A Small-Molecule Oral Agonist of the Human Glucagon-like Peptide-1 Receptor.

Peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) have revolutionized diabetes therapy, but their use has been limited because they require injection. Herein, we describe the discovery of the orally bioavailable, small-molecule, GLP-1R agonist PF-06882961 (danuglipron). A sensitized high-throughput screen was used to identify 5-fluoropyrimidine-based GLP-1R agonists that were optimized to promote endogenous GLP-1R signaling with nanomolar potency. Incorporation of a carboxylic acid moiety provided considerable GLP-1R potency gains with improved off-target pharmacology and reduced metabolic clearance, ultimately resulting in the identification of danuglipron. Danuglipron increased insulin levels in primates but not rodents, which was explained by receptor mutagensis studies and a cryogenic electron microscope structure that revealed a binding pocket requiring a primate-specific tryptophan 33 residue. Oral administration of danuglipron to healthy humans produced dose-proportional increases in systemic exposure (NCT03309241). This opens an opportunity for oral small-molecule therapies that target the well-validated GLP-1R for metabolic health.

Exenatide exerts a PKA-dependent positive inotropic effect in human atrial myocardium: GLP-1R mediated effects in human myocardium.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are a rapidly growing class of drugs developed for treating type-2 diabetes mellitus. Patients with diabetes carry an up to 5-fold greater mortality risk compared to non-diabetic patients, mainly as a result of cardiovascular diseases. Although beneficial cardiovascular effects have been reported, exact mechanisms of GLP-1R-agonist action in the heart, especially in human myocardium, are poorly understood. The effects of GLP-1R-agonists (exenatide, GLP-1(7-36)NH2, PF-06446009, PF-06446667) on cardiac contractility were tested in non-failing atrial and ventricular trabeculae from 72 patients. The GLP-1(7-36)NH2 metabolite, GLP-1(9-36)NH2, was also examined. In electrically stimulated trabeculae, the effects of compounds on isometric force were measured in the absence and presence of pharmacological inhibitors of signal transduction pathways. The role of ß-arrestin signaling was examined using a ß-arrestin partial agonist, PF-06446667. Expression levels were tested by immunoblots. Translocation of GLP-1R downstream molecular targets, Epac2, GLUT-1 and GLUT-4, were assessed by fluorescence microscopy. All tested GLP-1R-agonists significantly increased developed force in human atrial trabeculae, whereas GLP-1(9-36)NH2 had no effect. Exendin(9-39)NH2, a GLP-1R-antagonist, and H-89 blunted the inotropic effect of exenatide. In addition, exenatide increased PKA-dependent phosphorylation of phospholamban (PLB), GLUT-1 and Epac2 translocation, but not GLUT-4 translocation. Exenatide failed to enhance contractility in ventricular myocardium. Quantitative real-time PCR (qRT-PCR) revealed a significant higher GLP-1R expression in the atrium compared to ventricle. Exenatide increased contractility in a dose-dependent manner via GLP-1R/cAMP/PKA pathway and induced GLUT-1 and Epac2 translocation in human atrial myocardium, but had no effect in ventricular myocardium. Therapeutic use of GLP-1R-agonists may therefore impart beneficial effects on myocardial function and remodelling.

Latest approaches for the treatment of obesity.

INTRODUCTION: Obesity is a body weight disorder characterized by excess adiposity that increases the risk for developing co-morbidities such as type 2 diabetes. A large medical need exists for new anti-obesity treatments capable of promoting 10% or greater weight loss, with minimal side effects. AREAS COVERED: The authors describe the application of monogenic forms of rare obesity and genome-wide association studies in selecting critical pathways for drug discovery. Furthermore, they review in detail several pathways and pharmacological targets in the central nervous system (e.g., the leptin-melanocortin axis, the opioid system, GLP-1/GLP-1 system, and FGF21/FGFR1c/ß-Klotho axis) that play an important role in the regulation of feeding behavior and energy homeostasis. Special focus is given to new strategies that engage well-known targets via novel mechanisms in order to circumvent issues seen with previous drug candidates that failed in the clinic. Finally, the authors discuss the recent developments around fixed-dose combinations, targeted polypharmacology, and non-traditional combinations of drugs and devices. EXPERT OPINION: The future for new weight-loss approaches to treat obesity looks promising. Current therapies have shown modest effects on weight loss in the general obese population but will have greater impact in smaller homogeneous sub-populations of obese subjects using personalized medicine. Drug combinations that target multiple, complementary pathways have the potential to promote double-digit weight loss in a broader, heterogeneous patient population. Furthermore, the development of advanced subcutaneous delivery technologies has opened up opportunities to develop breakthrough peptide and biologic agents for the treatment of obesity.

MAP kinases couple hindbrain-derived catecholamine signals to hypothalamic adrenocortical control mechanisms during glycemia-related challenges.

Physiological responses to hypoglycemia, hyperinsulinemia, and hyperglycemia include a critical adrenocortical component that is initiated by hypothalamic control of the anterior pituitary and adrenal cortex. These adrenocortical responses ensure appropriate long-term glucocorticoid-mediated modifications to metabolism. Despite the importance of these mechanisms to disease processes, how hypothalamic afferent pathways engage the intracellular mechanisms that initiate adrenocortical responses to glycemia-related challenges are unknown. This study explores these mechanisms using network- and cellular-level interventions in in vivo and ex vivo rat preparations. Results show that a hindbrain-originating catecholamine afferent system selectively engages a MAP kinase pathway in rat paraventricular hypothalamic CRH (corticotropin-releasing hormone) neuroendocrine neurons shortly after vascular insulin and 2-deoxyglucose challenges. In turn, this MAP kinase pathway can control both neuroendocrine neuronal firing rate and the state of CREB phosphorylation in a reduced ex vivo paraventricular hypothalamic preparation, making this signaling pathway an ideal candidate for coordinating CRH synthesis and release. These results establish the first clear structural and functional relationships linking neurons in known nutrient-sensing regions with intracellular mechanisms in hypothalamic CRH neuroendocrine neurons that initiate the adrenocortical response to various glycemia-related challenges.

Dual regulation of anterograde and retrograde transmission by endocannabinoids.

Endocannabinoids (eCBs) are feedback messengers in the nervous system that act at the presynaptic nerve terminal to inhibit transmitter release. Here we report that in brain slices from rat, eCBs are released from vasopressin (VP) neurons in the paraventricular nucleus of the hypothalamus following coincident bursts of presynaptic and postsynaptic spiking. eCBs transiently depress glutamate release from excitatory terminals and, in doing so, prevent the synapses from undergoing long-term depression (LTD). Specifically, we show that blockade of CB1 receptors unmasks LTD following coincident presynaptic and postsynaptic activity. This LTD is presynaptic in nature, but requires the release of the opioid peptide dynorphin from the postsynaptic neuron. Dynorphin release and subsequent LTD require the activation of postsynaptic metabotropic glutamate receptors (mGluRs). Our findings indicate that eCBs, by transiently depressing glutamate release, limit mGluR activation and indirectly gate release of dynorphin from the postsynaptic neuron. We propose that eCBs, in addition to their well described role in the rapid modulation of transmitter release from the nerve terminal, also regulate the release of other retrograde transmitters and thus encode for multiple temporal windows of synaptic plasticity.

Short-term plasticity impacts information transfer at glutamate synapses onto parvocellular neuroendocrine cells in the paraventricular nucleus of the hypothalamus.

Glutamatergic synaptic inputs onto parvocellular neurosecretory cells (PNCs) in the paraventricular nucleus of the hypothalamus (PVN) regulate the hypothalamic-pituitary-adrenal (HPA) axis responses to stress and undergo stress-dependent changes in their capacity to transmit information. In spite of their pivotal role in regulating PNCs, relatively little is known about the fundamental rules that govern transmission at these synapses. Furthermore, since salient information in the nervous system is often transmitted in bursts, it is also important to understand the short-term dynamics of glutamate transmission under basal conditions. To characterize these properties, we obtained whole-cell patch clamp recordings from PNCs in brain slices from postnatal day 21-35 male Sprague-Dawley rats and examined EPSCs. EPSCs were elicited by electrically stimulating glutamatergic afferents along the periventricular aspect. In response to a paired-pulse stimulation protocol, EPSCs generally displayed a robust short-term depression that recovered within 5 s. Similarly, trains of synaptic stimuli (5-50 Hz) resulted in a frequency-dependent depression until a near steady state was achieved. Application of inhibitors of AMPA receptor (AMPAR) desensitization or the low-affinity, competitive AMPAR antagonist failed to affect the depression due to paired-pulse and trains of synaptic stimulation indicating that this use-dependent short-term synaptic depression has a presynaptic locus of expression. We used cumulative amplitude profiles during trains of stimulation and variance-mean analysis to estimate synaptic parameters. Finally, we report that these properties contribute to hamper the efficiency with which high frequency synaptic inputs generate spikes in PNCs, indicating that these synapses operate as effective low-pass filters in basal conditions.

Opposing actions of endothelin-1 on glutamatergic transmission onto vasopressin and oxytocin neurons in the supraoptic nucleus.

Endothelin (ET-1) given centrally has many reported actions on hormonal and autonomic outputs from the CNS. However, it is unclear whether these effects are due to local ischemia via its vasoconstrictor properties or to a direct neuromodulatory action. ET-1 stimulates the release of oxytocin (OT) and vasopressin (VP) from supraoptic magnocellular (MNCs) neurons in vivo; therefore, we asked whether ET-1 modulates the excitatory inputs onto MNCs that are critical in sculpting the activity of these neurons. To investigate whether ET-1 modulates excitatory synaptic transmission, we obtained whole-cell recordings and analyzed quantal glutamate release onto MNCs in the supraoptic nucleus (SON). Neurons identified as VP-containing neurosecretory cells displayed a decrease in quantal frequency in response to ET-1 (10-100 pm). This decrease was mediated by ET(A) receptor activation and production of a retrograde messenger that targets presynaptic cannabinoid-1 receptors. In contrast, neurons identified as OT-containing MNCs displayed a transient increase in quantal glutamate release in response to ET-1 application via ET(B) receptor activation. Application of TTX to block action potential-dependent glutamate release inhibited the excitatory action of ET-1 in OT neurons. There were no changes in quantal amplitude in either MNC type, suggesting that the effects of ET-1 were via presynaptic mechanisms. A gliotransmitter does not appear to be involved as ET-1 failed to elevate astrocytic calcium in the SON. Our results demonstrate that ET-1 differentially modulates glutamate release onto VP- versus OT-containing MNCs, thus implicating it in the selective regulation of neuroendocrine output from the SON.

Stress-induced priming of glutamate synapses unmasks associative short-term plasticity.

Exposure to a stressor sensitizes or 'primes' the hypothalamic-pituitary-adrenal axis to a subsequent novel stressor. The synaptic mechanisms underlying this priming, however, are not known. We found that exposing a rat to a single stressor primed glutamate synapses in the paraventricular nucleus of the hypothalamus and allowed them to undergo a short-term potentiation (STP) following a burst of high-frequency afferent activity. This transient potentiation requires a corticotrophin-releasing hormone-dependent depression of postsynaptic NMDA receptors (NMDARs). The long-term depression of NMDAR function after stress prevented the vesicular release of an inhibitory retrograde messenger that, in control conditions, arrests STP. Following stress, STP manifested as an increase in the release probability of glutamate that was sufficient to induce multivesicular release. Our findings indicate that the priming of synapses to express STP is a synaptic correlate to stress-induced behavioral and neuroendocrine sensitization.

Repeated stress impairs endocannabinoid signaling in the paraventricular nucleus of the hypothalamus.

Endocannabinoids (eCBs) are ubiquitous retrograde signaling molecules in the nervous system that are recruited in response to robust neuronal activity or the activation of postsynaptic G-protein-coupled receptors. Physiologically, eCBs have been implicated as important mediators of the stress axis and they may contribute to the rapid feedback inhibition of the hypothalamic-pituitary-adrenal axis (HPA) by circulating corticosteroids (CORTs). Understanding the relationship between stress and eCBs, however, is complicated by observations that eCB signaling is itself sensitive to stress. The mechanisms that link stress to changes in synaptic eCB signaling and the impact of these changes on CORT-mediated negative feedback have not been resolved. Here, we show that repetitive immobilization stress, in juvenile male rats, causes a functional downregulation of CB(1) receptors in the paraventricular nucleus of the hypothalamus (PVN). This loss of CB(1) receptor signaling, which requires the activation of genomic glucocorticoid receptors, impairs both activity and receptor-dependent eCB signaling at GABA and glutamate synapses on parvocellular neuroendocrine cells in PVN. Our results provide a plausible mechanism for how stress can lead to alterations in CORT-mediated negative feedback and may contribute to the development of plasticity of HPA responses.

Metaplasticity of hypothalamic synapses following in vivo challenge.

Neural networks that regulate an organism's internal environment must sense perturbations, respond appropriately, and then reset. These adaptations should be reflected as changes in the efficacy of the synapses that drive the final output of these homeostatic networks. Here we show that hemorrhage, an in vivo challenge to fluid homeostasis, induces LTD at glutamate synapses onto hypothalamic magnocellular neurosecretory cells (MNCs). LTD requires the activation of postsynaptic alpha2-adrenoceptors and the production of endocannabinoids that act in a retrograde fashion to inhibit glutamate release. In addition, both hemorrhage and noradrenaline downregulate presynaptic group III mGluRs. This loss of mGluR function allows high-frequency activity to potentiate these synapses from their depressed state. These findings demonstrate that noradrenaline controls a form of metaplasticity that may underlie the resetting of homeostatic networks following a successful response to an acute physiological challenge.

Microglial activation and TNFalpha production mediate altered CNS excitability following peripheral inflammation.

Peripheral inflammation leads to a number of centrally mediated physiological and behavioral changes. The underlying mechanisms and the signaling pathways involved in these phenomena are not yet well understood. We hypothesized that peripheral inflammation leads to increased neuronal excitability arising from a CNS immune response. We induced inflammation in the gut by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) to adult male rats. To examine the excitability of the brain in vivo, we administered pentylenetetrazole (PTZ; a GABAergic antagonist) intravenously to evoke clonic seizures. Rats treated with TNBS showed increased susceptibility to PTZ seizures that was strongly correlated with the severity and progression of intestinal inflammation. In vitro hippocampal slices from inflamed, TNBS-treated rats showed increased spontaneous interictal burst firing following application of 4-aminopyridine, indicating increased intrinsic excitability. The TNBS-treated rats exhibited a marked, reversible inflammatory response within the hippocampus, characterized by microglial activation and increases in tumor necrosis factor alpha (TNFalpha) levels. Central antagonism of TNFalpha using a monoclonal antibody or inhibition of microglial activation by i.c.v. injection of minocycline prevented the increase in seizure susceptibility. Moreover, i.c.v. infusion of TNFalpha in untreated rats for 4 days also increased seizure susceptibility and thus mimicked the changes in seizure threshold observed with intestinal inflammation. Our finding of a microglia-dependent TNFalpha-mediated increase in CNS excitability provides insight into potential mechanisms underlying the disparate neurological and behavioral changes associated with chronic inflammation.

Endogenous modulators of synaptic transmission: cannabinoid regulation in the supraoptic nucleus.

The magnocellular neurons of the hypothalamic supraoptic nucleus (SON) are a major source of both systemic and central release of the neurohypophyseal peptides, oxytocin (OXT) and arginine-vasopressin (AVP). Both OXT and AVP are released from the somatodendritic compartment of magnocellular neurons and act within the SON to modulate the electrophysiological function of these cells. Cannabinoids (CBs) affect hormonal output and the SON may represent a neural substrate through which CBs exert specific physiological and behavioural effects. Dynamic modulation of synaptic inputs is a fundamental mechanism through which neuronal output is controlled. Dendritically released OXT acts on autoreceptors to generate endocannabinoids (eCBs) which modify both excitatory and inhibitory inputs to OXT neurons through actions on presynaptic CB receptors. As such, OXT and eCBs cooperate to shape the electrophysiological properties of magnocellular OXT neurons, regulating the physiological function of this nucleus. Further study of eCB signalling in the SON, including its interaction with AVP neurons, promises to extend our understanding of the synaptic regulation of SON physiological function.

Muscarinic enhancement of R-type calcium currents in hippocampal CA1 pyramidal neurons.

The "toxin-resistant" R-type Ca2+ channels are expressed widely in the CNS and distributed mainly in apical dendrites and spines. They play important roles in regulating signal transduction and intrinsic properties of neurons, but the modulation of these channels in the mammalian CNS has not been studied. In this study we used whole-cell patch-clamp recordings and found that muscarinic activation enhances R-type, but does not affect T-type, Ca2+ currents in hippocampal CA1 pyramidal neurons after N, P/Q, and L-type Ca2+ currents selectively were blocked. M1/M3 cholinergic receptors mediated the muscarinic stimulation of R-type Ca2+ channels. The signaling pathway underlying the R-type enhancement was independent of intracellular [Ca2+] changes and required the activation of a Ca(2+)-independent PKC pathway. Furthermore, we found that the enhancement of R-type Ca2+ currents resulted in the de novo appearance of Ca2+ spikes and in remarkable changes in the firing pattern of R-type Ca2+ spikes, which could fire repetitively in the theta frequency. Therefore, muscarinic enhancement of R-type Ca2+ channels could play an important role in modifying the dendritic response to synaptic inputs and in the intrinsic resonance properties of neurons.

Galanin modulates neuronal and synaptic properties in the rat supraoptic nucleus in a use and state dependent manner.

The magnocellular neurons of the hypothalamic supraoptic nucleus (SON) synthesize and secrete oxytocin (OXT) and vasopressin (AVP) from their dendrites. These peptides, and several other neurotransmitters, have been shown to modulate afferent glutamatergic neurotransmission in the SON. The neuropeptide, galanin (GAL) is also localized in SON magnocellular neurons and in afferent fibers in the nucleus. We show that GAL dose-dependently reduces evoked excitatory postsynaptic currents (eEPSCs), alters paired pulse ratio and decreases mEPSC frequency, but not amplitude or decay kinetics in both OXT and AVP neurons. GAL therefore modulates excitatory neurotransmission at a likely presynaptic receptor. Neither OXT/AVP, GABA(B) nor cannabinoid antagonists blocked this effect. A GAL2/3 agonist mimicked GAL's action while GAL1 antagonist did not block GAL's effect, suggesting that GAL2/3 receptors mediate the presynaptic effect. In nondehydrated rats GAL causes a small postsynaptic response, as assessed by input resistance measurements. When the rats were water deprived for 2 days the presynaptic response to GAL was unaltered; however, the postsynaptic decrease in input resistance and hyperpolarization was increased, an effect consistent with a previously described increase in GAL1 receptor expression in dehydration. A GAL1 receptor antagonist blocked the postsynaptic effects. Last, when a train of eEPSCs was elicited, GAL was found to inhibit the earlier events in a train but not the latter. This indicates that GAL may modulate a single synaptic event more effectively than trains of synaptic inputs, thereby acting as a high-pass filter.

ATP released from astrocytes during swelling activates chloride channels.

ATP release from astrocytes contributes to calcium ([Ca(2+)]) wave propagation and may modulate neuronal excitability. In epithelial cells and hepatocytes, cell swelling causes ATP release, which leads to the activation of a volume-sensitive Cl(-) current (I(Cl,swell)) through an autocrine pathway involving purinergic receptors. Astrocyte swelling is counterbalanced by a regulatory volume decrease, involving efflux of metabolites and activation of I(Cl,swell) and K(+) currents. We used whole cell patch-clamp recordings in cultured astrocytes to investigate the autocrine role of ATP in the activation of I(Cl,swell) by hypo-osmotic solution (HOS). Apyrase, an ATP/ADP nucleotidase, inhibited HOS-activated I(Cl,swell), whereas ATP and the P2Y agonists, ADPbetaS and ADP, induced Cl(-) currents similar to I(Cl,swell). Neither the P2U agonist, UTP nor the P2X agonist, alpha,beta-methylene ATP, were effective. BzATP was less effective than ATP, suggesting that P2X7 receptors were not involved. P2 purinergic antagonists, suramin, RB2, and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) reversibly inhibited activation of I(Cl,swell), suggesting that ATP-activated P2Y1 receptors. Thus ATP release mediates I(Cl,swell) in astrocytes through the activation of P2Y1-like receptors. The multidrug resistance protein (MRP) transport inhibitors probenicid, indomethacin, and MK-571 all potently inhibited I(Cl.swell). ATP release from astrocytes in HOS was observed directly using luciferin-luciferase and MK-571 reversibly depressed this HOS-induced ATP efflux. We conclude that ATP release via MRP and subsequent autocrine activation of purinergic receptors contributes to the activation of I(Cl,swell) in astrocytes by HOS-induced swelling.