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Hypophysiotropic thyrotropin-releasing hormone (TRH) neurons function as metabolic sensors that regulate the thyroid axis and energy homeostasis. Less is known about the role of other hypothalamic TRH neurons. As central administration of TRH decreases food intake and increases histamine in the tuberomammillary nuclei (TMN), and TMN histamine neurons are densely innervated by TRH fibers from an unknown origin, we mapped the location of TRH neurons that project to the TMN. The retrograde tracer, cholera toxin B subunit (CTB), was injected into the TMN E1–E2, E4–E5 subdivisions of adult Sprague–Dawley male rats. TMN projecting neurons were observed in the septum, preoptic area, bed nucleus of the stria terminalis (BNST), perifornical area, anterior paraventricular nucleus, peduncular and tuberal lateral hypothalamus (TuLH), suprachiasmatic nucleus and medial amygdala. However, CTB/pro-TRH178-199 double-labeled cells were only found in the TuLH. The specificity of the retrograde tract-tracing result was confirmed by administering the anterograde tracer, Phaseolus vulgaris leuco-agglutinin (PHAL) into the TuLH. Double-labeled PHAL-pro-TRH boutons were identified in all subdivisions of the TMN. TMN neurons double-labeled for histidine decarboxylase (Hdc)/PHAL, Hdc/Trh receptor (Trhr), and Hdc/Trh. Further confirmation of a TuLH-TRH neuronal projection to the TMN was established in a transgenic mouse that expresses Cre recombinase in TRH-producing cells following microinjection of a Cre recombinase-dependent AAV that expresses mCherry into the TuLH. We conclude that, in rodents, the TRH innervation of TMN originates in part from TRH neurons in the TuLH, and that this TRH population may contribute to regulate energy homeostasis through histamine Trhr-positive neurons of the TMN.

Cocaine- and amphetamine-regulated transcript (CART) peptides are involved in several physiological and pathological processes, but their mechanism of action is unrevealed due to the lack of identified receptor(s). We provided evidence for the antihyperalgesic effect of CART(55-102) by inhibiting dipeptidyl-peptidase 4 (DPP4) in astrocytes and consequently reducing neuroinflammation in the rat spinal dorsal horn in a carrageenan-evoked inflammation model. Both naturally occurring CART(55-102) and CART(62-102) peptides are present in the spinal cord. CART(55-102) is not involved in acute nociception but regulates spinal pain transmission during peripheral inflammation. While the full-length peptide with a globular motif contributes to hyperalgesia, its N-terminal inhibits this process. Although the anti-hyperalgesic effects of CART(55-102), CART(55-76), and CART(62-76) are blocked by opioid receptor antagonists in our inflammatory models, but not in neuropathic Seltzer model, none of them bind to any opioid or G-protein coupled receptors. DPP4 interacts with Toll-like receptor 4 (TLR4) signalling in spinal astrocytes and enhances the TLR4-induced expression of interleukin-6 and tumour necrosis factor alpha contributing to inflammatory pain. Depending on the state of inflammation, CART(55-102) is processed in the spinal cord, resulting in the generation of biologically active isoleucine-proline-isoleucine (IPI) tripeptide, which inhibits DPP4, leading to significantly decreased glia-derived cytokine production and hyperalgesia.

Thyroid hormone (TH) signaling is a prerequisite of normal tissue function. Environmental pollutants with the potential to disrupt endocrine functions represent an emerging threat to human health and agricultural production. We used our Thyroid Hormone Action Indicator (THAI) mouse model to study the effects of tetrabromobisphenol A (TBBPA; 150 mg/bwkg/day orally for 6 days) and diclazuril (10.0 mg/bwkg/day orally for 5 days), a known and a potential hormone disruptor, respectively, on local TH economy. Tissue-specific changes of TH action were assessed in 90-day-old THAI mice by measuring the expression of a TH-responsive luciferase reporter in tissue samples and by in vivo imaging (14-day-long treatment accompanied with imaging on day 7, 14 and 21 from the first day of treatment) in live THAI mice. This was followed by promoter assays to elucidate the mechanism of the observed effects. TBBPA and diclazuril impacted TH action differently and tissue-specifically. TBBPA disrupted TH signaling in the bone and small intestine and impaired the global TH economy by decreasing the circulating free T4 levels. In the promoter assays, TBBPA showed a direct stimulatory effect on the hdio3 promoter, indicating a potential mechanism for silencing TH action. In contrast, diclazuril acted as a stimulator of TH action in the liver, skeletal muscle and brown adipose tissue without affecting the Hypothalamo-Pituitary-Thyroid axis. Our data demonstrate distinct and tissue-specific effects of TBBPA and diclazuril on local TH action and prove that the THAI mouse is a novel mammalian model to identify TH disruptors and their tissue-specific effects.

Background A mouse with hepatocyte-specific deiodinase type II inactivation (Alb-D2KO) is resistant to diet-induced obesity, hepatic steatosis, and hypertriglyceridemia due to perinatal epigenetic modifications in the liver. This phenotype is linked to low levels of Zfp125, a hepatic transcriptional repressor that promotes liver steatosis by inhibiting genes involved in packaging and secretion of very-low-density lipoprotein. Methods Here, we used chronic and binge ethanol (EtOH) in mice to cause liver steatosis. Results The EtOH treatment causes a 2.3-fold increase in hepatic triglyceride content; Zfp125 levels were approximately 50% higher in these animals. In contrast, Alb-D2KO mice did not develop EtOH-induced liver steatosis. They also failed to elevate Zfp125 to the same levels, despite being on the EtOH-containing diet for the same period of time. Their phenotype was associated with 1.3- to 2.9-fold up-regulation of hepatic genes involved in lipid transport and export that are normally repressed by Zfp125, that is, Mttp, Abca1, Ldlr, Apoc1, Apoc3, Apoe, Apoh, and Azgp1. Furthermore, genes involved in the EtOH metabolic pathway, that is, Aldh2 and Acss2, were also 1.6- to 3.1-fold up-regulated in Alb-D2KO EtOH mice compared with control animals kept on EtOH. Conclusions EtOH consumption elevates expression of Zfp125. Alb-D2KO animals, which have lower levels of Zfp125, are much less susceptible to EtOH-induced liver steatosis.

Myogenesis is positively regulated by thyroid hormone (triiodothyronine [T3]), which is amplified by the type 2 deiodinase (D2) activation of thyroxine to T3. Global inactivation of the Dio2 gene impairs skeletal muscle (SKM) differentiation and regeneration in response to muscle injury. Given that newborn and adult mice with late developmental SKM Dio2 disruption do not develop a significant phenotype, it was hypothesized that D2 plays an early role in this process.This was tested in mice with SKM disruption of Dio2 driven by two early developmental promoters: MYF5 and MYOD.MYF5 myoblasts in culture differentiate normally into myotubes, despite loss of almost all D2 activity. Dio2 mRNA levels in developing SKM obtained from MYF5-D2KO embryos (E18.5) were about 54% of control littermates, but the expression of the T3-responsive genes Myh1 and 7 and Atp2a1 and 2 were not affected. In MYF5-D2KO and MYOD-D2KO neonatal hind-limb muscle, the expression of Myh1 and 7 and Atp2a2 remained unaffected, despite 60-70% loss in D2 activity and/or mRNA. Only in MYOD-D2KO neonatal muscle was there a 40% reduction in Atp2a1 mRNA. Postnatal growth of both mouse models and SKM function as assessed by exercise capacity and measurement of muscle strength were normal. Furthermore, an analysis of the adult soleus revealed no changes in the expression of T3-responsive genes, except for an about 18% increase in MYOD-D2KO SOL Myh7 mRNA.Two mouse models of early developmental disruption of Dio2 in myocyte precursor exhibit no significant SKM phenotype.

Key points In skeletal muscle, physical exercise and thyroid hormone mediate the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1a) expression that is crucial to skeletal muscle mitochondrial function. The expression of type 2 deiodinase (D2), which activates thyroid hormone in skeletal muscle is upregulated by acute treadmill exercise through a β-adrenergic receptor-dependent mechanism. Pharmacological block of D2 or disruption of the Dio2 gene in skeletal muscle fibres impaired acute exercise-induced PGC-1a expression. Dio2 disruption also impaired muscle PGC-1a expression and mitochondrial citrate synthase activity in chronically exercised mice. Abstract Thyroid hormone promotes expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1a), which mediates mitochondrial biogenesis and oxidative capacity in skeletal muscle (SKM). Skeletal myocytes express the type 2 deiodinase (D2), which generates 3,5,3′-triiodothyronine (T3), the active thyroid hormone. To test whether D2-generated T3 plays a role in exercise-induced PGC-1a expression, male rats and mice with SKM-specific Dio2 inactivation (SKM-D2KO or MYF5-D2KO) were studied. An acute treadmill exercise session (20 min at 70–75% of maximal aerobic capacity) increased D2 expression/activity (1.5- to 2.7-fold) as well as PGC-1a mRNA levels (1.5- to 5-fold) in rat soleus muscle and white gastrocnemius muscle and in mouse soleus muscle, which was prevented by pretreatment with 1 mg (100 g body weight)−1 propranolol or 6 mg (100 g body weight)−1 iopanoic acid (5.9- vs. 2.8-fold; P

The firefly luciferase reporter protein is a crucial tool for studies targeting a broad range of biological questions. Importantly, luciferase assays are also widely used to explore mechanisms underlying thyroid hormone dependent regulation of gene expression. However, it was demonstrated that the firefly luciferase reporter is subject to triiodothyronine (T3)-evoked, promoter independent downregulation that is mediated by the thyroid hormone receptor. Since this effect can interfere with readout accuracy, the study aimed to find luciferase reporters that are not susceptible to this phenomenon.Luciferase reporter constructs were generated under the control of a minimal thymidine kinase (TK) promoter and transiently transfected into JEG-3 cells to test their activity upon T3 treatment.Activity of the TK-(dCpG)Luc encoding a synthetic (dCpG)Luciferase and TK-NanoLuc expressing the NanoLuc reporter was not significantly changed by T3 treatment while the firefly luciferase control was suppressed by ∼2.6-fold. T3 also downregulated the activity of Renilla luciferase by ∼30%.Novel types of luciferase reporters, especially the synthetic (dCpG)Luciferase, can be more accurate to study T3-regulated gene expression than the classical firefly luciferase reporter. Renilla luciferase, a popular transfection control of dual luciferase assays, should be used with caution in conditions with T3 treatment.

Thyroid hormone binds to nuclear receptors and regulates gene transcription. Here we report that in mice, at around the first day of life, there is a transient surge in hepatocyte type 2 deiodinase (D2) that activates the prohormone thyroxine to the active hormone triiodothyronine, modifying the expression of ∼165 genes involved in broad aspects of hepatocyte function, including lipid metabolism. Hepatocyte-specific D2 inactivation (ALB-D2KO) is followed by a delay in neonatal expression of key lipid-related genes and a persistent reduction in peroxisome proliferator-activated receptor-γ expression. Notably, the absence of a neonatal D2 peak significantly modifies the baseline and long-term hepatic transcriptional response to a high-fat diet (HFD). Overall, changes in the expression of approximately 400 genes represent the HFD response in control animals toward the synthesis of fatty acids and triglycerides, whereas in ALB-D2KO animals, the response is limited to a very different set of only approximately 200 genes associated with reverse cholesterol transport and lipase activity. A whole genome methylation profile coupled to multiple analytical platforms indicate that 10-20% of these differences can be related to the presence of differentially methylated local regions mapped to sites of active/suppressed chromatin, thus qualifying as epigenetic modifications occurring as a result of neonatal D2 inactivation. The resulting phenotype of the adult ALB-D2KO mouse is dramatic, with greatly reduced susceptibility to diet-induced steatosis, hypertriglyceridemia, and obesity.

The current treatment for patients with hypothyroidism is levothyroxine (L-T4) along with normalization of serum thyroid-stimulating hormone (TSH). However, normalization of serum TSH with L-T4 monotherapy results in relatively low serum 3,5,3′-triiodothyronine (T3) and high serum thyroxine/T3 (T4/T3) ratio. In the hypothalamus-pituitary dyad as well as the rest of the brain, the majority of T3 present is generated locally by T4 deiodination via the type 2 deiodinase (D2); this pathway is self-limited by ubiquitination of D2 by the ubiquitin ligase WSB-1. Here, we determined that tissue-specific differences in D2 ubiquitination account for the high T4/T3 serum ratio in adult thyroidectomized (Tx) rats chronically implanted with subcutaneous L-T4 pellets. While L-T4 administration decreased whole-body D2-dependent T4 conversion to T3, D2 activity in the hypothalamus was only minimally affected by L-T4. In vivo studies in mice harboring an astrocyte-specific Wsb1 deletion as well as in vitro analysis of D2 ubiquitination driven by different tissue extracts indicated that D2 ubiquitination in the hypothalamus is relatively less. As a result, in contrast to other D2-expressing tissues, the hypothalamus is wired to have increased sensitivity to T4. These studies reveal that tissue-specific differences in D2 ubiquitination are an inherent property of the TRH/TSH feedback mechanism and indicate that only constant delivery of L-T4 and L-T3 fully normalizes T3-dependent metabolic markers and gene expression profiles in Tx rats.

Type 2 deiodinase (D2) converts the prohormone thyroxine (T4) to the metabolically active molecule 3,5,3′-triiodothyronine (T3), but its global inactivation unexpectedly lowers the respiratory exchange rate (respiratory quotient [RQ]) and decreases food intake. Here we used FloxD2 mice to generate systemically euthyroid fat-specific (FAT), astrocyte-specific (ASTRO), or skeletal-muscle-specific (SKM) D2 knockout (D2KO) mice that were monitored continuously. The ASTRO-D2KO mice also exhibited lower diurnal RQ and greater contribution of fatty acid oxidation to energy expenditure, but no differences in food intake were observed. In contrast, the FAT-D2KO mouse exhibited sustained (24 h) increase in RQ values, increased food intake, tolerance to glucose, and sensitivity to insulin, all supporting greater contribution of carbohydrate oxidation to energy expenditure. Furthermore, FAT-D2KO animals that were kept on a high-fat diet for 8 weeks gained more body weight and fat, indicating impaired brown adipose tissue (BAT) thermogenesis and/or inability to oxidize the fat excess. Acclimatization of FAT-D2KO mice at thermoneutrality dissipated both features of this phenotype. Muscle D2 does not seem to play a significant metabolic role given that SKM-D2KO animals exhibited no phenotype. The present findings are unique in that they were obtained in systemically euthyroid animals, revealing that brain D2 plays a dominant albeit indirect role in fatty acid oxidation via its sympathetic control of BAT activity. D2-generated T3 in BAT accelerates fatty acid oxidation and protects against diet-induced obesity.

Background: An in-depth understanding of the fundamental principles that regulate thyroid hormone homeostasis is critical for the development of new diagnostic and treatment approaches for patients with thyroid disease. Summary: Important clinical practices in use today for the treatment of patients with hypothyroidism, hyperthyroidism, or thyroid cancer are the result of laboratory discoveries made by scientists investigating the most basic aspects of thyroid structure and molecular biology. In this document, a panel of experts commissioned by the American Thyroid Association makes a series of recommendations related to the study of thyroid hormone economy and action. These recommendations are intended to promote standardization of study design, which should in turn increase the comparability and reproducibility of experimental findings. Conclusions: It is expected that adherence to these recommendations by investigators in the field will facilitate progress towards a better understanding of the thyroid gland and thyroid hormone dependent processes.

Type II deiodinase (D2) activates thyroid hormone by converting thyroxine (T4) to 3,5,3′-triiodothyronine (T3). This allows plasma T4 to signal a negative feedback loop that inhibits production of thyrotropin-releasing hormone (TRH) in the mediobasal hypothalamus (MBH) and thyroid-stimulating hormone (TSH) in the pituitary. To determine the relative contributions of these D2 pathways in the feedback loop, we developed 2 mouse strains with pituitary- and astrocyte-specific D2 knockdown (pit-D2 KO and astro-D2 KO mice, respectively). The pit-D2 KO mice had normal serum T3 and were systemically euthyroid, but exhibited an approximately 3-fold elevation in serum TSH levels and a 40% reduction in biological activity. This was the result of elevated serum T4 that increased D2-mediated T3 production in the MBH, thus decreasing Trh mRNA. That tanycytes, not astrocytes, are the cells within the MBH that mediate T4-to-T3 conversion was defined by studies using the astro-D2 KO mice. Despite near-complete loss of brain D2, tanycyte D2 was preserved in astro-D2 KO mice at levels that were sufficient to maintain both the T4-dependent negative feedback loop and thyroid economy. Taken together, these data demonstrated that the hypothalamic-thyroid axis is wired to maintain normal plasma T3 levels, which is achieved through coordination of T4-to-T3 conversion between thyrotrophs and tanycytes.

Interleukin (IL)-6 deficient mice develop mature-onset obesity. Furthermore, i.c.v. administration of IL-6 increases energy expenditure, suggesting that IL-6 centrally regulates energy homeostasis. To investigate whether it would be possible for IL-6 to directly influence the energy homeostasis via hypothalamic regulation in humans and rodents, we mapped the distribution of the ligand binding IL-6 receptor α (IL-6Rα) in this brain region. In the human hypothalamus, IL-6Rα-immunoreactivity was detected in perikarya and first-order dendrites of neurones. The IL-6Rα-immunoreactive (-IR) neurones were observed posterior to the level of the interventricular foramen. There, IL-6Rα-IR neurones were located in the lateral hypothalamic, perifornical, dorsal and posterior hypothalamic areas, the hypothalamic dorsomedial nucleus and in the zona incerta. In the caudal part of the hypothalamus, the density of the IL-6Rα-IR neurones gradually increased. Double-labelling immunofluorescent studies demonstrated that IL-6Rα immunoreactivity was localised in the same neurones as the orexigenic neuropeptide, melanin-concentrating hormone (MCH). By contrast, IL-6Rα-immunoreactivity was not observed in the orexin B-IR neurones. To determine whether the observed expression of IL-6Rα is evolutionary conserved, we studied the co-localisation of IL-6Rα with MCH and orexin in the mouse hypothalamus, where IL-6Rα-immunoreactivity was present in numerous MCH-IR and orexin-IR neurones. Our data demonstrate that the MCH neurones of the human hypothalamus, as well as the MCH and orexin neurones of the mouse hypothalamus, contain IL-6Rα. This opens up the possibility that IL-6 influences the energy balance through the MCH neurones in humans, and both MCH and orexin neurones in mice.

Thyroid hormone receptor (TR) and liver X-receptor (LXR) are the master regulators of lipid metabolism. Remarkably, a mouse with a targeted deletion of both LXRα and LXRβ is resistant to western diet-induced obesity, and exhibits ectopic liver expression of the thyroid hormone activating type 2 deiodinase (D2). We hypothesized that LXR/retinoid X-receptor (RXR) signaling inhibits hepatic D2 expression, and studied this using a luciferase reporter containing the human DIO2 (hDIO2) promoter in HepG2 cells. Given that, in contrast to mammals, the chicken liver normally expresses D2, the chicken DIO2 (cDIO2) promoter was also studied. 22(R)-OH-cholesterol negatively regulated hDIO2 in a dose-dependent manner (100 μM, approximately twofold), while it failed to affect the cDIO2 promoter. Truncations in the hDIO2 promoter identified the region −901 to −584 bp as critical for negative regulation. We also investigated if 9-cis retinoic acid (9-cis RA), the ligand for the heterodimeric partner of TR and LXR, RXR, could regulate the hDIO2 promoter. Notably, 9-cis RA repressed the hDIO2 luciferase reporter (1 μM, approximately fourfold) in a dose-dependent manner, while coexpression of an inactive mutant RXR abolished this effect. However, it is unlikely that RXR homodimers mediate the repression of hDIO2 since mutagenesis of a DR-1 at −506 bp did not interfere with 9-cis RA-mediated repression. Our data indicate that hDIO2 transcription is negatively regulated by both 22(R)-OH-cholesterol and 9-cis RA, which is consistent with LXR/RXR involvement. In vivo, the inhibition of D2-mediated tri-iodothyronine (T3) production by cholesterol/9-cis RA could function as a feedback loop, given that T3 decreases hepatic cholesterol levels.

Thyroid hormone activation is catalyzed by two deiodinases, D1 and D2. Whereas D1 is a stable plasma membrane protein, D2 is resident in the endoplasmic reticulum (ER) and has a 20-min half-life due to selective ubiquitination and proteasomal degradation. Here we have shown that stable retention explains D2 residency in the ER, a mechanism that is nevertheless over-ridden by fusion to the long-lived plasma membrane protein, sodium-iodine symporter. Fusion to D2, but not D1, dramatically shortened sodium-iodine symporter half-life through a mechanism dependent on an 18-amino acid D2-specific instability loop. Similarly, the D2-specific loop-mediated protein destabilization was also observed after D2, but not D1, was fused to the stable ER resident protein SEC62. This indicates that the instability loop in D2, but not its subcellular localization, is the key determinant of D2 susceptibility to ubiquitination and rapid turnover rate. Our data also show that the 6 N-terminal amino acids, but not the 12 C-terminal ones, are the ones required for D2 recognition by WSB-1.

The type 3 iodothyronine selenodeiodinase (D3) is an integral membrane protein that inactivates thyroid hormones. By using immunofluorescence cytochemistry confocal microscopy of live or fixed cells transiently expressing FLAG-tagged human D3 or monkey hepatocarcinoma cells expressing endogenous D3, we identified D3 in the plasma membrane. It co-localizes with Na,K-ATPase alpha, with the early endosomal marker EEA-1 and clathrin, but not with two endoplasmic reticulum resident proteins. Most of the D3 molecule is extracellular and can be biotinylated with a cell-impermeant probe. There is constant internalization of D3 that is blocked by sucrose or methyl-beta-cyclodextrin-containing medium. Exposing cells to a weak base such as primaquine increases the pool of internalized D3, suggesting that D3 is recycled between plasma membrane and early endosomes. Such recycling could account for the much longer half-life of D3 (12 h) than the thyroxine activating members of the selenodeiodinase family, type 1 (D1; 8 h) or type 2 (D2; 2 h) deiodinase. The extracellular location of D3 gives ready access to circulating thyroid hormones, explaining its capacity for rapid inactivation of circulating thyroxine and triiodothyronine in patients with hemangiomas and its blockade of the access of maternal thyroid hormones to the human fetus.

Thyroid hormone plays a crucial role in the development and function of the nervous system. In order to bind to its nuclear receptor and regulate gene transcription thyroxine needs to be activated in the brain. This activation occurs via conversion of thyroxine to T3, which is catalyzed by the type 2 iodothyronine deiodinase (D2) in glial cells, in astrocytes, and tanycytes in the mediobasal hypothalamus. We discuss how thyroid hormone affects glial cell function followed by an overview on the fine-tuned regulation of T3 generation by D2 in different glial subtypes. Recent evidence on the direct paracrine impact of glial D2 on neuronal gene expression underlines the importance of glial-neuronal interaction in thyroid hormone regulation as a major regulatory pathway in the brain in health and disease.

The cDNA of an uncoupling protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird uncoupling protein (HmUCP) cDNA was amplified from pectoral muscle (flight muscle) using RT-PCR and primers for conserved domains of various known UCP homologs. The rapid amplification of cDNA ends (RACE) method was used to complete the cloning of the 5' and 3' ends of the open reading frame. The HmUCP coding region contains 915 nucleotides, and the deduced protein sequence consists of 304 amino acids, being approximately 72, 70, and 55% identical to human UCP3, UCP2, and UCP1, respectively. The uncoupling activity of this novel protein was characterized in yeast. In this expression system, the 12CA5-tagged HmUCP fusion protein was detected by Western blot in the enriched mitochondrial fraction. Similarly to rat UCP1, HmUCP decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3',3-dihexyloxacarbocyanine iodide. The HmUCP mRNA is primarily expressed in skeletal muscle, but high levels can also be detected in heart and liver, as assessed by Northern blot analysis. Lowering the room's temperature to 12-14 degrees C triggered the cycle torpor/rewarming, typical of hummingbirds. Both in the pectoral muscle and heart, HmUCP mRNA levels were 1.5- to 3.4-fold higher during torpor. In conclusion, this is the first report of an UCP homolog in birds. The data indicate that HmUCP has the potential to function as an UCP and could play a thermogenic role during rewarming.

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. Supplementary Information The online version contains supplementary material available at 10.1007/s00429-020-02189-1.