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Pharmacological activation of the hypothalamic glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) promotes weight loss and improves glucose tolerance. This demonstrates that the hypothalamic GLP-1R is sufficient but does not show whether it is necessary for the effects of exogenous GLP-1R agonists (GLP-1RA) or endogenous GLP-1 on these parameters. To address this, we crossed mice harboring floxed Glp1r alleles to mice expressing Nkx2.1-Cre to knock down Glp1r expression throughout the hypothalamus (GLP-1RKD). We also generated mice lacking Glp1r expression specifically in two GLP-1RA-responsive hypothalamic feeding nuclei/cell types, the paraventricular nucleus (GLP-1RKD) and proopiomelanocortin neurons (GLP-1RKD). Chow-fed GLP-1RKD mice exhibited increased food intake and energy expenditure with no net effect on body weight. When fed a high-fat diet, these mice exhibited normal food intake but elevated energy expenditure, yielding reduced weight gain. None of these phenotypes were observed in GLP-1RKD and GLP-1RKD mice. The acute anorectic and glucose tolerance effects of peripherally dosed GLP-1RA exendin-4 and liraglutide were preserved in all mouse lines. Chronic liraglutide treatment reduced body weight in chow-fed GLP-1RKD mice, but this effect was attenuated with high-fat diet feeding. In sum, classic homeostatic control regions are sufficient but not individually necessary for the effects of GLP-1RA on nutrient homeostasis.
© 2017 by the American Diabetes Association.
The regulated release of anorexigenic α-melanocyte stimulating hormone (α-MSH) and orexigenic Agouti-related protein (AgRP) from discrete hypothalamic arcuate neurons onto common target sites in the central nervous system has a fundamental role in the regulation of energy homeostasis. Both peptides bind with high affinity to the melanocortin-4 receptor (MC4R); existing data show that α-MSH is an agonist that couples the receptor to the Gαs signalling pathway, while AgRP binds competitively to block α-MSH binding and blocks the constitutive activity mediated by the ligand-mimetic amino-terminal domain of the receptor. Here we show that, in mice, regulation of firing activity of neurons from the paraventricular nucleus of the hypothalamus (PVN) by α-MSH and AgRP can be mediated independently of Gαs signalling by ligand-induced coupling of MC4R to closure of inwardly rectifying potassium channel, Kir7.1. Furthermore, AgRP is a biased agonist that hyperpolarizes neurons by binding to MC4R and opening Kir7.1, independently of its inhibition of α-MSH binding. Consequently, Kir7.1 signalling appears to be central to melanocortin-mediated regulation of energy homeostasis within the PVN. Coupling of MC4R to Kir7.1 may explain unusual aspects of the control of energy homeostasis by melanocortin signalling, including the gene dosage effect of MC4R and the sustained effects of AgRP on food intake.
Melanocortin-4 receptor (MC4R) is critical for energy homeostasis, and the paraventricular nucleus of the hypothalamus (PVN) is a key site of MC4R action. Most studies suggest that leptin regulates PVN neurons indirectly, by binding to receptors in the arcuate nucleus or ventromedial hypothalamus and regulating release of products like α-melanocyte-stimulating hormone (α-MSH), neuropeptide Y (NPY), glutamate, and GABA from first-order neurons onto the MC4R PVN cells. Here, we investigate mechanisms underlying regulation of activity of these neurons under various metabolic states by using hypothalamic slices from a transgenic MC4R-GFP mouse to record directly from MC4R neurons. First, we show that in vivo leptin levels regulate the tonic firing rate of second-order MC4R PVN neurons, with fasting increasing firing frequency in a leptin-dependent manner. We also show that, although leptin inhibits these neurons directly at the postsynaptic membrane, α-MSH and NPY potently stimulate and inhibit the cells, respectively. Thus, in contrast with the conventional model of leptin action, the primary control of MC4R PVN neurons is unlikely to be mediated by leptin action on arcuate NPY/agouti-related protein and proopiomelanocortin neurons. We also show that the activity of MC4R PVN neurons is controlled by the constitutive activity of the MC4R and that expression of the receptor mRNA and α-MSH sensitivity are both stimulated by leptin. Thus, leptin acts multinodally on arcuate nucleus/PVN circuits to regulate energy homeostasis, with prominent mechanisms involving direct control of both membrane conductances and gene expression in the MC4R PVN neuron.
Fasting-induced suppression of thyroid hormone levels is an adaptive response to reduce energy expenditure in both humans and mice. This suppression is mediated by the hypothalamic-pituitary-thyroid axis through a reduction in TRH levels expressed in neurons of the paraventricular nucleus of the hypothalamus (PVN). TRH gene expression is positively regulated by leptin. Whereas decreased leptin levels during fasting lead to a reduction in TRH gene expression, the mechanisms underlying this process are still unclear. Indeed, evidence exists that TRH neurons in the PVN are targeted by leptin indirectly via the arcuate nucleus, whereas correlative evidence for a direct action exists as well. Here we provide both in vivo and in vitro evidence that the activity of hypothalamic-pituitary-thyroid axis is regulated by both direct and indirect leptin regulation. We show that both leptin and α-MSH induce significant neuronal activity mediated through a postsynaptic mechanism in TRH-expressing neurons of PVN. Furthermore, we provide in vivo evidence indicating the contribution of each pathway in maintaining serum levels of thyroid hormone.
Phosphatidylinositol 3-OH-kinase (PI3K) and STAT3 are signal transduction molecules activated by leptin in brain areas controlling food intake. To investigate their role in leptin-mediated inhibition of hypothalamic neuropeptide Y (Npy) and agouti-related peptide (Agrp) gene expression, male Sprague-Dawley rats (n = 5/group) were either fed ad libitum or subjected to a 52-h fast. At 12-h intervals, the PI3K inhibitor LY-294002 (LY, 1 nmol) or vehicle was injected intracerebroventricularly (ICV) as a pretreatment, followed 1 h later by leptin (3 microg icv) or vehicle. Fasting increased hypothalamic Npy and Agrp mRNA levels (P < 0.05), and ICV leptin administration prevented this increase. As predicted, LY pretreatment blocked this inhibitory effect of leptin, such that Npy and Agrp levels in LY-leptin-treated animals were similar to fasted controls. By comparison, leptin-mediated activation of hypothalamic STAT3 signaling, as measured by induction of both phospho-STAT3 immunohistochemistry and suppressor of cytokine signaling-3 (Socs3) mRNA, was not significantly attenuated by ICV LY pretreatment. Because NPY/AgRP neurons project to the hypothalamic paraventricular nucleus (PVN), we next investigated whether leptin activation of PVN neurons is similarly PI3K dependent. Compared with vehicle, leptin increased the number of c-Fos positive cells within the parvocellular PVN (P = 0.001), and LY pretreatment attenuated this effect by 35% (P = 0.043). We conclude that leptin requires intact PI3K signaling both to inhibit hypothalamic Npy and Agrp gene expression and activate neurons within the PVN. In addition, these data suggest that leptin activation of STAT3 is insufficient to inhibit expression of Npy or Agrp in the absence of PI3K signaling.
The nucleus accumbens of the rat consists of several subregions that can be distinguished on the basis of histochemical markers. For example, the calcium-binding protein calbindin D28k is a useful marker of the core compartment of the nucleus accumbens. Calretinin, another calcium-binding protein, is found in a dense fibre plexus in the accumbal shell and septal pole regions. The source of the accumbal calretinin innervation is not known. We examined the distribution of calretinin in the nucleus accumbens and used tract-tracing and lesion methods to determine the source of this calretinin innervation. Intense calretinin immunoreactivity was present in the medial shell, but the density of calretinin axons diminished sharply in the ventrolateral shell. Regions of dense calretinin immunostaining and those areas with calbindin-like immunoreactive cell bodies were generally segregated in the nucleus accumbens, although some overlap in the transition region between the core and shell was seen. Small clusters of calretinin-immunoreactive fibres were seen in the core, where they were restricted to calbindin-negative patches. Injections of the anterograde tracer biotinylated dextran amine into the paraventricular thalamic nucleus labelled fibres in calretinin-rich regions of the accumbens. Conversely, injections of Fluoro-gold into the accumbal shell retrogradely labelled numerous cells in the paraventricular thalamic nucleus that were calretinin-immunoreactive. Electrolytic lesions of the paraventricular thalamic nucleus reduced calretinin levels in the shell by approximately 80%. These data indicate that the calretinin innervation of the nucleus accumbens is derived primarily from the thalamic paraventricular nucleus, and marks accumbal territories that are largely complementary to those defined by calbindin.
Transgenic mice expressing an Escherichia coli beta-galactosidase reporter gene under the control of 3 kilobases of human proenkephalin gene 5'-flanking sequence and 1.2 kilobases of 3'-flanking sequence exhibited an anatomically correct pattern of basal and stress-regulated transgene expression within the hypothalamus. Acute osmotic stress and hypovolemia induced transgene expression in neurons within both the paraventricular and supraoptic nuclei. Chronic osmotic stress resulted in dramatic induction of transgene expression in both nuclei. These results demonstrate that the information required for correct hypothalamic expression and stress regulation of the proenkephalin gene is contained within our fusion construct.