Topography of the GLP-1/GLP-1 receptor system in the spinal cord of male mice.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are now commonly used to treat type 2 diabetes and obesity. GLP-1R signaling in the spinal cord has been suggested to account for the mild tachycardia caused by GLP-1R agonists, and may also be involved in the therapeutic effects of these drugs. However, the neuroanatomy of the GLP-1/GLP-1R system in the spinal cord is still poorly understood. Here we applied in situ hybridization and immunohistochemistry to characterize this system, and its relation to cholinergic neurons. GLP-1R transcript and protein were expressed in neuronal cell bodies across the gray matter, in matching distribution patterns. GLP-1R-immunolabeling was also robust in dendrites and axons, especially in laminae II-III in the dorsal horn. Cerebrospinal fluid-contacting neurons expressed GLP-1R protein at exceedingly high levels. Only small subpopulations of cholinergic neurons expressed GLP-1R, including a subset of sympathetic preganglionic neurons at the rostral tip of the intermediolateral nucleus. GLP-1 axons innervated all regions where GLP-1R neurons were distributed, except laminae II-III. Scattered preproglucagon (Gcg) mRNA-expressing neurons were identified in the cervical and lumbar enlargements. The results will facilitate further studies on how GLP-1 regulates the sympathetic system and other autonomic and somatic functions via the spinal cord.

GLP-1 Receptor Signaling Has Different Effects on the Perikarya and Axons of the Hypophysiotropic Thyrotropin-Releasing Hormone Synthesizing Neurons in Male Mice.

Background: Glucagon-like peptide 1 (GLP-1) is involved in the regulation of energy and glucose homeostasis. As GLP-1 has similar effects on the energy homeostasis as the hypophysiotropic thyrotropin-releasing hormone (TRH) neurons that regulate the hypothalamic-pituitary-thyroid (HPT) axis, we raised the possibility that the TRH neurons are involved in the mediation of the effects of GLP-1. Therefore, the relationship and interaction of the GLP-1 system and the TRH neurons of the hypothalamic paraventricular nucleus (PVN) were studied. Methods: To examine the anatomical and functional relationship of TRH neurons and the GLP-1 system in the PVN, immunocytochemistry, in situ hybridization, in vitro patch-clamp electrophysiology, metabolic phenotyping, and explant experiments were performed. Results: Our data demonstrate that the TRH neurons of the PVN are innervated by GLP-1 producing neurons and express the GLP-1 receptor (GLP-1R). However, not only do the GLP-1-innervated TRH neurons express GLP-1R but the receptor is also present in the axons of the hypophysiotropic TRH neurons in the blood-brain barrier free median eminence (ME) suggesting that peripherally derived GLP-1 may also influence the TRH neurons. In vitro, GLP-1 increased the firing rate of TRH neurons and depolarized them. In addition, GLP-1 directly stimulated the GABAergic input of a population of TRH neurons. Furthermore, GLP-1 inhibited the release of TRH from the hypophysiotropic axons in the ME. In vivo, peripheral GLP-1R agonist administration markedly inhibited the food intake and the energy expenditure, but had no effect on the TRH expression in the PVN and resulted in lower circulating free T4 levels. Conclusions: Our results indicate that GLP-1R activation has a direct stimulatory effect on TRH neurons in the PVN, but the activation of GLP-1R may also inhibit TRH neurons by facilitating their inhibitory inputs or by inhibiting the axon terminals of these cells in the ME. The innervation of TRH neurons by GLP-1 neurons suggests that TRH neurons might be influenced by both circulating GLP-1 and by GLP-1 neurons of the nucleus tractus solitarii. The lack of GLP-1R agonist-induced regulation of TRH neurons in vivo suggests that the HPT axis does not mediate the GLP-1R agonist-induced weight loss.

A previously uncharacterized Factor Associated with Metabolism and Energy (FAME/C14orf105/CCDC198/1700011H14Rik) is related to evolutionary adaptation, energy balance, and kidney physiology.

In this study we use comparative genomics to uncover a gene with uncharacterized function (1700011H14Rik/C14orf105/CCDC198), which we hereby name FAME (Factor Associated with Metabolism and Energy). We observe that FAME shows an unusually high evolutionary divergence in birds and mammals. Through the comparison of single nucleotide polymorphisms, we identify gene flow of FAME from Neandertals into modern humans. We conduct knockout experiments on animals and observe altered body weight and decreased energy expenditure in Fame knockout animals, corresponding to genome-wide association studies linking FAME with higher body mass index in humans. Gene expression and subcellular localization analyses reveal that FAME is a membrane-bound protein enriched in the kidneys. Although the gene knockout results in structurally normal kidneys, we detect higher albumin in urine and lowered ferritin in the blood. Through experimental validation, we confirm interactions between FAME and ferritin and show co-localization in vesicular and plasma membranes.

The metabolic stress response: Adaptation to acute-, repeated- and chronic challenges in mice.

There is a strong relationship between stress and metabolism. Because acute traumatic- and chronic stress events are often accompanied with metabolic pathophysiology, it is important to understand the details of the metabolic stress response. In this study we directly compared metabolic effects of acute stress with chronic repeated- and chronic unpredictable stress in mouse models. All types of adversities increased energy expenditure, chronic stress exposure decreased body weight gain, locomotor activity and differentially affected fuel utilization. During chronic exposure to variable stressors, carbohydrates were the predominant fuels, whereas fatty acids were catabolized in acutely and repeatedly restrained animals. Chronic exposure to variable stressors in unpredictable manner provoked anxiety. Our data highlight differences in metabolic responses to acute- repeated- and chronic stressors, which might affect coping behavior and underlie stress-induced metabolic and psychopathologies.

Hypothalamic Nesfatin-1 Resistance May Underlie the Development of Type 2 Diabetes Mellitus in Maternally Undernourished Non-obese Rats.

Intrauterine growth retardation (IUGR) poses a high risk for developing late-onset, non-obese type 2 diabetes (T2DM). The exact mechanism underlying this phenomenon is unknown, although the contribution of the central nervous system is recognized. The main hypothalamic nuclei involved in the homeostatic regulation express nesfatin-1, an anorexigenic neuropeptide and identified regulator of blood glucose level. Using intrauterine protein restricted rat model (PR) of IUGR, we investigated, whether IUGR alters the function of nesfatin-1. We show that PR rats develop fat preference and impaired glucose homeostasis by adulthood, while the body composition and caloric intake of normal nourished (NN) and PR rats are similar. Plasma nesfatin-1 levels are unaffected by IUGR in both neonates and adults, but pro-nesfatin-1 mRNA expression is upregulated in the hypothalamus of adult PR animals. We find that centrally injected nesfatin-1 inhibits the fasting induced neuronal activation in the hypothalamic arcuate nucleus in adult NN rats. This effect of nesfatin-1 is not seen in PR rats. The anorexigenic effect of centrally injected nesfatin-1 is also reduced in adult PR rats. Moreover, chronic central nesfatin-1 administration improves the glucose tolerance and insulin sensitivity in NN rats but not in PR animals. Birth dating of nesfatin-1 cells by bromodeoxyuridine (BrDU) reveals that formation of nesfatin-1 cells in the hypothalamus of PR rats is disturbed. Our results suggest that adult PR rats acquire hypothalamic nesfatin-1-resistance, probably due to the altered development of the hypothalamic nesfatin-1 cells. Hypothalamic nesfatin-1-resistance, in turn, may contribute to the development of non-obese type T2DM.

Expression of glucagon-like peptide 1 receptor in neuropeptide Y neurons of the arcuate nucleus in mice.

Glucagon-like peptide 1 (GLP-1) and its agonists exert anorexigenic effect at least partly via acting on GLP-1 receptors (GLP-1R) in the arcuate nucleus (ARC). While the anorexigenic, proopiomelanocortin (POMC) neurons of the ARC were shown previously to express GLP-1R, the putative GLP-1R-content of the orexigenic, neuropeptide Y (NPY) neurons remained so far undetected. As GLP-1R is abundant in the ventromedial ARC, where NPY neurons are located; here, we address the possibility that GLP-1 can act directly on the orexigenic NPY system via GLP-1R. Double-labeling immunocytochemistry and in situ hybridization were performed on tissues of adult male mice to detect GLP-1R in NPY neurons. In double-immunolabeled preparations, GLP-1R-immunoreactivity was observed in NPY neurons and in axons ensheathing the majority of NPY neurons. Ultrastructural studies confirmed that GLP-1R-immunoreactivity is associated with the outer membrane of NPY perikarya as well as with axons forming symmetric type, inhibitory synapses on NPY-containing neurons. Double-labeling in situ hybridization experiments demonstrated the expression of GLP-1R mRNA in approximately 20% of NPY mRNA-containing neurons of the ARC. In summary, our data demonstrate the presence of GLP-1R protein and mRNA in NPY neurons of ARC and also reveal the innervation of NPY neurons by GLP-1R-containing inhibitory neurons. These observations suggest that GLP-1 signaling can influence NPY neurons both directly and indirectly. Furthermore, GLP-1 signaling on energy homeostasis appears to involve both direct and indirect effects of GLP-1 on the orexigenic NPY neurons, in addition to the previously known effects via the anorexigenic POMC neuronal system.

Tanycyte specific ablation of diacylglycerol lipase alpha stimulates the hypothalamic-pituitary-thyroid axis by decreasing the endocannabinoid mediated inhibition of TRH release.

In addition to the hypophysiotropic thyrotropin-releasing hormone (TRH)-synthesizing neurons, a glial cell type, the tanycytes, also play a role in the regulation of the hypothalamic-pituitary-thyroid (HPT) axis. Tanycytes modulate the feedback regulation of the axis by regulating the local thyroid hormone availability in the median eminence where the hypophysiotropic axons terminate. Recently, we showed that tanycytes produce diacylglycerol lipase alpha (DAGLα), the synthesizing enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG) that inhibits the release of TRH from the hypophysiotropic terminals in median eminence explants. To determine the importance of the endocannabinoid production of tanycytes, adult male Rax-CreERT2//DAGLαfl/fl mice were treated with tamoxifen to induce a tanycyte specific decrease of DAGLα expression (T-DAGLα KO). The effect of this genetic manipulation on the activity of the HPT axis was determined. Tanycyte specific decrease of DAGLα expression resulted in an approximately 2-fold increase of TSHß mRNA level that was accompanied by increased levels of circulating free T4. The TRH mRNA level was, however, not influenced by the genetic manipulation. In addition to the effects on the HPT axis, the T-DAGLα KO mice showed increased fat mass ratio and decreased blood glucose levels. These data indicate that when endocannabinoid release of tanycytes is decreased, the disinhibition of the TRH release induces increased TSH synthesis and higher circulating T4 levels. Thus it suggests that in wild-type mice, tanycytes exert a tonic inhibitory effect on the TRH release of hypophysiotropic axons. Furthermore, the endocannabinoid release of tanycytes also influences glucose homeostasis and fat deposition.

Distribution and ultrastructural localization of the glucagon-like peptide-1 receptor (GLP-1R) in the rat brain.

Glucagon-like peptide-1 (GLP-1) inhibits food intake and regulates glucose homeostasis. These actions are at least partly mediated by central GLP-1 receptor (GLP-1R). Little information is available, however, about the subcellular localization and the distribution of the GLP-1R protein in the rat brain. To determine the localization of GLP-1R protein in the rat brain, immunocytochemistry was performed at light and electron microscopic levels. The highest density of GLP-1R-immunoreactivity was observed in the circumventricular organs and regions in the vicinity of these areas like in the arcuate nucleus (ARC) and in the nucleus tractus solitarii (NTS). In addition, GLP-1R-immunreactive (IR) neuronal profiles were also observed in a number of telencephalic, diencephalic and brainstem areas and also in the cerebellum. Ultrastructural examination of GLP-1R-immunoreactivity in energy homeostasis related regions showed that GLP-1R immunoreactivity is associated with the membrane of perikarya and dendrites but GLP-1R can also be observed inside and on the surface of axon varicosities and axon terminals. In conclusion, in this study we provide a detailed map of the GLP-1R-IR structures in the CNS. Furthermore, we demonstrate that in addition to the perikaryonal and dendritic distribution, GLP-1R is also present in axonal profiles suggesting a presynaptic action of GLP-1. The very high concentration of GLP-1R-profiles in the circumventricular organs and in the ARC and NTS suggests that peripheral GLP-1 may influence brain functions via these brain areas.

Glucagon-Like Peptide-1 Regulates the Proopiomelanocortin Neurons of the Arcuate Nucleus both Directly and Indirectly via Presynaptic Action.

Glucagon-like peptide-1 (GLP-1) exerts its anorexigenic effect at least partly via the proopiomelanocortin (POMC) neurons of the arcuate (ARC) nucleus. These neurons are known to express GLP-1 receptor (GLP-1R). The aim of the study was to determine whether in addition to its direct effect, GLP-1 also modulates how neuronal inputs can regulate the POMC neurons by acting on presynaptic terminals, ultrastructural and electrophysiological studies were performed on tissues of adult male mice. GLP-1R-immunoreactivity was associated with the cell membrane of POMC neurons and with axon terminals forming synapses on these cells. The GLP-1 analog exendin 4 (Ex4) markedly increased the firing rate of all examined POMC neurons and depolarized these cells. These effects of Ex4 were prevented by intracellular administration of the G-protein blocker guanosine 5'-[ß-thio]diphosphate trilithium salt (GDP-ß-S). Ex4 also influenced the miniature postsynaptic currents (mPSCs) and evoked PSCs of POMC neurons. Ex4 increased the frequency of miniature excitatory PSCs (EPSCs) and the amplitude of the evoked EPSCs in half of the POMC neurons. Ex4 increased the frequency of miniature inhibitory PSCs (IPSCs) and the amplitudes of the evoked IPSCs in one-third of neurons. These effects of Ex4 were not influenced by intracellular GDP-ß-S, indicating that GLP-1 signaling directly stimulates a population of axon terminals innervating the POMC neurons. The different Ex4 responsiveness of their mPSCs indicates the heterogeneity of the POMC neurons of the ARC. In summary, our data demonstrate that in addition to its direct excitatory effect on the POMC neurons, GLP-1 signaling also facilitates the presynaptic input of these cells by acting on presynaptically localized GLP-1R.

A Glial-Neuronal Circuit in the Median Eminence Regulates Thyrotropin-Releasing Hormone-Release via the Endocannabinoid System.

Based on the type-I cannabinoid receptor (CB1) content of hypophysiotropic axons and the involvement of tanycytes in the regulation of the hypothalamic-pituitary-thyroid (HPT) axis, we hypothesized that endocannabinoids are involved in the tanycyte-induced regulation of TRH release in the median eminence (ME). We demonstrated that CB1-immunoreactive TRH axons were associated to DAGLα-immunoreactive tanycyte processes in the external zone of ME and showed that endocannabinoids tonically inhibit the TRH release in this tissue. We showed that glutamate depolarizes the tanycytes, increases their intracellular Ca2+ level and the 2-AG level of the ME via AMPA and kainite receptors and glutamate transport. Using optogenetics, we demonstrated that glutamate released from TRH neurons influences the tanycytes in the ME. In summary, tanycytes regulate TRH secretion in the ME via endocannabinoid release, whereas TRH axons regulate tanycytes by glutamate, suggesting the existence of a reciprocal microcircuit between tanycytes and TRH terminals that controls TRH release.

Semaglutide lowers body weight in rodents via distributed neural pathways.

Semaglutide, a glucagon-like peptide 1 (GLP-1) analog, induces weight loss, lowers glucose levels, and reduces cardiovascular risk in patients with diabetes. Mechanistic preclinical studies suggest weight loss is mediated through GLP-1 receptors (GLP-1Rs) in the brain. The findings presented here show that semaglutide modulated food preference, reduced food intake, and caused weight loss without decreasing energy expenditure. Semaglutide directly accessed the brainstem, septal nucleus, and hypothalamus but did not cross the blood-brain barrier; it interacted with the brain through the circumventricular organs and several select sites adjacent to the ventricles. Semaglutide induced central c-Fos activation in 10 brain areas, including hindbrain areas directly targeted by semaglutide, and secondary areas without direct GLP-1R interaction, such as the lateral parabrachial nucleus. Automated analysis of semaglutide access, c-Fos activity, GLP-1R distribution, and brain connectivity revealed that activation may involve meal termination controlled by neurons in the lateral parabrachial nucleus. Transcriptomic analysis of microdissected brain areas from semaglutide-treated rats showed upregulation of prolactin-releasing hormone and tyrosine hydroxylase in the area postrema. We suggest semaglutide lowers body weight by direct interaction with diverse GLP-1R populations and by directly and indirectly affecting the activity of neural pathways involved in food intake, reward, and energy expenditure.

Neuronal connections of the central amygdalar nucleus with refeeding-activated brain areas in rats.

Following fasting, satiety is accompanied by neuronal activation in brain areas including the central amygdalar nucleus (CEA). Since CEA is known to inhibit food intake, we hypothesized that CEA contributes to the termination of meal during refeeding. To better understand the organization of this satiety-related circuit, the interconnections of the CEA with refeeding-activated neuronal groups were elucidated using retrograde (cholera toxin-ß subunit, CTB) and anterograde (phaseolus vulgaris leucoagglutinin, PHA-L) tracers in male rats. C-Fos-immunoreactivity was used as marker of neuronal activation. The refeeding-activated input of the CEA primarily originated from the paraventricular thalamic, parasubthalamic and parabrachial nuclei. Few CTB-c-Fos double-labeled neurons were detected in the prefrontal cortex, lateral hypothalamic area, nucleus of the solitary tract (NTS) and the bed nuclei of the stria terminalis (BNST). Only few refeeding-activated proopiomelanocortin-producing neurons of the arcuate nucleus projected to the CEA. Anterograde tract tracing revealed a high density of PHAL-labeled axons contacted with refeeding-activated neurons in the BNST, lateral hypothalamic area, parasubthalamic, paraventricular thalamic and parabrachial nuclei and NTS; a low density of labeled axons was found in the paraventricular hypothalamic nucleus. Chemogenetic activation of the medial CEA (CEAm) inhibited food intake during the first hour of refeeding, while activation of lateral CEA had no effect. These data demonstrate the existence of reciprocal connections between the CEA and distinct refeeding-activated hypothalamic, thalamic and brainstem nuclei, suggesting the importance of short feedback loops in the regulation of satiety and importance of the CEAm in the regulation of food intake during refeeding.

Localization of connexin 43 gap junctions and hemichannels in tanycytes of adult mice.

Tanycytes are specialized glial cells lining the lateral walls and the floor of the third ventricle behind the optic chiasm. In addition to functioning as barrier cells, they also have an important role in the regulation of neuroendocrine axes and energy homeostasis. To determine whether tanycytes communicate with each other via Connexin 43 (Cx43) gap junctions, individual tanycytes were loaded with Lucifer yellow (LY) through a patch pipette. In all cases, LY filled a larger group of tanycytes as well as blood vessels adjacent to tanycyte processes. The Cx43-blocker, carbenoxolone, inhibited spreading of LY. The greatest density of Cx43-immunoreactive spots was observed in the cell membrane of α-tanycyte cell bodies. Cx43-immunoreactivity was also present in the membrane of ß-tanycyte cell bodies, but in lower density. Processes of both types of tanycytes also contained Cx43-immunoreactivity. At the ultrastructural level, Cx43-immunoreactivity was present in the cell membrane of all types of tanycytes including their ventricular surface, but gap junctions were more frequent among α-tanycytes. Cx43-immunoreactivity was also observed in the cell membrane between contacting tanycyte endfeet processes, and between tanycyte endfeet process and axon varicosities in the external zone of the median eminence and capillaries in the arcuate nucleus and median eminence. These results suggest that gap junctions are present not only among tanycytes, but also between tanycytes and the axons of hypophysiotropic neurons. Cx43 hemichannels may also facilitate the transport between tanycytes and extracellular fluids, including the cerebrospinal fluid, extracellular space of the median eminence and bloodstream.

Variable proopiomelanocortin expression in tanycytes of the adult rat hypothalamus and pituitary stalk.

It is generally believed that proopiomelanocortin (POMC) is expressed exclusively by neurons in the adult rodent brain. Unbeknownst to most researchers, however, Pomc in situ hybridization studies in the rat show specific labeling in the ventral wall of the hypothalamic third ventricle, which is formed by specialized ependymal cells, called tanycytes. Here we characterized this non-neuronal POMC expression in detail using in situ hybridization and immunohistochemical techniques, and report two unique characteristics. First, POMC mRNA and precursor protein expression in non-neuronal cells varies to a great degree as to the extent and abundance of expression. In brains with low-level expression, POMC mRNA and protein was largely confined to a population of tanycytes within the infundibular stalk/caudal median eminence, termed here γ tanycytes, and a subset of closely located ß and α2 tanycytes. In brains with high-level expression, POMC mRNA and protein was observed in the vast majority of α2, ß, and γ tanycytes. This variability was observed in both adult males and females; of 41 rats between 8 and 15 weeks of age, 17 had low-, 9 intermediate-, and 15 high-level POMC expression in tanycytes. Second, unlike other known POMC-expressing cells, tanycytes rarely contained detectable levels of adrenocorticotropin or α-melanocyte-stimulating hormone. The results indicate either a dynamic spatiotemporal pattern whereby low and high POMC syntheses in tanycytes occur periodically in each brain, or marked interindividual differences that may persist throughout adulthood. Future studies are required to examine these possibilities and elucidate the physiologic importance of POMC in tanycytes. J. Comp. Neurol. 525:411-441, 2017. © 2016 Wiley Periodicals, Inc.

Gonadotropin-releasing hormone neurones innervate kisspeptin neurones in the female mouse brain.

Kisspeptin (KP) neurones in the rostral periventricular area of the third ventricle (RP3V) and arcuate nucleus (Arc) are important elements in the neuronal circuitry regulating gonadotropin-releasing hormone (GnRH) secretion. KP and co-synthesised neuropeptides/neurotransmitters act directly on GnRH perikarya and processes. GnRH neurones not only form the final output pathway regulating the reproductive functions of the anterior pituitary gland, but also provide neuronal input to sites within the hypothalamus. The current double-label immunohistochemical studies investigated whether GnRH-immunoreactive (IR) projections to the RP3V and/or Arc establish morphological connections with KP-IR neurones at these sites. To optimise visualisation of KP immunoreactivity in, respectively, the RP3V and Arc, ovariectomised (OVX) oestrogen-treated and OVX oil-treated female mice were studied. Confocal laser microscopic analysis of immunofluorescent specimens revealed GnRH-IR axon varicosities in apposition to approximately 25% of the KP-IR neurones in the RP3V and 50% of the KP-IR neurones in the Arc. At the ultrastructural level, GnRH-IR neurones were seen to establish asymmetric synaptic contacts, which usually reflect excitatory neurotransmission, with KP-IR neurones in both the RP3V and Arc. Together with previous data, these findings indicate reciprocal connectivity between both of the KP cell populations and the GnRH neuronal system. The functional significance of the GnRH-IR input to the two separate KP cell populations requires electrophysiological investigation.