Philip J. Larsen

Distribution of glucagon-like peptide-1 and other preproglucagon-derived peptides in the rat hypothalamus and brainstem

Central administration of the preproglucagon-derived peptide glucagon-like peptide-1 significantly inhibits ingestion of food and water, and glucagon-like peptide-1 binding sites are present in a multitude of central areas involved in the regulation of ingestional behaviour. To evaluate further the neuroanatomical organization of central glucagon-like peptide-1 containing neuronal circuits with potential implications on ingestional behaviour, we carried out a series of experiments in the rat demonstrating the topographical sites of synthesis and processing of the preproglucagon precursor followed by a chromatographic analysis of the processed fragments. In situ hybridization histochemistry revealed that preproglucagon encoding messenger RNA was expressed in a single population of neurons in the caudal portion of the nucleus of the solitary tract. Gel chromatographic analysis of hypothalamic and brainstem tissue extracts revealed that the preproglucagon precursor is processed in a fashion similar to that seen in the small intestine, preferentially giving rise to glicentin, glucagon-like peptide-1 and glucagon-like peptide-2. This single brain site of glucagon-like peptide-1 synthesis was subsequently confirmed by immunohistochemical demonstration of glucagon-like peptide-1-immunoreactive perikarya in the central and caudal parts of the nucleus of the solitary tract. Numerous sites containing glucagon-like peptide-1 immunoreactive fibres were, however, discovered in the forebrain including hypothalamic, thalamic and cortical areas. The densest innervation by glucagon-like peptide-1 immunoreactive nerve fibres was seen in the hypothalamic dorsomedial and paraventricular nuclei, but numerous glucagon-like peptide-1 immunoreactive fibres were also seen throughout the periventricular strata of the third ventricle. Dual-labelling immunohistochemistry for tyrosine hydroxylase and glucagon-like peptide-1 gave no evidence for co-localization of catecholamines and glucagon-like peptide-1 in neurons of the lower brainstem. To identify neurons of the nucleus of the solitary tract that project to the hypothalamic paraventricular nucleus, the retrograde tracer FluoroGold was injected into this hypothalamic target and dual immunocytochemical identification of glucagon-like peptide-1 and tyrosine hydroxylase-positive neurons was performed on brainstem sections containing retrogradely labelled perikarya. From this experiment it was seen that many of the retrogradely labelled neurons in the central portion of the nucleus of the solitary tract are catecholaminergic, while none is glucagon-like peptide-1 immunoreactive. In contrast, most of the retrogradely labelled neurons of the caudal portion of the nucleus of the solitary tract contain glucagon-like peptide-1. These observations further substantiate that glucagon-like peptide-1 neurons of the solitary tract constitute a distinct non-catecholaminergic cell group which projects to many targets, one of which is the hypothalamic paraventricular nucleus.

Distribution of GLP‐1 Binding Sites in the Rat Brain: Evidence that Exendin‐4 is a Ligand of Brain GLP‐1 Binding Sites

The distribution and biochemical properties of glucagon‐like peptide (GLP)‐1(7–36)amide (GLP‐1) binding sites in the rat brain were investigated. By receptor autoradiography of tissue sections, the highest densities of [125I]GLP‐1 binding sites were identified in the lateral septum, the subfornical organ (SFO), the thalamus, the hypothalamus, the interpenduncular nucleus, the posterodorsal tegmental nucleus, the area postrema (AP), the inferior olive and the nucleus of the solitary tract (NTS). Binding studies with [125I][Tyr39]exendin‐4, a GLP‐1 receptor agonist, showed an identical distribution pattern of binding sites. Binding specificity and affinity was investigated using sections of the brainstem containing the NTS. Binding of [125I]GLP‐1 to the NTS was inhibited concentration‐dependently by unlabelled GLP‐1 and [Tyr39]exendin‐4 with K1 values of 3.5 and 9.4 nM respectively. Cross‐linking of hypothalamic membranes with [125I]GLP‐1 or [125I][Tyr39]exendin‐4 identified a single ligand‐binding protein complex with a molecular mass of 63 000 Da. The fact that no GLP‐1 binding sites were detected in the cortex but that they were detected in the phylogenetically oldest parts of the brain emphasizes that GLP‐1 may be involved in the regulation of vital functions. In conclusion, the biochemical data support the idea that the central GLP‐1 receptor resembles the peripheral GLP‐1 receptor. Furthermore, the presence of GLP‐1 binding sites in the circumventricular organs suggests that these may be receptors which act as the target for both peripheral blood‐borne GLP‐1 and GLP‐1 in the nervous system.

The proglucagon-derived peptide, glucagon-like peptide-2, is a neurotransmitter involved in the regulation of food intake

The dorsomedial hypothalamic nucleus harbors leptin sensitive neurons and is intrinsically connected to hypothalamic nuclei involved in feeding behavior. However, it also receives ascending input from the visceroceptive neurons of the brainstem. We have identified a unique glucagon-like-peptide-2 containing neuronal pathway connecting the nucleus of the solitary tract with the dorsomedial hypothalamic nucleus. A glucagon-like-peptide-2 fiber plexus targets neurons expressing its receptor within the dorsomedial hypothalamic nucleus. Pharmacological and behavioral studies confirmed that glucagon-like-peptide-2 signaling is a specific transmitter inhibiting rodent feeding behavior and with potential long-term effects on body weight homeostasis. The glucagon-like-peptide-1 receptor antagonist, Exendin (9–39) is also a functional antagonist of centrally applied glucagon-like-peptide-2.

Regional Distribution of Putative NPY Y1 Receptors and Neurons Expressing Y1 mRNA in Forebrain Areas of the Rat Central Nervous System

Using monoiodinated radioligands of peptide YY (PYY), and the recently introduced neuropeptide Y (NPY) analogue [Leu31,Pro34]NPY, receptor binding sites of the Y1 and Y2 type were localized in the rat brain by quantitative in vitro autoradiography. The binding specificity and affinity of both radiolabeled ligands were analysed by ligand binding studies employing rat brain membrane homogenates from cerebral cortex and hippocampus. Using in situ hybridization histochemistry, the regional distribution and cellular localization of mRNA encoding the Y1 receptor were investigated in rat brain sections and compared to the distribution of Y1‐specific binding sites. PYY binds to both Y1 and Y2 receptors, while long C‐terminal fragments such as NPY13–36 and NPY16–36 bind preferentially to Y2 receptors. [Leu31, Pro34]NPY is a specific agonist for Y1 receptors. Highest densities of [125I]PYY binding sites were found in the cerebral cortex, the thalamus, the lateral septum, the hippocampus and the mesencephalic dopaminergic areas. In order to block putative Y2 receptors, a series of [125I]PYY binding experiments was performed in the presence of NPY13–36 (1 μM), a Y2 preferring C‐terminal fragment. High densities of binding sites remained present in the cerebral cortex, the thalamus and the medial mammillary nucleus when NPY13–36 was present in the incubation medium. Furthermore, these areas were highly enriched with [125l][Leu31, Pro34]NPY binding sites. In contrast, the hippocampal complex had its binding capacity reduced by ‐50%, while the lateral septum and mesencephalic dopaminergic areas had their binding capacities reduced even further. Linear regression analysis showed a high degree of correspondence between [125l][Leu31, Pro34]NPY binding and that obtained with [125I]PYY in the presence of 1 μM NPY13–36, suggesting that the two independent approaches to visualizing Y1 binding sites are comparable. In situ hybridization histochemistry revealed high levels of Y1 mRNA in the granular cell layer of the hippocampal dentate gyrus, several thalamic nuclei and the hypothalamic arcuate nucleus. Moderate levels of Y1 mRNA were seen in the frontoparietal cortex, several thalamic nuclei, the hippocampal pyramidal layers, the subiculum, the olfactory tubercle, the claustrum and a number of hypothalamic nuclei. The mesencephalon, the amygdala and most basal ganglia showed very low levels of hybridization. The present study further clarifies the anatomical distribution of multiple NPY binding sites within the central nervous system of the rat, and extends earlier suggestions that Y1 and Y2 receptor types are present within the central nervous system.

Evidence for arginine vasopressin as the primary activator of the HPA axis during adjuvant-induced arthritis.

1 Adjuvant‐induced arthritis (AA) is an experimental inflammation of the joints that results in chronic activation of the hypothalamo‐pituitary‐adrenal (HPA) axis. 2 In this study the role of hypothalamic corticotrophin‐releasing factor (CRF) and arginine vasopressin (AVP) in the regulation of the HPA axis in this condition both in Sprague‐Dawley (SD), and Piebald‐Viral‐Glaxo (PVG) rats has been further characterized. 3 The increase in AVP peptide content of portal blood (as early as day 11), just prior to the onset of arthritis is confirmed and further increases, peaking at day 16 are shown, coincident with the progression of inflammation in the PVG rats. 4 The increase in AVP is associated with a significant increase in the expression of AVP but not CRF mRNAs in the medial parvocellular division of the hypothalamic paraventricular nucleus (PVN) of arthritic SD rats. 5 In the presence of maximal inflammation of SD rats there was a significant decrease in the maximum binding of [125I]‐Tyr‐oCRF to anterior pituitary membranes, whereas AVP receptor concentration in anterior pituitary membranes from both PVG and SD rats showed a significant increase with respect to controls. 6 The basal adrenocorticotrophin (ACTH) secretion in vitro was similar in both control and arthritic SD rats but that from arthritic PVG rat pituitaries was significantly greater than the respective controls (436±91 v 167±23 pg/tube). The ACTH response of pituitaries of arthritic PVG rats to CRF or the combination of CRF and AVP was significantly higher compared with the controls, although the ACTH response of arthritic SD rat pituitaries was unchanged. 7 The results are consistent with the view that activation of the parvocellular vasopressin system has an important role in the adaptation of the HPA axis to experimentally‐induced chronic stress of arthritis.

Long-term characterization of the diet-induced obese and diet-resistant rat model: a polygenetic rat model mimicking the human obesity syndrome.

The availability of useful animal models reflecting the human obesity syndrome is crucial in the search for novel compounds for the pharmacological treatment of obesity. In the current study, we have performed an extensive characterization of the obesity syndrome in a polygenetic animal model, namely the selectively bred diet-induced obese (DIO) and diet-resistant (DR) rat strains. We show that they constitute useful models of the human obesity syndrome. DIO and DR rats were fed either a high-energy (HE) or a standard chow (Chow) diet from weaning to 9 months of age. Metabolic characterization including blood biochemistry and glucose homeostasis was examined at 2, 3, 6, and 9 months of age. Furthermore, in 6-month-old HE-fed DIO rats, the anti-obesity effects of liraglutide and sibutramine were examined in a 28-day study. Only HE-fed DIO rats developed visceral obesity, hyperleptinemia, hyperinsulinemia, and dyslipidemia, and showed a worsening of glucose tolerance over time. In line with the hyperlipidemic profile, a severe hepatic fat infiltration was observed in DIO rats at 6 months of age. The effects of liraglutide and sibutramine were tested in 6-month-old DIO rats. Both compounds effectively reduced food intake and body weight in DIO rats. Liraglutide furthermore improved glucose tolerance when compared with sibutramine. Our data highlights the usefulness of a polygenetic animal model for screening of compounds affecting food intake, body weight, and glucose homeostasis. Furthermore, the results underscore the effectiveness of GLP-1 mimetics both as anti-diabetes and anti-obesity agents.

Characterization of brainstem preproglucagon projections to the paraventricular and dorsomedial hypothalamic nuclei.

In the brain preproglucagon expression is limited to a cluster of neurons in the caudal part of the nucleus of the solitary tract (NTS) as well as a smaller number of neurons that extend laterally from the NTS through the dorsal reticular area into the A1 area. These neurons process preproglucagon to glucagon-like peptide-1 (GLP-1), GLP-2, oxyntomodulin and glicentin. The neurons project mainly to the hypothalamus, where especially two nuclei involved in appetite regulation--the paraventricular (PVN) and dorsomedial (DMH) hypothalamic nuclei--are heavily endowed with GLP-immunoreactive nerve fibres. To gain further insight into this neurocircuitry, we injected the retrograde tracers cholera toxin, subunit B (ChB) and Fluorogold (FG) into the PVN and the DMH, respectively. Of thirty-five injected rats, six had successful injections that predominantly restricted within the boundaries of the PVN and DMH. Hindbrain sections from these rats were triple labelled for ChB, FG and GLP-2. A total of 24+/-1% of the PVN-projecting NTS-neurons contained GLP-2-ir whereas 67+/-4% of the DMH-projecting neurons were also stained for GLP-2, suggesting that the NTS-projections to the DMH arise mainly from preproglucagon neurons. Approximately 20% of backfilled cells in the NTS contained both retrograde tracers, therefore presumably representing neurons projecting to both the PVN and the DMH. The results of the present study demonstrate that the majority of the preproglucagon-expressing neurons in the NTS project in a target-specific manner to the hypothalamus. It is therefore possible that individual subgroups of GLP-containing neurons can mediate different physiological responses.

Direct projection from the suprachiasmatic nucleus to hypophysiotrophic corticotropin-releasing factor immunoreactive cells in the paraventricular nucleus of the hypothalamus demonstrated by means ofPhaseolus vulgaris-leucoagglutinin tract tracing

The diurnal rhythm of the activity of the hypothalamo-pituitary-adrenal axis is generated by the circadian pacemaker located in the suprachiasmatic nuclei (SCN). However, the neuronal circuit connecting the SCN with the neurosecretory corticotropin-releasing factor (CRF) neurons in the paraventricular nucleus of the hypothalamus is not clear. To investigate the existence of a direct link between the SCN and the CRF neurons in the PVN we combined microiontopheretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) into the SCN with immunohistochemical detection of CRF in adrenalectomized male rats. The majority of the PHA-L-ir axons originating from the SCN terminated in the subparaventricular area. A minor contingent of fibers continued into the PVN proper, involving the medial and dorsal parvicellular subnuclei of the PVN. All PHA-L injections involving the entire SCN gave rise to PHA-L positive fibers endowed with boutons en passage and terminal boutons contacting CRF positive cell bodies in the PVN. Notably, varicosities on the PHA-L labelled fibers were present in close proximity to cell bodies and proximal dendrites of a subportion of the CRF neurons located in the periphery of the CRF cell cluster. The present study provides the first evidence to suggest a direct connection between the SCN and the CRF producing neurons of the hypothalamo-pituitary-adrenocortical axis in the PVN. Considering the sparse number of PHA-L-ir varicosities in close proximity to the CRF-ir cells, it seems likely that this direct pathway constitutes but a part of a projection system from the SCN, possibly involving multisynaptic pathways, influencing the hypothalamo-pituitary-adrenocortical axis.

Electroconvulsive shocks increase the expression of neuropeptide Y (NPY) mRNA in the piriform cortex and the dentate gyrus

Repeated electroconvulsive stimulations represent one treatment modality for depressive disorders, but the mechanism leading to its effect is largely unknown. Studies of humans and rats have indicated that neuropeptide Y (NPY) is involved in major depression and anxiety. The purpose of the present investigation was to detect changes in the expression of preproNPY mRNA in the limbic cortex of rats exposed to electroconvulsive shocks (ECS) daily for 14 days. Twenty-four hours after the last ECS, the animals were sacrificed, brain sections were hybridized with a synthetic oligonucleotide probe complimentary to rat preproNPY mRNA. Semi-quantitative in situ hybridization histochemistry revealed an about ten-fold increase of preproNPY mRNA levels over the dentate gyrus and the piriform cortex in animals exposed to ECS compared to sham-treated controls. In the dentate gyrus dipped sections showed that the increase of gene expression took place in individual neurons in the polymorph layer. In the piriform cortex a moderate increase in the number of grains was observed over many individual cells in the pyramidal layer. These data show that the expression of preproNPY mRNA is markedly increased in specific brains regions after ECS, but whether this increase is a result of the ECS-induced seizures per se, or rather should be regarded as a protective adaptation to changes in neuronal activity pattern remains to be established.

Origin of projections from the midbrain raphe nuclei to the hypothalamic paraventricular nucleus in the rat: A combined retrograde and anterograde tracing study

A number of neuronal functions governed by the hypothalamic paraventricular nucleus are influenced by serotonin, and it is generally believed that the moderate density of serotonin-immunoreactive fibres and terminals within the paraventricular nucleus originates from the midbrain dorsal and median raphe nuclei. To further evaluate the intricate anatomy of projections from brain stem raphe nuclei of the rat, a combination of retrograde and anterograde tracing experiments were conducted to determine the medullary raphe nuclei projection to the paraventricular nucleus. Rhodamine-labelled latex microspheres, Cholera toxin subunit B and FluoroGold we used as retrograde tracers. Intracerebroventricular injections into the third ventricle of all retrograde tracers labelled a distinct population of neurons in the dorsal raphe situated in the subependymal stratum adjacent to the cerebral aqueduct indicating that these cells take up the tracer from the cerebrospinal fluid. Very few retrogradely labelled neurons were seen in the median raphe after i.c.v. administration of the tracers. Retrograde tracers delivered into the medial part of the paraventricular nucleus labelled no further cells in the midbrain dorsal and median raphe nuclei, whereas a substantial number of retrogradely labelled cells emerged in the pontine raphe magnus. However, when the retrograde tracers were delivered into the lateral part of the paraventricular nucleus, avoiding leakage of the tracer into the ventricle, very few labelled neurons were seen in the dorsal and median raphe, whereas the prominent labelling of raphe magnus neurons persisted. The anatomical organization of nerve fibres terminating in the area of the paraventricular nucleus originating from midbrain raphe nuclei was studied in a series of anterograde tracing experiments using the plant lectin Phaseolus vulgaris leucoagglutinin. Injections delivered into the dorsal raphe or median raphe labelled but a few fibres in the paraventricular nucleus proper. A high number of fine calibered nerve fibres overlying the ependyma adjacent to the paraventricular nucleus was, however, seen after the injections into the subependymal rostral part of the dorsal raphe. Injections delivered into the raphe magnus gave rise to a dense plexus of terminating fibres in the parvicellular parts of the paraventricular nucleus and moderately innervated the posterior magnocellular part of the paraventricular nucleus as well as the magnocellular supraoptic nucleus. Concomitant visualization of serotonin-immunoreactive neurons and retrograde FluoroGold-tracing from the paraventricular nucleus revealed that none of the serotonergic neurons of the raphe magnus projects to this nucleus, while a few of the neurons putatively projecting to the paraventricular nucleus from the median raphe are serotonergic. The current observations suggest that the raphe magnus constitute by far the largest raphe input to the paraventricular nucleus and strongly questions the earlier held view that most raphe fibres innervating the paraventricular nucleus are derived from the midbrain dorsal and median raphe. However, the source of serotonergic innervation of the paraventricular nucleus remains elusive.