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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.

Large-scale mass spectrometry-based peptidomics for drug discovery is relatively unexplored because of challenges in peptide degradation and identification following tissue extraction. Here we present a streamlined analytical pipeline for large-scale peptidomics. We developed an optimized sample preparation protocol to achieve fast, reproducible and effective extraction of endogenous peptides from sub-dissected organs such as the brain, while diminishing unspecific protease activity. Each peptidome sample was analysed by high-resolution tandem mass spectrometry and the resulting data set was integrated with publically available databases. We developed and applied an algorithm that reduces the peptide complexity for identification of biologically relevant peptides. The developed pipeline was applied to rat hypothalamus and identifies thousands of neuropeptides and their post-translational modifications, which is combined in a resource format for visualization, qualitative and quantitative analyses. Neuropeptide research is challenged by technical difficulties in identifying new bioactive peptides. Here the authors present an analytical pipeline for large-scale peptidomics applied to the rat hypothalamus, identifying thousands of endogenous neuropeptides and their post-translational modifications.

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.

The brainstem dorsal vagal complex (DVC) is known to regulate energy balance and is the target of appetite-suppressing hormones, such as glucagon-like peptide 1 (GLP-1). Here we provide a comprehensive genetic map of the DVC and identify neuronal populations that control feeding. Combining bulk and single-nucleus gene expression and chromatin profiling of DVC cells, we reveal 25 neuronal populations with unique transcriptional and chromatin accessibility landscapes and peptide receptor expression profiles. GLP-1 receptor (GLP-1R) agonist administration induces gene expression alterations specific to two distinct sets of Glp1r neurons—one population in the area postrema and one in the nucleus of the solitary tract that also expresses calcitonin receptor ( Calcr ). Transcripts and regions of accessible chromatin near obesity-associated genetic variants are enriched in the area postrema and the nucleus of the solitary tract neurons that express Glp1r and/or Calcr , and activating several of these neuronal populations decreases feeding in rodents. Thus, DVC neuronal populations associated with obesity predisposition suppress feeding and may represent therapeutic targets for obesity. Ludwig et al. map transcription and chromatin accessibility in single cells across the brainstem dorsal vagal complex, thereby identifying neuronal populations, including some that control feeding.

In rodent models of type 2 diabetes (T2D), sustained remission of hyperglycemia can be induced by a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1), and the mediobasal hypothalamus (MBH) was recently implicated as the brain area responsible for this effect. To better understand the cellular response to FGF1 in the MBH, we sequenced >79,000 single-cell transcriptomes from the hypothalamus of diabetic Lep ob/ob mice obtained on Days 1 and 5 after icv injection of either FGF1 or vehicle. A wide range of transcriptional responses to FGF1 was observed across diverse hypothalamic cell types, with glial cell types responding much more robustly than neurons at both time points. Tanycytes and ependymal cells were the most FGF1-responsive cell type at Day 1, but astrocytes and oligodendrocyte lineage cells subsequently became more responsive. Based on histochemical and ultrastructural evidence of enhanced cell-cell interactions between astrocytes and Agrp neurons (key components of the melanocortin system), we performed a series of studies showing that intact melanocortin signaling is required for the sustained antidiabetic action of FGF1. These data collectively suggest that hypothalamic glial cells are leading targets for the effects of FGF1 and that sustained diabetes remission is dependent on intact melanocortin signaling. In rodent models of type 2 diabetes, sustained remission of hyperglycemia can be induced by FGF1 action in the mediobasal hypothalamus. Here, the authors show that FGF1-injection is followed by marked changes in glial cell populations and that the sustained glycemic response is dependent on intact melanocortin signaling.

AimsTo investigate the influence of the dose in the FITC-Dextran 4kDa (FD-4) permeability test in an obese mouse model, we tested the bodyweight dose regimen and a lean body mass-based dose regimen in high fat diet (HFD) mice and low fat diet (LFD) mice. We hypothesized that the FD-4 permeation result would be dose-dependent.MethodsThe two dose regimens were compared in HFD and LFD mice. Furthermore, we conducted a dose-response study to test the effect of a low or high dose of FD-4 in weight-stratified lean mice. Gene analysis of tight junctions was also carried out.ResultsThe FD-4 intestinal permeability test was dose-dependent as we found a significant increase in plasma levels of FD-4 in obese mice with the bodyweight dose regimen. However, this difference was not detectable with the lean body mass dose regimen, even with variability-adjusted group sizes. However, the qPCR analysis revealed a decrease in tight junction gene expression in obese mice. Furthermore, we found a dose-dependent significant increase in FD-4 measured in plasma samples in lean mice. No significant difference in intestinal weight was observed between lean and obese mice.ConclusionEvaluation of the intestinal permeability by FD-4 with the typical bodyweight dose regimen in obese mice will be confounded by the significant difference in dose given when compared to a lean control group. If the test dose is based on lean body mass, no significant difference in intestinal permeability is observed, even with large group sizes. Furthermore, we showed a dose-dependent difference in plasma FD-4 levels in lean mice. Therefore, we conclude that the dose should be based on lean body mass for the FD-4 permeability test if mice with considerable obesity differences are to be compared or to use another test with fixed doses.

One of the major histopathological hallmarks of Alzheimer's disease (AD) is cerebral deposits of extracellular β-amyloid peptides. Preclinical studies have pointed to glucagon-like peptide 1 (GLP-1) receptors as a potential novel target in the treatment of AD. GLP-1 receptor agonists, including exendin-4 and liraglutide, have been shown to promote plaque-lowering and mnemonic effects of in a number of experimental models of AD. Transgenic mouse models carrying genetic mutations of amyloid protein precursor (APP) and presenilin-1 (PS1) are commonly used to assess the pharmacodynamics of potential amyloidosis-lowering and pro-cognitive compounds. In this study, effects of long-term liraglutide treatment were therefore determined in two double APP/PS1 transgenic mouse models of Alzheimer's disease carrying different clinical APP/PS1 mutations, i.e. the 'London' (hAPPLon/PS1A246E) and 'Swedish' mutation variant (hAPPSwe/PS1ΔE9) of APP, with co-expression of distinct PS1 variants. Liraglutide was administered in 5 month-old hAPPLon/PS1A246E mice for 3 months (100 or 500 ng/kg/day, s.c.), or 7 month-old hAPPSwe/PS1ΔE9 mice for 5 months (500 ng/kg/day, s.c.). In both models, regional plaque load was quantified throughout the brain using stereological methods. Vehicle-dosed hAPPSwe/PS1ΔE9 mice exhibited considerably higher cerebral plaque load than hAPPLon/PS1A246E control mice. Compared to vehicle-dosed transgenic controls, liraglutide treatment had no effect on the plaque levels in hAPPLon/PS1A246E and hAPPSwe/PS1ΔE9 mice. In conclusion, long-term liraglutide treatment exhibited no effect on cerebral plaque load in two transgenic mouse models of low- and high-grade amyloidosis, which suggests differential sensitivity to long-term liraglutide treatment in various transgenic mouse models mimicking distinct pathological hallmarks of AD.

Background The metabolic syndrome encompasses a state of inflammation and metabolic dysfunction, possibly mediated via a disturbed intestinal barrier. Glucagon‐like peptide‐1 receptor agonists (GLP‐1RAs), such as liraglutide, have shown promising anti‐inflammatory effects beyond glucose lowering and weight loss, but the underlying mechanism remains to be elucidated. We hypothesised that GLP‐1RAs improve the intestinal barrier function and overall inflammatory status by direct gene activation in mucus‐secreting Brunner's glands in the mouse duodenum, known for their high density of glucagon‐like peptide‐1 receptors (GLP‐1Rs). Methods Using bulk RNA sequencing, in situ hybridisation, and immunohistochemistry, we analysed the change in the genetic phenotype of mouse Brunner's gland cells following GLP‐1R activation by liraglutide. Results We show that liraglutide induces a novel and robust upregulation of the gene for the Cystic fibrosis transmembrane conductance regulator, Cftr, in Brunner's glands as a part of an overall genetic phenotype involved in ion channel activity, mucus secretion, and hydration via GLP‐1R activation. Additionally, we found a robust upregulation of the genes Muc5b, Il33, Ren1, and Vldlr in Brunner's glands. Conclusion Collectively, our results imply an enhanced mucus response from Brunner's glands following GLP‐1R activation, which might play a role in the effect of GLP‐1. RNA sequencing, in situ hybridisation, and immunohistochemistry show that the glucagon‐like peptide‐1 receptor agonist liraglutide upregulates Cftr, Muc5b, Il33, Ren1, and Vldlr in Brunner's glands of mice.

Liraglutide is a glucagon‐like peptide‐1 receptor (GLP‐1R) agonist marketed for the treatment of type 2 diabetes. Besides lowering blood glucose, liraglutide reduces bodyweight, and has recently also been approved for the obesity indication. Acutely, GLP‐1 markedly reduces gastric emptying, and this effect was previously believed to at least partly explain the effect on bodyweight loss. However, recent studies in both humans and animals have shown that GLP‐1R agonists, such as liraglutide, that lead to pharmacological concentrations for 24 h/day only have a minor effect on gastric emptying; such an effect is unlikely to have lasting effects on appetite reduction. Liraglutide has been shown to have direct effects in the arcuate nucleus of the rodent brain, activating pro‐opiomelanocortin neurons and increasing levels of the cocaine‐ and amphetamine‐stimulated transcript neuropeptide messenger ribonucleic acid, which correlate nicely to clinical studies where liraglutide was shown to increase feelings of satiety. However, despite the lack of a GLP‐1R on agouti‐related peptide/neuropeptide Y neurons, liraglutide also was able to prevent a hunger associated increase in agouti‐related peptide and neuropeptide Y neuropeptide messenger ribonucleic acid, again with a strong correlation to clinical studies that document reduced hunger feelings in patients while taking liraglutide. Studies using fluorescent labeled liraglutide, as well as other GLP‐1R agonists, and analysis using single‐plane illumination microscopy show that such medium‐sized peptide‐based compounds can directly access not only circumventricular organs of the brain, but also directly access discrete regions in the hypothalamus. The direct effects of long‐acting GLP‐1R agonists in the hypothalamus are likely to be an important new pathway in understanding GLP‐1R agonist mediated weight loss. GLP‐1R agonists are marketed for the treatment of type 2 diabetes, and liraglutide is also approved as a weight management agent. Studies using fluorescent labelled liraglutide as well as other GLP‐1R agonists and analysis using single plane illumination microscopy (SPIM) show that such medium size peptide based compounds can directly access not only circumventricular organs of the brain but also directly access discrete regions in the hypothalamus. The direct effects of long‐acting GLP‐1R agonists in the hypothalamus are likely to be an important new pathway in understanding GLP‐1R agonist mediated weight loss.

The GLP‐1 receptor (GLP‐1R) in the kidney is expressed exclusively in vascular smooth muscle cells in arteries and arterioles. Downstream effects of the activation of the renal vascular GLP‐1R are elusive but may involve regulation of the renin‐angiotensin‐aldosterone system (RAAS). The expression of Ren1 in the mouse renal vasculature was investigated by in situ hybridization after a single subcutaneous dose of liraglutide, semaglutide and after repeated injections of liraglutide. Single and repeated exposure to GLP‐1R agonists induced expression of Ren1 in the renal vascular smooth muscle cell compartment compared with vehicle injected controls (p < .0001) for both semaglutide and liraglutide. The present data show a robust induction of Ren1 expression in the vascular smooth muscle cells of the kidney after single and repeated GLP‐1R activation and this renin recruitment may be involved in the effects of GLP‐1R agonist treatment on kidney disease. Single and repeated injections with long acting GLP‐1R agonists leads to induction of Ren1 transcription from VSMC in renal vasculature in mice.