Leptin mediates the increase in blood pressure associated with obesity.

Obesity is associated with increased blood pressure (BP), which in turn increases the risk of cardiovascular diseases. We found that the increase in leptin levels seen in diet-induced obesity (DIO) drives an increase in BP in rodents, an effect that was not seen in animals deficient in leptin or leptin receptors (LepR). Furthermore, humans with loss-of-function mutations in leptin and the LepR have low BP despite severe obesity. Leptin's effects on BP are mediated by neuronal circuits in the dorsomedial hypothalamus (DMH), as blocking leptin with a specific antibody, antagonist, or inhibition of the activity of LepR-expressing neurons in the DMH caused a rapid reduction of BP in DIO mice, independent of changes in weight. Re-expression of LepRs in the DMH of DIO LepR-deficient mice caused an increase in BP. These studies demonstrate that leptin couples changes in weight to changes in BP in mammalian species.

Novel 5-aryloxypyrimidine SEN1576 as a candidate for the treatment of Alzheimer's disease.

Prefibrillar assembly of amyloid-ß (Aß) is a major event underlying the development of neuropathology and dementia in Alzheimer's disease (AD). This study determined the neuroprotective properties of an orally bioavailable Aß synaptotoxicity inhibitor, SEN1576. Binding of SEN1576 to monomeric Aß 1-42 was measured using surface plasmon resonance. Thioflavin-T and MTT assays determined the ability of SEN1576 to block Aß 1-42-induced aggregation and reduction in cell viability, respectively. In vivo long-term potentiation (LTP) determined effects on synaptic toxicity induced by intracerebroventricular (i.c.v.) injection of cell-derived Aß oligomers. An operant behavioural schedule measured effects of oral administration following i.c.v. injection of Aß oligomers in normal rats. SEN1576 bound to monomeric Aß 1-42, protected neuronal cells exposed to Aß 1-42, reduced deficits in in vivo LTP and behaviour. SEN1576 exhibits the necessary features of a drug candidate for further development as a disease modifying treatment for the early stages of AD-like dementia.

Pharmacodynamic effects of a D-amino acid oxidase inhibitor indicate a spinal site of action in rat models of neuropathic pain.

Inhibition of d-amino acid oxidase (DAAO) activity is a potential target for the treatment of chronic pain. Here we characterized the effects of systemic administration of the DAAO inhibitor 4H-furo[3,2-b]pyrrole-5-carboxylic acid (SUN) in rat models of neuropathic and inflammatory pain. Oral administration of SUN dose dependently attenuated tactile allodynia induced by ligation of the L5 spinal nerve (SNL) and similarly reversed thermal hyperalgesia produced by chronic constriction injury. In addition, SUN was efficacious against complete Freund's adjuvant-induced thermal hyperalgesia. In these models, maximal reversal of pain-related behaviors corresponded with maximum rates of increase in brain and plasma d-serine concentrations, indicative of full inhibition of DAAO activity. To investigate the possible site(s) of action, we recorded spontaneous nerve activity and mechanically evoked responses of central spinal cord dorsal horn neurons and compared these with spontaneous activity of peripheral dorsal root filaments in anesthetized SNL model animals. Oral SUN reduced spontaneous activity in both central and peripheral recordings at doses and pretreatment times that corresponded to reduced mechanical allodynia in behavioral experiments. After intravenous administration of SUN, the onset of action for this central effect was rapid (maximal effects within 30 minutes), but was abolished by severing afferent inputs to the dorsal horn. Overall, these results indicate that inhibition of DAAO in peripheral afferent spinal circuits reduced spontaneous neuronal activity to attenuate pain-related behaviors in rat models of neuropathic and inflammatory pain.

Pro-opiomelanocortin neurons in the nucleus of the solitary tract mediate endorphinergic endogenous analgesia in mice.

ABSTRACT: The nucleus of the solitary tract (NTS) contains pro-opiomelanocortin (POMC) neurons that are 1 of the 2 major sources of ß-endorphin in the brain. The functional role of these NTS POMC neurons in nociceptive and cardiorespiratory function is debated. We have shown that NTS POMC optogenetic activation produces bradycardia and transient apnoea in a working heart-brainstem preparation and chemogenetic activation with an engineered ion channel (PSAM) produced opioidergic analgesia in vivo. To better define the role of the NTS POMC neurons in behaving animals, we adopted in vivo optogenetics (ChrimsonR) and excitatory/inhibitory chemogenetic DREADD (hM3Dq/hM4Di) strategies in POMC-Cre mice. We show that optogenetic activation of NTS POMC neurons produces time-locked, graded, transient bradycardia and bradypnoea in anaesthetised mice that is naloxone sensitive (1 mg/kg, i.p.), suggesting a role of ß-endorphin. Both optogenetic and chemogenetic activation of NTS POMC neurons produces sustained thermal analgesia in behaving mice that can be blocked by naloxone. It also produced analgesia in an inflammatory pain model (carrageenan) but not in a neuropathic pain model (tibial nerve transection). Inhibiting NTS POMC neurons does not produce any effect on basal nociception but inhibits stress-induced analgesia (unlike inhibition of arcuate POMC neurons). Activation of NTS POMC neuronal populations in conscious mice did not cause respiratory depression, anxiety, or locomotor deficit (in open field) or affective preference. These findings indicate that NTS POMC neurons play a key role in the generation of endorphinergic endogenous analgesia and can also regulate cardiorespiratory function.

The N-methylated peptide SEN304 powerfully inhibits Aß(1-42) toxicity by perturbing oligomer formation.

Oligomeric forms of ß-amyloid (Aß) have potent neurotoxic activity and are the primary cause of neuronal injury and cell death in Alzheimer's disease (AD). Compounds that perturb oligomer formation or structure may therefore be therapeutic for AD. We previously reported that d-[(chGly)-(Tyr)-(chGly)-(chGly)-(mLeu)]-NH(2) (SEN304) is able to inhibit Aß aggregation and toxicity, shown primarily by thioflavin T fluorescence and MTT (Kokkoni, N. et al. (2006) N-Methylated peptide inhibitors of ß-amyloid aggregation and toxicity. Optimisation of inhibitor structure. Biochemistry 45, 9906-9918). Here we extensively characterize how SEN304 affects Aß(1-42) aggregation and toxicity, using biophysical assays (thioflavin T, circular dichroism, SDS-PAGE, size exclusion chromatography, surface plasmon resonance, traveling wave ion mobility mass spectrometry, electron microscopy, ELISA), toxicity assays in cell culture (MTT and lactate dehydrogenase in human SH-SHY5Y cells, mouse neuronal cell death and synaptophysin) and long-term potentiation in a rat hippocampal brain slice. These data, with dose response curves, show that SEN304 is a powerful inhibitor of Aß(1-42) toxicity, particularly effective at preventing Aß inhibition of long-term potentiation. It can bind directly to Aß(1-42), delay ß-sheet formation and promote aggregation of toxic oligomers into a nontoxic form, with a different morphology that cannot bind thioflavin T. SEN304 appears to work by inducing aggregation, and hence removal, of Aß oligomers. It is therefore a promising lead compound for Alzheimer's disease.

Metabolic sensing in AgRP neurons integrates homeostatic state with dopamine signalling in the striatum.

Agouti-related peptide (AgRP) neurons increase motivation for food, however, whether metabolic sensing of homeostatic state in AgRP neurons potentiates motivation by interacting with dopamine reward systems is unexplored. As a model of impaired metabolic-sensing, we used the AgRP-specific deletion of carnitine acetyltransferase (Crat) in mice. We hypothesised that metabolic sensing in AgRP neurons is required to increase motivation for food reward by modulating accumbal or striatal dopamine release. Studies confirmed that Crat deletion in AgRP neurons (KO) impaired ex vivo glucose-sensing, as well as in vivo responses to peripheral glucose injection or repeated palatable food presentation and consumption. Impaired metabolic-sensing in AgPP neurons reduced acute dopamine release (seconds) to palatable food consumption and during operant responding, as assessed by GRAB-DA photometry in the nucleus accumbens, but not the dorsal striatum. Impaired metabolic-sensing in AgRP neurons suppressed radiolabelled 18F-fDOPA accumulation after ~30 min in the dorsal striatum but not the nucleus accumbens. Impaired metabolic sensing in AgRP neurons suppressed motivated operant responding for sucrose rewards during fasting. Thus, metabolic-sensing in AgRP neurons is required for the appropriate temporal integration and transmission of homeostatic hunger-sensing to dopamine signalling in the striatum.

Selective activation of Gαob by an adenosine A1 receptor agonist elicits analgesia without cardiorespiratory depression.

The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, benzyloxy-cyclopentyladenosine (BnOCPA), is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPA's unique and exquisitely selective activation of Gob among the six Gαi/o subtypes, and in the absence of ß-arrestin recruitment. BnOCPA thus demonstrates a highly-specific Gα-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective Gα agonism.

Suppression of Corticostriatal Circuit Activity Improves Cognitive Flexibility and Prevents Body Weight Loss in Activity-Based Anorexia in Rats.

BACKGROUND: The ability to adapt behavior to changing environmental circumstances, or cognitive flexibility, is impaired in multiple psychiatric conditions, including anorexia nervosa (AN). Exaggerated prefrontal cortical activity likely underpins the inflexible thinking and rigid behaviors exhibited by patients with AN. A better understanding of the neural basis of cognitive flexibility is necessary to enable treatment approaches that may target impaired executive control. METHODS: Utilizing the activity-based anorexia (ABA) model and touchscreen operant learning paradigms, we investigated the neurobiological link between pathological weight loss and cognitive flexibility. We used pathway-specific chemogenetics to selectively modulate activity in neurons of the medial prefrontal cortex (mPFC) projecting to the nucleus accumbens shell (AcbSh) in female Sprague Dawley rats. RESULTS: DREADD (designer receptor exclusively activated by designer drugs)-based inhibition of the mPFC-AcbSh pathway prevented weight loss in ABA and improved flexibility during early reversal learning by reducing perseverative responding. Modulation of activity within the mPFC-AcbSh pathway had no effect on running, locomotor activity, or feeding under ad libitum conditions, indicating the specific involvement of this circuit in conditions of dysregulated reward. CONCLUSIONS: Parallel attenuation of weight loss in ABA and improvement of cognitive flexibility following suppression of mPFC-AcbSh activity align with the relationship between disrupted prefrontal function and cognitive rigidity in AN patients. The identification of a neurobiological correlate between cognitive flexibility and pathological weight loss provides a unique insight into the executive control of feeding behavior. It also highlights the utility of the ABA model for understanding the biological bases of cognitive deficits in AN and provides context for new treatment strategies.

Neuron-specific responses to acetylcholine within the spinal dorsal horn circuits of rodent and primate.

Excitatory and inhibitory neurotransmission within the spinal dorsal horn is tightly controlled to regulate transmission of nociceptive signals to the brain. One aspect of this control is modulation of neuronal activity through cholinergic signaling. Nociceptive neurons in the dorsal horn express both nicotinic and muscarinic cholinergic receptors and activation of these receptors reduces pain in humans, while inhibition leads to nociceptive hypersensitivity. At a cellular level, acetylcholine (ACh) has diverse effects on excitability which is dependent on the receptor and neuronal subtypes involved. In the present study we sought to characterize the electrophysiological responses of specific subsets of lamina II interneurons from rat and marmoset spinal cord. Neurons were grouped by morphology and by action potential firing properties. Whole-cell voltage-clamp recordings from lamina II dorsal horn neurons of adult rats showed that bath applied acetylcholine increased, decreased or had no effect on spontaneous synaptic current activity in a cell-type specific manner. ACh modulated inhibitory synaptic activity in 80% of neurons, whereas excitatory synaptic activity was affected in less than 50% of neurons. In whole-cell current clamp recordings, brief somatic application of ACh induced cell-type specific responses in 79% of rat lamina II neurons, which included: depolarization and action potential firing, subthreshold membrane depolarization, biphasic responses characterized by transient depolarization followed by hyperpolarization and membrane hyperpolarization alone. Similar responses were seen in marmoset lamina II neurons and the properties of each neuron group were consistent across species. ACh-induced hyperpolarization was blocked by the muscarinic antagonist atropine and all forms of acetylcholine-induced depolarization were blocked by the nicotinic antagonist mecamylamine. The cholinergic system plays an important role in regulating nociception and this study contributes to our understanding of how circuit activity is controlled by ACh at a cellular level in primate and rodent spinal cord.

Alkyne-Bridged α-Conotoxin Vc1.1 Potently Reverses Mechanical Allodynia in Neuropathic Pain Models.

Several Conus-derived venom peptides are promising lead compounds for the management of neuropathic pain, with α-conotoxins being of particular interest. Modification of the interlocked disulfide framework of α-conotoxin Vc1.1 has been achieved using on-resin alkyne metathesis. Although introduction of a metabolically stable alkyne motif significantly disrupts backbone topography, the structural modification generates a potent and selective GABAB receptor agonist that inhibits Cav2.2 channels and exhibits dose-dependent reversal of mechanical allodynia in a behavioral rat model of neuropathic pain. The findings herein support the hypothesis that analgesia can be achieved via activation of GABABRs expressed in dorsal root ganglion (DRG) sensory neurons.

Norepinephrine can act via alpha(2)-adrenoceptors to reduce the hyper-excitability of spinal dorsal horn neurons following chronic nerve injury.

BACKGROUND/PURPOSE: Rats display behavioral signs of neuropathic pain lasting for months in the chronic constriction injury (CCI) model. During intrathecal anesthesia, the administered drugs mainly diffuse directly into the superficial neurons in the spinal dorsal horn. This study aimed to investigate the effect of bath application of norepinephrine on whole cell patch clamp recordings from spinal cord slices of CCI rats with allodynia. METHODS: An assessment of paw withdrawal threshold in response to mechanical stimulation was performed on the operated side on the day before surgery and was repeated after recovery from anesthesia and on the 7(th) and 14(th) days after surgery. Spinal cord slice preparations containing dorsal horn neurons were obtained from both sham-operated rats and CCI rats (after the 14(th) postoperative day behavior test). RESULTS: Compared with normal controls, CCI rats had significantly lower levels of both hyperpolarization and spike threshold in single action potentials recorded from lamina I and II neurons of the spinal dorsal horn. In contrast, a series of action potential recordings showed that the percentage of spiking neurons of the spinal dorsal horn of CCI rats were significantly higher than those of normal controls. The CCI-induced reduction in hyperpolarization, as well as the increased numbers of spinal dorsal horn spiking neurons could be significantly reduced by norepinephrine application. The norepinephrine-induced increased hyperpolarization and input resistance could be abolished by the application of an alpha(2)-adrenoceptor antagonist (idazoxan; 200 nM). CONCLUSION: The results suggest that chronic nerve injury may induce neuropathic pain by increasing the excitability of spinal dorsal horn neurons. This excitability can be reduced by norepinephrine.

α-Conotoxin Peptidomimetics: Probing the Minimal Binding Motif for Effective Analgesia.

Several analgesic α-conotoxins have been isolated from marine cone snails. Structural modification of native peptides has provided potent and selective analogues for two of its known biological targets-nicotinic acetylcholine and γ-aminobutyric acid (GABA) G protein-coupled (GABAB) receptors. Both of these molecular targets are implicated in pain pathways. Despite their small size, an incomplete understanding of the structure-activity relationship of α-conotoxins at each of these targets has hampered the development of therapeutic leads. This review scrutinises the N-terminal domain of the α-conotoxin family of peptides, a region defined by an invariant disulfide bridge, a turn-inducing proline residue and multiple polar sidechain residues, and focusses on structural features that provide analgesia through inhibition of high-voltage-activated Ca2+ channels. Elucidating the bioactive conformation of this region of these peptides may hold the key to discovering potent drugs for the unmet management of debilitating chronic pain associated with a wide range of medical conditions.

A Neural basis for Octanoic acid regulation of energy balance.

OBJECTIVES: Nutrient sensing by hypothalamic neurons is critical for the regulation of food intake and energy expenditure. We aimed to identify long- and medium-chain fatty acid species transported into the brain, their effects on energy balance, and the mechanisms by which they regulate activity of hypothalamic neurons. METHODS: Simultaneous blood and cerebrospinal fluid (CSF) sampling was undertaken in rats and metabolic analyses using radiolabeled fatty acid tracers were performed on mice. Electrophysiological recording techniques were used to investigate signaling mechanisms underlying fatty acid-induced changes in activity of pro-opiomelanocortin (POMC) neurons. RESULTS: Medium-chain fatty acid (MCFA) octanoic acid (C8:0), unlike long-chain fatty acids, was rapidly transported into the hypothalamus of mice and almost exclusively oxidized, causing rapid, transient reductions in food intake and increased energy expenditure. Octanoic acid differentially regulates the excitability of POMC neurons, activating these neurons directly via GPR40 and inducing inhibition via an indirect non-synaptic, purine, and adenosine receptor-dependent mechanism. CONCLUSIONS: MCFA octanoic acid is a central signaling nutrient that targets POMC neurons via distinct direct and indirect signal transduction pathways to instigate changes in energy status. These results could explain the beneficial health effects that accompany MCFA consumption.

Feeding association between the nucleus of the solitary tract and the ventral tegmental area.

Male Sprague-Dawley rats were fitted with two cannulae in the VTA and one cannula in the NTS for co-administration of the micro-opioid receptor agonist DAMGO in one site and the opioid antagonist naltrexone in the other. Injection of DAMGO into the VTA or the NTS stimulated feeding. The increase in food intake after DAMGO injection into the VTA was decreased following injection of naltrexone into the NTS. Furthermore, the increase in food intake after DAMGO injection into the NTS was decreased following injection of naltrexone into the VTA. These results suggest an opioid-mediated feeding association between the VTA and NTS.

Glucose availability regulates ghrelin-induced food intake in the ventral tegmental area.

Information about metabolic status arrives in the brain in the form of a complex milieu of circulating signalling factors, including glucose and fatty acids, ghrelin, leptin and insulin. The specific interactions between humoural factors, brain sites of action and how they influence behaviour are largely unknown. We have previously observed interactions between glucose availability and the actions of ghrelin mediated via the agouti-related peptide neurones of the hypothalamus. In the present study, we examine whether these effects generalise to another ghrelin-sensitive brain nucleus, the ventral tegmental area (VTA). We altered glucose availability by injecting mice with glucose or 2-deoxyglucose i.p. to induce hyperglycaemia and glucopenia, respectively. Thirty minutes later, we injected ghrelin in the VTA. Glucose administration suppressed intra-VTA ghrelin-induced feeding. Leptin, a longer-term signal of positive energy balance, did not affect intra-VTA ghrelin-induced feeding. 2-Deoxyglucose and ghrelin both increased food intake in their own right and, together, they additively increased feeding. These results add support to the idea that calculation of metabolic need depends on multiple signals across multiple brain regions and identifies that VTA circuits are sensitive to the integration of signals reflecting internal homeostatic state and influencing food intake.

The development of insulin resistance in Type 2 diabetes: insights from knockout studies.

Diabetes is a common endocrine disorder, primarily characterised by elevated plasma glucose levels. The disease affects all age groups worldwide. Most patients suffer from Type 2 diabetes, which is mainly due to insulin resistance. It is thought that changes in insulin signalling pathways underlie the development of insulin resistance. This article aims to review recent studies that have elucidated the role of individual proteins in these insulin signalling pathways. These studies have been undertaken using two strategies, one employing mice carrying a global null mutation of particular gene-encoding proteins by the homologous recombination method and another strategy using mice with tissue-specific insulin receptor and/or GLUT4 knockout by the Cre-loxP system. The various phenotypes of these knockout mice, and the light they shed on the etiology of insulin resistance, are discussed. By advancing our understanding of the complex molecular mechanisms underlying insulin resistance, these knock-out models may help us to develop more effective treatments for Type 2 diabetes.

Orexigen-sensitive NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus.

The hypothalamic arcuate nucleus (ARC) integrates and responds to satiety and hunger signals and forms the origins of the central neural response to perturbations in energy balance. Here we show that rat ARC neurons containing neuropeptide Y (NPY) and agouti-related protein (AgRP), which are conditional pacemakers, are activated by orexigens and inhibited by the anorexigen leptin. We propose a neuron-specific signaling mechanism through which central and peripheral signals engage the central neural anabolic drive.

Glucose Availability Predicts the Feeding Response to Ghrelin in Male Mice, an Effect Dependent on AMPK in AgRP Neurons.

Metabolic feedback from the periphery to the brain results from a dynamic physiologic fluctuation of nutrients and hormones, including glucose and fatty acids, ghrelin, leptin, and insulin. The specific interactions between humoral factors and how they influence feeding is largely unknown. We hypothesized that acute glucose availability may alter how the brain responds to ghrelin, a hormonal signal of energy availability. Acute glucose administration suppressed a range of ghrelin-induced behaviors as well as gene expression changes in hypothalamic neuropeptide Y (NPY) and agouti-related peptide (AgRP) neurons after ghrelin administration. Knockdown of the energy-sensing molecule AMP-activated protein kinase (AMPK) in AgRP neurons resulted in loss of the glucose effect, and mice responded as though pretreated with saline. Conversely, 2-deoxyglucose (2-DG), which decreases glucose availability, potentiated ghrelin-induced feeding and increased hypothalamic NPY mRNA levels. AMPK knockdown did not alter the additive effect of 2-DG and ghrelin on feeding. Our findings support the idea that computation of energy status is dynamic, is informed by multiple signals, and responds to acute fluctuations in metabolic state. These observations are broadly relevant to the investigation of neuroendocrine control of feeding and highlight the underappreciated complexity of control within these systems.

Insulin regulates POMC neuronal plasticity to control glucose metabolism.

Hypothalamic neurons respond to nutritional cues by altering gene expression and neuronal excitability. The mechanisms that control such adaptive processes remain unclear. Here we define populations of POMC neurons in mice that are activated or inhibited by insulin and thereby repress or inhibit hepatic glucose production (HGP). The proportion of POMC neurons activated by insulin was dependent on the regulation of insulin receptor signaling by the phosphatase TCPTP, which is increased by fasting, degraded after feeding and elevated in diet-induced obesity. TCPTP-deficiency enhanced insulin signaling and the proportion of POMC neurons activated by insulin to repress HGP. Elevated TCPTP in POMC neurons in obesity and/or after fasting repressed insulin signaling, the activation of POMC neurons by insulin and the insulin-induced and POMC-mediated repression of HGP. Our findings define a molecular mechanism for integrating POMC neural responses with feeding to control glucose metabolism.

Mitochondrial uncoupling in the melanocortin system differentially regulates NPY and POMC neurons to promote weight-loss.

OBJECTIVE: The mitochondrial uncoupling agent 2,4-dinitrophenol (DNP), historically used as a treatment for obesity, is known to cross the blood-brain-barrier, but its effects on central neural circuits controlling body weight are largely unknown. As hypothalamic melanocortin neuropeptide Y/agouti-related protein (NPY/AgRP) and pro-opiomelanocortin (POMC) neurons represent key central regulators of food intake and energy expenditure we investigated the effects of DNP on these neurons, food intake and energy expenditure. METHOD: C57BL/6 and melanocortin-4 receptor (MC4R) knock-out mice were administered DNP intracerebroventricularly (ICV) and the metabolic changes were characterized. The specific role of NPY and POMC neurons and the ionic mechanisms mediating the effects of uncoupling were examined with in vitro electrophysiology performed on NPY hrGFP or POMC eGFP mice. RESULTS: Here we show DNP-induced differential effects on melanocortin neurons including inhibiting orexigenic NPY and activating anorexigenic POMC neurons through independent ionic mechanisms coupled to mitochondrial function, consistent with an anorexigenic central effect. Central administration of DNP induced weight-loss, increased BAT thermogenesis and browning of white adipose tissue, and decreased food intake, effects that were absent in MC4R knock-out mice and blocked by the MC4R antagonist, AgRP. CONCLUSION: These data show a novel central anti-obesity mechanism of action of DNP and highlight the potential for selective melanocortin mitochondrial uncoupling to target metabolic disorders.