Probes for INS

Abstract

Objective

The prevalence of obesity and related co-morbidities is reaching pandemic proportions. Today, the most effective obesity treatments are glucagon-like peptide 1 (GLP-1) analogs and bariatric surgery. Interestingly, both intervention paradigms have been associated with adaptive growth responses in the gut; however, intestinotrophic mechanisms associated with or secondary to medical or surgical obesity therapies are poorly understood. Therefore, the objective of this study was to assess the local basal endogenous and pharmacological intestinotrophic effects of glucagon-like peptides and bariatric surgery in mice.

Methods

We used in situ hybridization to provide a detailed and comparative anatomical map of the local distribution of GLP-1 receptor (Glp1r), GLP-2 receptor (Glp2r), and preproglucagon (Gcg) mRNA expression throughout the mouse gastrointestinal tract. Gut development in GLP-1R-, GLP-2R-, or GCG-deficient mice was compared to their corresponding wild-type controls, and intestinotrophic effects of GLP-1 and GLP-2 analogs were assessed in wild-type mice. Lastly, gut volume was determined in a mouse model of vertical sleeve gastrectomy (VSG).

Results

Comparison of Glp1r, Glp2r, and Gcg mRNA expression indicated a widespread, but distinct, distribution of these three transcripts throughout all compartments of the mouse gastrointestinal tract. While mice null for Glp1r or Gcg showed normal intestinal morphology, Glp2r^−/− mice exhibited a slight reduction in small intestinal mucosa volume. Pharmacological treatment with GLP-1 and GLP-2 analogs significantly increased gut volume. In contrast, VSG surgery had no effect on intestinal morphology.

Conclusion

The present study indicates that the endogenous preproglucagon system, exemplified by the entire GCG gene and the receptors for GLP-1 and GLP-2, does not play a major role in normal gut development in the mouse. Furthermore, elevation in local intestinal and circulating levels of GLP-1 and GLP-2 achieved after VSG has limited impact on intestinal morphometry. Hence, although exogenous treatment with GLP-1 and GLP-2 analogs enhances gut growth, the contributions of endogenously-secreted GLP-1 and GLP-2 to gut growth may be more modest and highly context-dependent.

The activation of resident microglial cells, alongside the infiltration of peripheral macrophages, are key neuroinflammatory responses to traumatic brain injury (TBI) that are directly associated with neuronal death. Sexual disparities in response to TBI have been previously reported; however it is unclear whether a sex difference exists in neuroinflammatory progression after TBI. We exposed male and female mice to moderate-to-severe controlled cortical impact injury and studied glial cell activation in the acute and chronic stages of TBI using immunofluorescence and in situ hybridization analysis. We found that the sex response was completely divergent up to 7 days postinjury. TBI caused a rapid and pronounced cortical microglia/macrophage activation in male mice with a prominent activated phenotype that produced both pro- (IL-1β and TNFα) and anti-inflammatory (Arg1 and TGFβ) cytokines with a single-phase, sustained peak from 1 to 7 days. In contrast, TBI caused a less robust microglia/macrophage phenotype in females with biphasic pro-inflammatory response peaks at 4 h and 7 days, and a delayed anti-inflammatory mRNA peak at 30 days. We further report that female mice were protected against acute cell loss after TBI, with male mice demonstrating enhanced astrogliosis, neuronal death, and increased lesion volume through 7 days post-TBI. Collectively, these findings indicate that TBI leads to a more aggressive neuroinflammatory profile in male compared with female mice during the acute and subacute phases postinjury. Understanding how sex affects the course of neuroinflammation following brain injury is a vital step toward developing personalized and effective treatments for TBI.

Glutamatergic neurons that express pre-proglucagon (PPG) and are immunopositive (+) for glucagon-like peptide-1 (i.e., GLP-1+ neurons) are located within the caudal nucleus of the solitary tract (cNTS) and medullary reticular formation in rats and mice. GLP-1 neurons give rise to an extensive central network in which GLP-1 receptor (R) signaling suppresses food intake, attenuates rewarding, increases avoidance, and stimulates stress responses, partly via . GLP-1R signaling within the cNTS. In mice, noradrenergic (A2) cNTS neurons express GLP-1R, whereas PPG neurons do not. In the present study, confocal microscopy in rats confirmed that prolactin-releasing peptide (PrRP)+ A2 neurons are closely apposed by GLP-1+ axonal varicosities. Surprisingly, GLP-1+ appositions were also observed on dendrites of PPG/GLP-1+ neurons in both species, and electron microscopy in rats revealed that GLP-1+ boutons form asymmetric synaptic contacts with GLP-1+ dendrites. However, RNAscope confirmed that rat GLP-1 neurons do not express GLP-1R mRNA. Similarly, Ca2+ imaging of somatic and dendritic responses in mouse ex vivo slices confirmed that PPG neurons do not respond directly to GLP-1, and a mouse cross-breeding strategy revealed that fewer than 1% of PPG neurons co-express GLP-1R. Collectively, these data suggest that GLP-1R signaling pathways modulate the activity of PrRP+ A2 neurons, and also reveal a local "feed-forward" synaptic network among GLP-1 neurons that apparently does not utilize GLP-1R signaling. This local GLP-1 network may instead use glutamatergic signaling to facilitate dynamic and potentially selective recruitment of GLP-1 neural populations that shape behavioral and physiological responses to internal and external challenges.

Objective

Adult obesity risk is influenced by alterations to fetal and neonatal environments. Modifying neonatal gut or neurohormone signaling pathways can have negative metabolic consequences in adulthood. Here we characterize the effect of neonatal activation of glucagon like peptide-1 (GLP-1) receptor (GLP1R) signaling on adult adiposity and metabolism.

Methods

Wild type C57BL/6 mice were injected with 1 nmol/kg Exendin-4 (Ex-4), a GLP1R agonist, for 6 consecutive days after birth. Growth, body composition, serum analysis, energy expenditure, food intake, and brain and fat pad histology and gene expression were assessed at multiple time points through 42 weeks. Similar analyses were conducted in a Glp1r conditional allele crossed with a Sim1Cre deleter strain to produce Sim1Cre;Glp1r^loxP/loxP mice and control littermates.

Results

Neonatal administration of Ex-4 reduced adult body weight and fat mass, increased energy expenditure, and conferred protection from diet-induced obesity in female mice. This was associated with induction of brown adipose genes and increased noradrenergic fiber density in parametrial white adipose tissue (WAT). We further observed durable alterations in orexigenic and anorexigenic projections to the paraventricular hypothalamic nucleus (PVH). Genetic deletion of Glp1r in the PVH by Sim1-Cre abrogated the impact of neonatal Ex-4 on adult body weight, WAT browning, and hypothalamic architecture.

Conclusion

These observations suggest that the acute activation of GLP1R in neonates durably alters hypothalamic architecture to limit adult weight gain and adiposity, identifying GLP1R as a therapeutic target for obesity prevention.

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-1RKDΔNkx2.1cre). We also generated mice lacking Glp1r expression specifically in two GLP-1RA-responsive hypothalamic feeding nuclei/cell types, the paraventricular nucleus (GLP-1RKDΔSim1cre) and proopiomelanocortin neurons (GLP-1RKDΔPOMCcre). Chow -fed GLP-1RKDΔNkx2.1cre mice exhibited increased food intake and energy expenditure with no net effect on body weight. When fed a high fat diet (HFD), these mice exhibited normal food intake but elevated energy expenditure, yielding reduced weight gain. None of these phenotypes were observed in GLP-1RKDΔSim1creand GLP-1RKDΔPOMCcre 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-1RKDΔNkx2.1cre mice, but this effect was attenuated upon HFD feeding. In sum, classical homeostatic control regions are sufficient but not individually necessary for the effects of GLP-1RA on nutrient homeostasis.

Light Sheet Fluorescence Microscopy (LSFM) of whole organs, in particular the brain, offers a plethora of biological data imaged in 3D. This technique is however often hindered by cumbersome non-automated analysis methods. Here we describe an approach to fully automate the analysis by integrating with data from the Allen Institute of Brain Science (AIBS), to provide precise assessment of the distribution and action of peptide-based pharmaceuticals in the brain. To illustrate this approach, we examined the acute central nervous system effects of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide. Peripherally administered liraglutide accessed the hypothalamus and brainstem, and led to activation in several brain regions of which most were intersected by projections from neurons in the lateral parabrachial nucleus. Collectively, we provide a rapid and unbiased analytical framework for LSFM data which enables quantification and exploration based on data from AIBS to support basic and translational discovery.

Understanding the neural framework behind appetite control is fundamental to developing effective therapies to combat the obesity epidemic. The paraventricular hypothalamus (PVH) is critical for appetite regulation, yet, the real-time, physiological response properties of PVH neurons to nutrients are unknown. Using a combination of fiber photometry, electrophysiology, immunohistochemistry, and neural manipulation strategies, we determined the population dynamics of four molecularly delineated PVH subsets implicated in feeding behavior: glucagon-like peptide 1 receptor (PVHGlp1r), melanocortin-4 receptor (PVHMc4r), oxytocin (PVHOxt), and corticotropin-releasing hormone (PVHCrh). We identified both calorie- and state-dependent sustained activity increases and decreases in PVHGlp1r and PVHCrh populations, respectively, while observing transient bulk changes of PVHMc4r, but no response in PVHOxt, neurons to food. Furthermore, we highlight the role of PVHGlp1r neurons in orchestrating acute feeding behavior, independent of the anti-obesity drug liraglutide, and demonstrate the indispensability of PVHGlp1r and PVHMc4r, but not PVHOxt or PVHCrh neurons, in body weight maintenance.