Glucagon-like peptide-1 (GLP-1) signalling in the brain: From neural circuits and metabolism to therapeutics.

Glucagon-like-peptide-1 (GLP-1) derived from gut enteroendocrine cells and a discrete population of neurons in the caudal medulla acts through humoral and neural pathways to regulate satiety, gastric motility and pancreatic endocrine function. These physiological attributes contribute to GLP-1 having a potent therapeutic action in glycaemic regulation and chronic weight management. In this review, we provide an overview of the neural circuits targeted by endogenous versus exogenous GLP-1 and related drugs. We also highlight candidate subpopulations of neurons and cellular mechanisms responsible for the acute and chronic effects of GLP-1 and GLP-1 receptor agonists on energy balance and glucose metabolism. Finally, we present potential future directions to translate these findings towards the development of effective therapies for treatment of metabolic disease. LINKED ARTICLES: This article is part of a themed issue on GLP1 receptor ligands (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.4/issuetoc.

Time and metabolic state-dependent effects of GLP-1R agonists on NPY/AgRP and POMC neuronal activity in vivo.

OBJECTIVE: Long-acting glucagon-like peptide-1 receptor agonists (GLP-1RAs), like liraglutide and semaglutide, are viable treatments for diabetes and obesity. Liraglutide directly activates hypothalamic proopiomelanocortin (POMC) neurons while indirectly inhibiting Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons ex vivo. While temporal control of GLP-1R agonist concentration as well as accessibility to tissues/cells can be achieved with relative ease ex vivo, in vivo this is dependent upon the pharmacokinetics of these agonists and relative penetration into structures of interest. Thus, whether liraglutide or semaglutide modifies the activity of POMC and NPY/AgRP neurons in vivo as well as mechanisms required for any changes in cellular activity remains undefined. METHODS: In order to resolve this issue, we utilized neuron-specific transgenic mouse models to examine changes in the activity of POMC and NPY/AgRP neurons after injection of either liraglutide or semaglutide (intraperitoneal - I.P. and subcutaneous - S·C.). POMC and NPY/AgRP neurons were targeted for patch-clamp electrophysiology as well as in vivo fiber photometry. RESULTS: We found that liraglutide and semaglutide directly activate and increase excitatory tone to POMC neurons in a time-dependent manner. This increased activity of POMC neurons required GLP-1Rs in POMC neurons as well as a downstream mixed cation channel comprised of TRPC5 subunits. We also observed an indirect upregulation of excitatory input to POMC neurons originating from glutamatergic cells that also required TRPC5 subunits. Conversely, GLP-1Ra's decreased excitatory input to and indirectly inhibited NPY/AgRP neurons through activation of K-ATP and TRPC5 channels in GABAergic neurons. Notably, the temporal activation of POMC and inhibition of NPY/AgRP neuronal activity after liraglutide or semaglutide was injected [either intraperitoneal (I.P.) or subcutaneous (S·C.)] was dependent upon the nutritional state of the animals (fed vs food-deprived). CONCLUSIONS: Our results support a mechanism of liraglutide and semaglutide in vivo to activate POMC while inhibiting NPY/AgRP neurons, which depends upon metabolic state and mirrors the pharmacokinetic profile of these compounds in vivo.

PERK in POMC neurons connects celastrol with metabolism.

ER stress and activation of the unfolded protein response in the periphery as well as the central nervous system have been linked to various metabolic abnormalities. Chemically lowering protein kinase R-like ER kinase (PERK) activity within the hypothalamus leads to decreased food intake and body weight. However, the cell populations required in this response remain undefined. In the current study, we investigated the effects of proopiomelanocortin-specific (POMC-specific) PERK deficiency on energy balance and glucose metabolism. Male mice deficient for PERK in POMC neurons exhibited improvements in energy balance on a high-fat diet, showing decreased food intake and body weight, independent of changes in glucose and insulin tolerances. The plant-based inhibitor of PERK, celastrol, increases leptin sensitivity, resulting in decreased food intake and body weight in a murine model of diet-induced obesity (DIO). Our data extend these observations by demonstrating that celastrol-induced improvements in leptin sensitivity and energy balance were attenuated in mice with PERK deficiency in POMC neurons. Altogether, these data suggest that POMC-specific PERK deficiency in male mice confers protection against DIO, possibly providing a new therapeutic target for the treatment of diabetes and metabolic syndrome.

Effects of metabolic state on the regulation of melanocortin circuits.

Dysfunction in neurophysiological systems that regulate food intake and metabolism are at least partly responsible for obesity and related comorbidities. An important component of this process is the hypothalamic melanocortin system, where an imbalance can result in severe obesity and deficits in glucose metabolism. Exercise offers many health benefits related to cardiovascular improvements, hunger control, and blood glucose homeostasis. However, the molecular mechanism underlying the exercise-induced improvements to the melanocortin system remain undefined. Here, we review the role of the melanocortin system to sense hormonal, nutrient, and neuronal signals of energy status. This information is then relayed onto secondary neurons in order to regulate physiological parameters, which promote proper energy and glucose balance. We also provide an overview on the effects of physical exercise to induce biophysical changes in the melanocortin circuit which may regulate food intake, glucose metabolism and improve overall metabolic health.

Direct and indirect effects of liraglutide on hypothalamic POMC and NPY/AgRP neurons - Implications for energy balance and glucose control.

OBJECTIVE: The long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist, liraglutide, stimulates insulin secretion and efficiently suppresses food intake to reduce body weight. As such, liraglutide is growing in popularity in the treatment of diabetes and chronic weight management. Within the brain, liraglutide has been shown to alter the activity of hypothalamic proopiomelanocortin (POMC) and Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons. Moreover, the acute activities of POMC and NPY neurons have been directly linked to feeding behavior, body weight, and glucose metabolism. Despite the increased usage of liraglutide and other GLP-1 analogues as diabetic and obesity interventions, the cellular mechanisms by which liraglutide alters the activity of metabolically relevant neuronal populations are poorly understood. METHODS: In order to resolve this issue, we utilized neuron-specific transgenic mouse models to identify POMC and NPY neurons for patch-clamp electrophysiology experiments. RESULTS: We found that liraglutide directly activated arcuate POMC neurons via TrpC5 channels, sharing a similar mechanistic pathway to the adipose-derived peptide leptin. Liraglutide also indirectly increases excitatory tone to POMC neurons. In contrast, liraglutide inhibited NPY/AgRP neurons through post-synaptic GABAA receptors and enhanced activity of pre-synaptic GABAergic neurons, which required both TrpC5 subunits and K-ATP channels. In support of an additive role of leptin and liraglutide in suppressing food intake, leptin potentiated the acute effects of liraglutide to activate POMC neurons. TrpC5 subunits in POMC neurons were also required for the intact pharmacological effects of liraglutide on food intake and body weight. Thus, the current study adds to recent work from our group and others, which highlight potential mechanisms to amplify the effects of GLP-1 agonists in vivo. Moreover, these data highlight multiple sites of action (both pre- and post-synaptic) for GLP-1 agonists on this circuit. CONCLUSIONS: Taken together, our results identify critical molecular mechanisms linking GLP-1 analogues in arcuate POMC and NPY/AgRP neurons with metabolism.

Acute effects of zinc and insulin on arcuate anorexigenic proopiomelanocortin neurons.

BACKGROUND AND PURPOSE: Acute insulin administration hyperpolarized, with concomitant decrease of firing rate, a subpopulation of arcuate proopiomelanocortin (POMC) and neuropeptide Y/agouti-related peptide cells. This rapid effect on cellular activity has been proposed as a cellular correlate of insulin effects on energy balance and glucose homoeostasis. Recent evidence suggests that zinc in mammalian insulin formulations is required for the insulin-induced inhibition of arcuate POMC neurons, while guinea pig insulin, which fails to bind zinc, activates POMC neurons in mice. Here, we tested the effects of zinc and insulin formations on arcuate POMC neurons. EXPERIMENTAL APPROACH: Effects of zinc and insulin formulations were assessed through whole-cell patch clamp recordings on transgenic mice in vitro. KEY RESULTS: Insulin formulations containing zinc hyperpolarized POMC neurons. Zinc also hyperpolarized arcuate POMC neurons, albeit at much higher concentration than found in various insulin formulations. Chelation of zinc inhibited the zinc-induced hyperpolarization of POMC neurons, whereas effects of insulin on POMC cellular activity were unchanged after chelation. Zinc-free insulin also hyperpolarized arcuate POMC neurons. Insulin failed to hyperpolarize POMC neurons deficient for insulin receptors, suggesting that insulin receptors are required for these effects. Activation of POMC neurons by guinea pig insulin was independent of insulin receptors but was inhibited by PDGF receptor antagonism or loss of TRPC5 channel subunits. CONCLUSIONS AND IMPLICATIONS: Together, these findings suggest that insulin inhibited arcuate POMC neurons independent of zinc and highlights a possible role of putative PDGF receptors in the acute effects of guinea pig insulin.

Cellular and synaptic reorganization of arcuate NPY/AgRP and POMC neurons after exercise.

OBJECTIVE: Hypothalamic Pro-opiomelanocortin (POMC) and Neuropeptide Y/Agouti-Related Peptide (NPY/AgRP) neurons are critical nodes of a circuit within the brain that sense key metabolic cues as well as regulate metabolism. Importantly, these neurons retain an innate ability to rapidly reorganize synaptic inputs and electrophysiological properties in response to metabolic state. While the cellular properties of these neurons have been investigated in the context of obesity, much less is known about the effects of exercise training. METHODS: In order to further investigate this issue, we utilized neuron-specific transgenic mouse models to identify POMC and NPY/AgRP neurons for patch-clamp electrophysiology experiments. RESULTS: Using whole-cell patch-clamp electrophysiology, we found exercise depolarized and increased firing rate of arcuate POMC neurons. The increased excitability of POMC neurons was concomitant with increased excitatory inputs to these neurons. In agreement with recent work suggesting leptin plays an important role in the synaptic (re)organization of POMC neurons, POMC neurons which express leptin receptors were more sensitive to exercise-induced changes in biophysical properties. Opposite to effects observed in POMC neurons, NPY neurons were shunted toward inhibition following exercise. CONCLUSIONS: Together, these data support a rapid reorganization of synaptic inputs and biophysical properties in response to exercise, which may facilitate adaptations to altered energy balance and glucose metabolism.

Phosphoinositide 3-Kinase Is Integral for the Acute Activity of Leptin and Insulin in Male Arcuate NPY/AgRP Neurons.

Neuropeptide Y (NPY)/Agouti-related protein (AgRP) neurons in the arcuate nucleus of the hypothalamus are part of a neuroendocrine feedback loop that regulates feeding behavior and glucose homeostasis. NPY/AgRP neurons sense peripheral signals (including the hormones leptin, insulin, and ghrelin) and integrate those signals with inputs from other brain regions. These inputs modify both long-term changes in gene transcription and acute changes in the electrical activity of these neurons, leading to a coordinated response to maintain energy and glucose homeostasis. However, the mechanisms by which the hormones insulin and leptin acutely modify the electrical activity of these neurons remain unclear. In this study, we show that loss of the phosphoinositide 3-kinase catalytic subunits p110α and p110ß in AgRP neurons abrogates the leptin- and insulin-induced inhibition of AgRP neurons. Moreover, continual disruption of p110α and p110ß in AgRP neurons results in increased weight gain. The increased adiposity was concomitant with a hypometabolic phenotype: decreased energy expenditure independent of changes in food intake. Deficiency of p110α and p110ß in AgRP neurons also impaired glucose homeostasis and insulin sensitivity. In summary, these data highlight the requirement of both p110α and p110ß in AgRP neurons for the proper regulation of energy balance and glucose homeostasis.

Substantial Targeting Advantage Achieved by Pulmonary Administration of Colistin Methanesulfonate in a Large-Animal Model.

Colistin, administered as its inactive prodrug colistin methanesulfonate (CMS), is often used in multidrug-resistant Gram-negative pulmonary infections. The CMS and colistin pharmacokinetics in plasma and epithelial lining fluid (ELF) following intravenous and pulmonary dosing have not been evaluated in a large-animal model with pulmonary architecture similar to that of humans. Six merino sheep (34 to 43 kg body weight) received an intravenous or pulmonary dose of 4 to 8 mg/kg CMS (sodium) or 2 to 3 mg/kg colistin (sulfate) in a 4-way crossover study. Pulmonary dosing was achieved via jet nebulization through an endotracheal tube cuff. CMS and colistin were quantified in plasma and bronchoalveolar lavage fluid (BALF) samples by high-performance liquid chromatography (HPLC). ELF concentrations were calculated via the urea method. CMS and colistin were comodeled in S-ADAPT. Following intravenous CMS or colistin administration, no concentrations were quantifiable in BALF samples. Elimination clearance was 1.97 liters/h (4% interindividual variability) for CMS (other than conversion to colistin) and 1.08 liters/h (25%) for colistin. On average, 18% of a CMS dose was converted to colistin. Following pulmonary delivery, colistin was not quantifiable in plasma and CMS was detected in only one sheep. Average ELF concentrations (standard deviations [SD]) of formed colistin were 400 (243), 384 (187), and 184 (190) mg/liter at 1, 4, and 24 h after pulmonary CMS administration. The population pharmacokinetic model described well CMS and colistin in plasma and ELF following intravenous and pulmonary administration. Pulmonary dosing provided high ELF and low plasma colistin concentrations, representing a substantial targeting advantage over intravenous administration. Predictions from the pharmacokinetic model indicate that sheep are an advantageous model for translational research.

Predicting the composition of red wine blends using an array of multicomponent Peptide-based sensors.

Differential sensing using synthetic receptors as mimics of the mammalian senses of taste and smell is a powerful approach for the analysis of complex mixtures. Herein, we report on the effectiveness of a cross-reactive, supramolecular, peptide-based sensing array in differentiating and predicting the composition of red wine blends. Fifteen blends of Cabernet Sauvignon, Merlot and Cabernet Franc, in addition to the mono varietals, were used in this investigation. Linear Discriminant Analysis (LDA) showed a clear differentiation of blends based on tannin concentration and composition where certain mono varietals like Cabernet Sauvignon seemed to contribute less to the overall characteristics of the blend. Partial Least Squares (PLS) Regression and cross validation were used to build a predictive model for the responses of the receptors to eleven binary blends and the three mono varietals. The optimized model was later used to predict the percentage of each mono varietal in an independent test set composted of four tri-blends with a 15% average error. A partial least square regression model using the mouth-feel and taste descriptive sensory attributes of the wine blends revealed a strong correlation of the receptors to perceived astringency, which is indicative of selective binding to polyphenols in wine.

Dimension reduction for classification with gene expression microarray data.

An important application of gene expression microarray data is classification of biological samples or prediction of clinical and other outcomes. One necessary part of multivariate statistical analysis in such applications is dimension reduction. This paper provides a comparison study of three dimension reduction techniques, namely partial least squares (PLS), sliced inverse regression (SIR) and principal component analysis (PCA), and evaluates the relative performance of classification procedures incorporating those methods. A five-step assessment procedure is designed for the purpose. Predictive accuracy and computational efficiency of the methods are examined. Two gene expression data sets for tumor classification are used in the study.