), and to test the hypothesis that, in febrile animals, vagotomy would block the preference for a higher ambient temperature (T(a)) as T(c) is rising. First, female Sprague-Dawley rats received IP injections of either saline or LPS (50 microg/kg), prior to placement inside a thermal gradient that offered subjects T(a) values between 7 and 45 degrees C. LPS injection caused significant increases in T(c) and selection of a higher T(a) as compared to saline administration. Second, groups of rats were vagotomized, sham-vagotomized or received no surgery, and then underwent the same gradient testing procedure. Vagotomy attenuated LPS-induced fever, but did not influence the concomitant behavioral thermoregulatory response. All groups selected comparable, higher T(a) values following LPS vs. saline. These data suggest that the reduction in the febrile response to LPS administration following vagotomy is not due to inhibition of the behavioral thermoregulatory response to the pyrogen. Rather, this behavioral response to LPS appears to be mediated by a nonvagal mechanism.

"Weighing in" on synergy: preclinical research on neurohormonal anti-obesity combinations.

Active weight loss and the maintenance of a weight-reduced state elicit potent counter-regulatory responses in multiple neurochemical pathways rendering monotherapy-based anti-obesity agents relatively ineffective. Herein, we highlight potential strategies for overcoming counter-regulatory responses to states of negative energy balance using combinatorial approaches. We discuss methodological and practical considerations for preclinical modeling of additive/synergistic weight loss combinations that have emerged in our translational research program aimed at identifying naturally occurring neurohormonal synergies. As an example of synergy, pharmacological and mechanistic findings with the combined administration of the beta-cell hormone amylin and the adipokine leptin are reviewed. Finally, we briefly discuss what the future landscape of neurohormonal anti-obesity combinations may hold.

Multi-hormonal weight loss combinations in diet-induced obese rats: therapeutic potential of cholecystokinin?

Cholecystokinin (CCK) acutely synergizes with amylin to suppress food intake in lean mice. To extend on these findings, the present studies sought to identify neural correlates for the interaction of amylin and CCK, as well as further understand the therapeutic potential of CCK-based combinations in obesity. First, c-Fos activation was assessed in various brain nuclei after a single intraperitoneal injection of amylin (5microg/kg) and/or CCK (5microg/kg). Amylin and CCK additively increased c-Fos within the area postrema (AP), predominantly in noradrenergic (e.g., dopamine-beta-hydroxylase-containing) cells. Next, amylin (100 or 300microg/kg/d) and/or CCK (100 or 300microg/kg/d) were subcutaneously infused for 7days in diet-induced obese (DIO) rats. Amylin treatment of DIO rats for 7days induced significant body weight loss. CCK, while ineffective alone, significantly enhanced body weight loss when co-administered with the higher dose of amylin. Finally, the addition of CCK (300microg/kg/d) to leptin (125microg/kg/d), and to the combination of amylin (50microg/kg/d) and leptin (125microg/kg/d), was also explored in DIO rats via sustained subcutaneous infusion for 14days. Infusion of amylin/leptin/CCK for 14days exerted significantly greater body weight loss, inhibition of food intake, and reduction in adiposity compared to amylin/leptin treatment alone in DIO rats. However, co-infusion of CCK and leptin was an ineffective weight loss regimen in this model. Whereas CCK agonism alone is ineffective at eliciting or maintaining weight loss, it durably augmented the food intake and body weight-lowering effects of amylin and amylin/leptin in a relevant disease model, and when combined with amylin, cooperatively activated neurons within the caudal brainstem.

Mechanisms of amylin/leptin synergy in rodent models.

The present studies aimed to identify mechanisms contributing to amylin/leptin synergy in reducing body weight and adiposity. We reasoned that if amylin/leptin harnessed complementary neuronal pathways, then in the leptin-sensitive state, amylin should augment leptin signaling/binding and that in the absence of endogenous amylin, leptin signaling should be diminished. Amylin (50 microg/kg, ip) amplified low-dose leptin-stimulated (15 microg/kg, ip) phosphorylated signal transducer and activator of transcription-3 signaling within the arcuate nucleus (ARC) in lean rats. Amylin (50 microg/kg x d) or leptin (125 microg/kg x d) infusion to lean rats decreased 28-d food intake (14 and 10%, respectively), body weight (amylin by 4.3%, leptin by 4.9%), and epididymal fat (amylin by 19%, leptin by 37%). Amylin/leptin co-infusion additively decreased food intake (by 26%) and reduced body weight (by 15%) and epididymal fat (by 78%; all P < 0.05 vs. all groups) in a greater than mathematically additive manner, consistent with synergy. Amylin increased leptin binding within the ventromedial hypothalamus (VMN) by 35% and dorsomedial hypothalamus by 47% (both P < 0.05 vs. vehicle). Amylin/leptin similarly increased leptin binding in the VMN by 40% and ARC by 70% (P < 0.05 vs. vehicle). In amylin-deficient mice, hypothalamic leptin receptor mRNA expression was reduced by 50%, leptin-stimulated phosphorylated signal transducer and activator of transcription-3 within ARC and VMN was reduced by 40%, and responsiveness to leptin's (1 mg/kg x d for 28 d) weight-reducing effects was attenuated (all P < 0.05 vs. wild-type controls). We suggest that amylin/leptin's marked weight- and fat-reducing effects are due to activation of intrinsic synergistic neuronal signaling pathways and further point to the integrated neurohormonal therapeutic potential of amylin/leptin agonism in obesity.

Enhanced amylin-mediated body weight loss in estradiol-deficient diet-induced obese rats.

In rodents, ovariectomy (OVX) elicits weight gain and diminished responsiveness to homeostatic signals. Here we characterized the response of obese OVX rats to peripheral amylin. Rats received sham surgery (SHAM), OVX, or OVX with hormonal replacement (17ß-estradiol, 2 µg per 4 d; OVX+E) and were infused with vehicle or amylin (50 µg/kg · d) for 28 d. Amylin reduced body weight (5.1 ± 1.1%) and food intake (10.9 ± 3.4%) in SHAM rats but was twice as efficacious in OVX rats in reducing weight (11.2 ± 1.9%) and food intake (23.0 ± 2.0%). There were no differences between amylin-treated SHAM and OVX+E rats. OVX decreased metabolic rate (∼24%) and increased respiratory exchange ratio relative to SHAM. Amylin partially normalized metabolic rate (13% increase) in OVX rats and decreased respiratory exchange ratio in OVX and SHAM rats. Regarding central mechanisms, amylin infusion corrected the OVX-induced decrease in hippocampal neurogenesis and increased immobility in the forced swim test. Additionally, amylin increased neurogenesis (∼2-fold) within the area postrema of OVX rats. To assess the contribution of endogenous leptin to amylin-mediated weight loss in OVX rats, amylin was administered to SHAM or OVX Zucker diabetic fatty rats. In SHAM rats, amylin infusion reduced food intake but not body weight, whereas in OVX Zucker diabetic fatty rats, food intake, body weight, and insulin were reduced. Overall, amylin induced greater body weight loss in the absence of estradiol via central and peripheral actions that did not require leptin. These findings support the clinical investigation of amylin in low estradiol (e.g. postmenopausal) states.

Differences in the urocortin 1 system between long-sleep and short-sleep mice.

There is evidence that the peptide urocortin 1 (Ucn1) may be involved in mediating some of the effects of ethanol. The purpose of the present study was to characterize Ucn1 immunoreactivity in mice selectively bred for either high or low sensitivity to ethanol-induced sedation, with additional differences in their response to ethanol-induced hypothermia. The brains of naïve male mice of the inbred long sleep/short sleep (ILS/ISS) selected lines were analyzed by immunohistochemistry. Significant differences were found between lines in the number of Ucn1-containing cells in the non-preganglionic Edinger-Westphal nucleus (npEW, the main source of Ucn1 in the brain); with the ISS mice having more cells. However, significant differences in the optical density of Ucn1 immunoreactivity in individual npEW cells and differences in cell area were also found between lines, with ILS mice having a greater density of Ucn1 per cell and having larger cells in the npEW. Importantly, the ILS mice also had a significantly greater number of Ucn1-positive terminal fibers than ISS mice in the lateral septum and the dorsal raphe nucleus, projection areas of Ucn1-containing neurons. These results suggest that the greater sensitivity of ILS than ISS mice to the hypothermic effects of ethanol could be mediated by stronger innervation of the dorsal raphe by Ucn1-containing fibers. In addition, these results lend further support to previous findings implicating Ucn1-containing projections from npEW to the dorsal raphe in ethanol-induced hypothermia.