Biology subjects

Beall, C.

Publications and source records attributed to Beall, C..

10 recordsLinked to original sources

bioRxiv2025

Mgl2+ cDC2 triggering of fungal allergic inflammation depends on a spore induced glycolytic shift fuelled by local availability of glucose

Fungal spores are a major cause of severe asthmatic disease. However, the precise events that cause individuals to become sensitised to spores are poorly understood. Mgl2+ type 2 conventional dendritic cells (Mgl2+ cDC2s) are critical in coordinating allergic airway inflammation in mice following repeated exposure to inhaled spores. Yet, whether these DCs are directly acquiring spores from the airway, and the downstream mechanism(s) upon fungal uptake causing DCs to trigger allergic inflammation are unknown. Here we find that spores are acquired by lung DCs after inhalation although these events are rare ([~] 0.5% of the cDC2 population). Transcriptomics on isolated spore+ Mgl2+ cDC2s, compared to spore- Mgl2+ cDC2s from the same environment, revealed that a major consequence of fungal uptake was a boost in metabolic activity. Single-cell metabolic profiling revealed this increase in Mgl2+ cDC2 metabolism upon spore acquisition was fuelled by a glycolytic shift. To pinpoint if nutrient availability and acquisition is an important determinant of this response, mass spectrometry-based metabolomics revealed that, during fungal allergic inflammation, the local airway nutrient environment is altered. To ascertain which of these could be fuelling DC responses, we identified which substrates that feed into glycolysis were crucial. Of these, we found that glucose availability acts as a key rheostat in shaping cDC2 responses to spores. These data highlight a crucial role for glycolytic metabolism in driving cDC2 responses to spores, which is governed by glucose availability, defining novel targets for future therapeutic development of fungal allergic inflammation.

immunology↗

bioRxiv2024

Validation of a refined protocol for mouse oral glucose tolerance testing without gavage

A glucose tolerance test (GTT) is routinely used to assess glucose homeostasis in clinical settings and in preclinical research studies using rodent models. The procedure assesses the ability of the body to clear glucose from the blood in a defined time after a bolus dose. In the human clinical setting, glucose is ingested via voluntary consumption of a glucose-sweetened drink. Typically, in the rodent GTT oral gavage (gavage-oGTT) or (more commonly) intraperitoneal injection (IPGTT) are used to administer the glucose bolus. Although used less frequently, likely due to investigator technical and experience barriers, the former is the more physiologically relevant as it integrates the gastrointestinal tract (GI), including release of key incretin hormones. However, orally gavaging glucose in the GTT is also not without its limitations: gavaging glucose straight into the stomach bypasses potentially critical early glucose-sensing via the mouth (cephalic phase) and associated physiological responses. Furthermore, gavaging is stressful on mice, and this by itself can increase blood glucose levels. We have developed and validated a refined protocol for mouse oral GTT which uses a voluntary oral glucose dosing method, micropipette-guided drug administration (MDA), without the need for water deprivation. This approach is simple and non-invasive. It is less stressful for the mice, as evidenced by lower circulating corticosterone levels 10 minutes after glucose-dosing compared to oral gavage. This is significant for animal and investigator welfare, and importantly minimising the confounding effect of stress on mouse glucose homeostasis. Using a randomised cross-over design, we have validated the MDA approach in the oGTT against oral gavage in male and female C57BL/6J and C57BL/6N mice. We show the ability of this method to detect changes in glucose tolerance in diet-induced obese animals. Compared to oral gavage there was lower inter-animal variation in the MDA-oGTT. In addition to being more representative of the human procedure, the MDA-oGTT is easy and has lower barriers to adoption than the gavage oGTT as it is non-invasive and requires no specialist equipment or operator training. The MDA-oGTT a more clinically representative, accessible, and refined replacement for the gavage-oGTT for mouse metabolic phenotyping, which is simple yet overcomes significant deficiencies in the current standard experimental approaches.

physiology↗

bioRxiv2023

Pharmacological AMP-activated protein kinase activation suppresses low glucose-dependent macrophage migration inhibitory factor release from macrophages

Aims/hypothesisAcute hypoglycemia promotes pro-inflammatory cytokine production, increasing risk for cardiovascular events in diabetes. AMP-activated protein kinase (AMPK) is regulated by and influences production of pro-inflammatory cytokines. We tested the mechanistic role of AMPK in low glucose induced changes in the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF), which is elevated in patients with diabetes. MethodsMacrophage cell line Raw264.7 cells, primary macrophage bone marrow derived macrophages obtained from wild type mice or AMPK {gamma}1 gain-of-function mice were utilized, as were AMPK1/2 knockout mouse embryonic fibroblasts (MEF). Allosteric AMPK activators PF-06409577 and BI-9774 were used, in conjunction with inhibitor SBI-0206965 were also used. We examined changes in protein phosphorylation/expression using western blotting, and protein localisation using immunofluorescence. Metabolic function was assessed using extracellular flux analyses and luciferase-based ATP assay. Cytokine release was quantified by ELISA. Oxidative stress was detected using a fluorescence-based ROS assay, and cell viability was examined using flow cytometry. ResultsMacrophages exposed to low glucose showed a transient and modest activation of AMPK and a metabolic shift towards increased oxidative phosphorylation. Low glucose induced oxidative stress and increased release of macrophage migration inhibitory factor (MIF). Pharmacological activation of AMPK by PF-06409577 and BI-9774 attenuated low glucose-induced MIF release, with a similar trend noted with genetic activation using AMPK{gamma}1 gain-of-function (D316A) mice, which produced a mild effect on low glucose-induced MIF release. Inhibition of NFB signalling diminished MIF release and AMPK activation modestly but significantly reduced low glucose-induced nuclear translocation of NFB. AMPK activation did not alter low glucose-induced oxidative stress in macrophages but application of AMPK inhibitor SBI-0206965 enhanced oxidative stress in macrophages and in AMPK knockout MEFs, suggesting an AMPK-independent mechanism Conclusions/interpretationTaken together, these data indicate that pharmacological AMPK activation suppresses release of MIF from macrophages. This is mediated by reduced activation of NFB signalling in response to low glucose-induced oxidative stress and suggests that pharmacological AMPK activation could be a useful strategy for mitigating hypoglycemia-induced inflammation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/562445v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1d3364borg.highwire.dtl.DTLVardef@1919395org.highwire.dtl.DTLVardef@704923org.highwire.dtl.DTLVardef@1d1cc59_HPS_FORMAT_FIGEXP M_FIG Tweet Low glucose induces pro-inflammatory MIF release from macrophages, and pharmacological AMPK activation suppresses the release of MIF. AMPK/ NFB signalling pathway is involved, which may be a new strategy to attenuate the pro-inflammatory response in hypoglycemia. C_FIG

pharmacology and toxicology↗

bioRxiv2023

Regulation of astrocyte metabolism by mitochondrial translocator protein 18kDa

The mitochondrial translocator protein 18kDa (TSPO) has been linked to a variety of functions from steroidogenesis to regulation of cellular metabolism and is an attractive therapeutic target for chronic CNS inflammation. Studies in the periphery using Leydig cells and hepatocytes, as well as work in microglia, indicate that the function of TSPO may vary between cells depending on their specialised roles. Astrocytes are critical for providing trophic and metabolic support in the brain as part of their role in maintaining brain homeostasis. Recent work has highlighted that TSPO expression increases in astrocytes under inflamed conditions and may drive astrocyte reactivity. However, relatively little is known about the role TSPO plays in regulating astrocyte metabolism and whether this protein is involved in immunometabolic processes in these cells. Using TSPO-deficient (TSPO-/-) mouse primary astrocytes in vitro (MPAs) and a human astrocytoma cell line (U373 cells), we performed metabolic flux analyses. We found that loss of TSPO reduced basal astrocyte respiration and increased the bioenergetic response to glucose reintroduction following glucopenia, while increasing fatty acid oxidation (FAO). Lactate production was significantly reduced in TSPO-/- astrocytes. Co-immunoprecipitation studies in U373 cells revealed that TSPO forms a complex with carnitine palmitoyltransferase 1a, which presents a mechanism wherein TSPO may regulate FAO in astrocytes. Compared to TSPO+/+ cells, inflammation induced by 3h lipopolysaccharide (LPS) stimulation of TSPO-/- MPAs revealed attenuated tumour necrosis factor release, which was enhanced in TSPO-/- MPAs at 24h LPS stimulation. Together these data suggest that while TSPO acts as a regulator of metabolic flexibility in astrocytes, loss of TSPO does not appear to modulate the metabolic response of astrocytes to inflammation, at least in response to the stimulus/time course used in this study.

neuroscience↗

bioRxiv2021

Astrocytes in the dorsal vagal complex are not activated by systemic glucoprivation and their chemogenetic activation does not elicit homeostatic glucoregulatory responses in mice

The dorsal vagal complex (DVC) is a brainstem site regulating diverse aspects of physiology including food intake and blood glucose homeostasis. Astrocytes are purported to play an active role in regulating DVC function and, by extension, physiological parameters. Previous work has demonstrated that DVC astrocytes directly sense hormones that regulate food intake and blood glucose and are critical for their effect. In addition, DVC astrocytes in ex vivo slices respond to low tissue glucose. The response of neurons, including catecholaminergic neurons, to low glucose is conditional on intact astrocyte signalling in slice preparations suggesting astrocytes are possibly the primary sensors of glucose deprivation (glucoprivation). Based on these findings we hypothesised that if DVC astrocytes act as glucoprivation sensors in vivo they would both show a response to systemic glucoprivation and drive physiological responses to restore blood glucose. We found that 2 hours of systemic glucoprivation induced neither FOS nor glial fibrillary acidic protein (GFAP)-immunoreactivity in DVC astrocytes, specifically those in the nucleus of the solitary tract (NTS). Furthermore, we found that while chemogenetic activation of DVC astrocytes suppressed food intake by reducing both meal size and meal number, this manipulation also suppressed food intake under conditions of glucoprivation. Chemogenetic activation of DVC astrocytes did not increase basal blood glucose nor protect against insulin-induced hypoglycaemia. In male mice chemogenetic DVC astrocyte activation did not alter glucose tolerance, in female mice the initial glucose excursion was reduced, suggesting enhanced glucose absorption. Taken together this suggests that as a whole-population DVC astrocytes do not function as glucoprivation sensors in vivo in mice. Instead, we propose that DVC astrocytes play an indispensable, homeostatic role to maintain the function of glucoregulatory neuronal circuitry.

neuroscience↗

bioRxiv2020

Analysis of the transcriptome and DNA methylome in response to acute and recurrent low glucose in human primary astrocytes

Aims/hypothesisRecurrent hypoglycaemia (RH) is a major side-effect of intensive insulin therapy for people with diabetes. Changes in hypoglycaemia sensing by the brain contribute to the development of impaired counterregulatory responses to and awareness of hypoglycaemia. Little is known about the intrinsic changes in human astrocytes in response to acute and recurrent low glucose (RLG) exposure. MethodsHuman primary astrocytes (HPA) were exposed to zero, one, three or four bouts of low glucose (0.1 mmol/l) for three hours per day for four days to mimic RH. On the fourth day, DNA and RNA were collected. Differential gene expression and ontology analyses were performed using DESeq2 and GOseq respectively. DNA methylation was assessed using the Infinium MethylationEPIC BeadChip platform. Results24 differentially expressed genes (DEGs) were detected (after correction for multiple comparisons). One bout of low glucose exposure had the largest effect on gene expression. Pathway analyses revealed that endoplasmic-reticulum (ER) stress-related genes such as HSPA5, XBP1, and MANF, involved in the unfolded protein response (UPR), were all significantly increased following LG exposure, which was diminished following RLG. There was little correlation between differentially methylated positions and changes in gene expression yet the number of bouts of LG exposure produced distinct methylation signatures. Conclusions/interpretationThese data suggest that exposure of human astrocytes to transient LG triggers activation of genes involved in the UPR linked to endoplasmic reticulum (ER) stress. Following RLG, the activation of UPR related genes was diminished, suggesting attenuated ER stress. This may be mediated by metabolic adaptations to better preserve intracellular and/or ER ATP levels, but this requires further investigation.

neuroscience↗

bioRxiv2019

Brain permeable AMPK activator R481 raises glycemia by autonomic nervous system activation and amplifies the counterregulatory response to hypoglycemia in rats

AMP-activated protein kinase (AMPK) is a critical cellular and whole body energy sensor activated by energy stress, including hypoglycemia, which is frequently experienced by people with diabetes. Previous studies using direct delivery of an AMPK activator to the ventromedial hypothalamus (VMH) in rodents increased hepatic glucose production. Moreover, recurrent glucoprivation in the hypothalamus leads to blunted AMPK activation and defective hormonal responses to subsequent hypoglycemia. These data suggest that amplifying AMPK activation may prevent or reduce frequency hypoglycemia in diabetes. We used a novel brain-permeable AMPK activator, R481, which potently increased AMPK phosphorylation in vitro. R481 significantly increased peak glucose levels during glucose tolerance tests in rats, which were attenuated by treatment with AMPK inhibitor SBI-0206965 and completely abolished by blockade of the autonomic nervous system. This occurred without altering insulin sensitivity measured by hyperinsulinemic-euglycemic clamps. Endogenous insulin secretion was not altered by R481 treatment. During hyperinsulinemic-hypoglycemic clamp studies, R481 treatment reduced exogenous glucose requirements and amplified peak glucagon levels during hypoglycemia. These data demonstrate that peripheral administration of the brain permeable AMPK activator R481 amplifies the counterregulatory response to hypoglycemia in rats, which could have clinical relevance for prevention of hypoglycemia.

neuroscience↗

bioRxiv2019

Regulation of food intake by astrocytes in the brainstem dorsal vagal complex

Food intake is controlled by the coordinated action of numerous brain regions but a complete understanding remains elusive. Of these brain regions the brainstem dorsal vagal complex (DVC) is the first site for integration of visceral synaptic and hormonal cues that act to inhibit food intake. The DVC consists of three nuclei: the nucleus of the solitary tract (NTS), area postrema (AP) and dorsal motor nucleus of the vagus (DMX). Targeted chemogenetic activation of appetite-responsive NTS neuronal populations causes short term decreases in food intake. Astrocytes are a class of glial cell which provide metabolic and structural support to neurons and play an active role in modulating neurotransmission. Within the hypothalamic arcuate nucleus (ARC) astrocytes are regulated by both positive and negative energy balance and express receptors for hormones that influence satiety and hunger. Chemogenetic activation of these ARC astrocytes alters food intake. Since NTS astrocytes respond to vagal stimulation, we hypothesised that they may be involved in mediating satiety. Here we show that NTS astrocytes show plastic alterations in morphology following excess food consumption and that chemogenetic activation of DVC astrocytes causes a decrease in food intake, by recruiting an appetite-inhibiting circuit, without producing aversion. These findings are the first using genetically-targeted manipulation of DVC astrocytes to demonstrate their role in the brains regulation of food intake.

neuroscience↗

bioRxiv2018

The genetic prehistory of the Andean highlands 7,000 Years BP though European contact

The peopling of the Andean highlands above 2500m in elevation was a complex process that included cultural, biological and genetic adaptations. Here we present a time series of ancient whole genomes from the Andes of Peru, dating back to 7,000 calendar years before present (BP), and compare them to 64 new genome-wide genetic variation datasets from both high and lowland populations. We infer three significant features: a split between low and high elevation populations that occurred between 9200-8200 BP; a population collapse after European contact that is significantly more severe in South American lowlanders than in highland populations; and evidence for positive selection at genetic loci related to starch digestion and plausibly pathogen resistance after European contact. Importantly, we do not find selective sweep signals related to known components of the human hypoxia response, which may suggest more complex modes of genetic adaptation to high altitude.\n\nOne Sentence SummaryAncient DNA from the Andes reveals a complex picture of human adaptation from early settlement to the colonial period.

genomics↗

bioRxiv2018

Human primary astrocytes increase basal fatty acid oxidation following recurrent low glucose to maintain intracellular nucleotide levels

Hypoglycemia is a major barrier to good glucose control in type 1 diabetes and frequent exposure to hypoglycemia can impair awareness to subsequent bouts of hypoglycemia. The neural changes that occur to reduce a persons awareness of hypoglycemia are poorly defined. Moreover, the molecular mechanisms by which glial cells contribute to hypoglycemia sensing and glucose counterregulation require further investigation. To test whether glia, specifically astrocytes, could detect changes in glucose, we utilized human primary astrocytes (HPA) and U373 astrocytoma cells and exposed them to recurrent low glucose (RLG) in vitro. This allowed measurement, with high specificity and sensitivity, of changes in cellular metabolism following RLG. We report that the AMP-activated protein kinase (AMPK) is activated over a pathophysiologically-relevant glucose concentration range. We observed an increased dependency on fatty acid oxidation for basal mitochondrial metabolism and hallmarks of mitochondrial stress including increased proton leak and reduced coupling efficiency. Relative to glucose availability, lactate release increased during low glucose but this was not modified by RLG, nor were glucose uptake or glycogen levels. Taken together, these data indicate that astrocyte mitochondria are dysfunctional following recurrent low glucose exposure, which could have implications for hypoglycemia glucose counterregulation and/or hypoglycemia awareness.

neuroscience↗