Biology subjects

Hughes, J. W.

Publications and source records attributed to Hughes, J. W..

12 recordsLinked to original sources

bioRxiv2026

Serotonin signals through ciliary 5-HT6 to control insulin secretion

In the pancreatic islet, serotonin (5-HT) functions basally to modulate glucagon secretion, but its contribution to beta-cell signaling and function remains unclear. Here, we demonstrate that the Gs-coupled serotonin receptor 6 (5-HT6), previously thought to be expressed almost exclusively in the brain, localizes to the primary cilium and plasma membrane of islet cells and augments insulin secretion. Utilizing a transgenic model of beta-cell cilia loss, along with approaches to perturb cilia-specific 5-HT6 function without disrupting the native cilium architecture, we show that 5-HT6 action in beta cells is dependent on the primary cilium. Critically, the plasma membrane pool of 5-HT6 cannot stimulate insulin release when the cilium is absent, representing an instance of whole-cell regulation by a cilia-localized GPCR. These findings establish subcellular localization as a determinant of GPCR function in beta cells and identify ciliary 5-HT6 signaling as a distinct pathway for modulating insulin secretion.

cell biology↗

bioRxiv2026

Compartmentalized glycolysis powers ATP production in primary cilia and engages mitochondria via the phosphoenolpyruvate cycle

Primary cilia are antenna-like sensory and signaling organelles present on most mammalian cells, including glucose-sensing pancreatic {beta}-cells. Here, we show that the local energetic demands of primary cilia require the ATP-producing enzyme pyruvate kinase, with loss of PKm1, but not PKm2, impairing ciliary glycolytic flux. While the entire glycolytic machinery localizes to cilia, our data indicate that mitochondria are a critical source of phosphoenolpyruvate (PEP), the high-energy glycolytic intermediate that drives the pyruvate kinase reaction. Abolishing PCK2, the mitochondrial enzyme that generates PEP, prevents cilia from sensing not only glucose but also the amino acids glutamine and leucine. Finally, by mislocalizing glycolysis, we demonstrate that primary cilia can utilize ATP generated within the cell body when glucose is limiting. These findings indicate that primary cilia, while possessing the capacity for local ATP generation, leverage a ciliary-mitochondrial signaling axis to meet their bioenergetic needs.

cell biology↗

bioRxiv2026

Primary cilia regulate GLP-1 signaling in pancreatic beta cells

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are mainstay therapies for diabetes and obesity, acting in part by enhancing glucose-dependent insulin secretion. While the primary cilium is a known signaling compartment for certain G-protein coupled receptors (GPCRs), its role in the {beta}-cell response to incretins remains undefined. Here, we show that primary cilia are essential for GLP-1R signaling. Loss of {beta}-cell cilia in mouse and human islets severely impaired GLP-1-potentiated insulin secretion, an effect preceded by blunted whole-cell cAMP and Ca{superscript 2} responses. Immunofluorescence and immunogold scanning electron microscopy revealed endogenous GLP-1R localized to the primary cilium. Critically, disrupting ciliary GPCR trafficking via Tulp3 knockdown - while preserving cilia structure - recapitulated the signaling and secretory deficits, demonstrating a specific requirement for the ciliary receptor pool. These findings establish the primary cilium as a non-redundant signaling compartment for GLP-1R and uncover a new layer of subcellular organization in incretin action in {beta} cells.

cell biology↗

bioRxiv2025

Amino acid sensing by the α-cell mitochondrial phosphoenolpyruvate cycle regulates intracellular Ca2+ levels without impacting glucagon secretion

Pancreatic islet -cells are increasingly recognized as amino acid sensors for the organism. Building on our prior work in {beta}-cells, we sought to determine whether the mitochondrial phosphoenolpyruvate (PEP) cycle is involved in -cell amino acid sensing. Three different methods were used to probe the PEP cycle, including pyruvate kinase activators (TEPP-46), and mice with -cell specific deletion of pyruvate kinase (PKM1/2-KO) or mitochondrial PEP carboxykinase (PCK2-KO). The mitochondrial fuels glutamine/leucine antagonized alanine/arginine-stimulated Ca2+ influx and glucagon secretion under hypoglycemic conditions. Both PKM1/2 and PCK2 were required for glutamine/leucine to close KATP channels and limit amino acid-stimulated membrane depolarization. The Ca2+ response to amino acids was suppressed by pyruvate kinase activation with TEPP-46, and enhanced by -cell deletion of pyruvate kinase or PCK2 - all without changing glucagon secretion. Finally, using diazoxide/KCl to probe the pathways downstream of membrane depolarization, we identified an essential role of the PEP cycle in homeostatically restoring intracellular Ca2+ levels. In sum, the -cell mitochondrial PEP cycle senses glutamine/leucine and inhibits KATP channels similarly to {beta}-cells, while restricting amino acid-stimulated membrane depolarization and Ca2+ influx. However, defying expectations, none of the amino acids tested, including alanine/arginine, regulate glucagon secretion by modulating membrane depolarization or intracellular Ca2+. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/656009v2_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@18a29f0org.highwire.dtl.DTLVardef@1c77041org.highwire.dtl.DTLVardef@9adc1dorg.highwire.dtl.DTLVardef@e91407_HPS_FORMAT_FIGEXP M_FIG Graphical abstract-cells as amino acids sensors. Arginine, alanine, and glutamine potentiate glucagon secretion, while leucine has a suppressive effect (left). Independently of glucagon secretion, glutamine and leucine suppress alanine and arginine-stimulated Ca2+ influx via the phosphoenolpyruvate (PEP) cycle, which closes KATP channels and suppresses Vm depolarization (right). C_FIG HighlightsO_LIOur studies identify a role for the -cell PEP cycle in sensing amino acids under hypoglycemic conditions. C_LIO_LIPyruvate kinase and PCK2 are required for glutamine/leucine to close -cell KATP channel and limit membrane depolarization and Ca2+ influx. C_LIO_LIGlutamine/leucine oppose alanine/arginine-stimulated Ca2+ influx and glucagon secretion. C_LIO_LIAll of the amino acids tested regulate glucagon secretion, but none do so by modulating membrane depolarization or intracellular Ca2+ levels. C_LI

cell biology↗

bioRxiv2024

Expansion microscopy of axonemal dyneins in islet primary cilia

Primary cilia are vital sensory organelles whose structures are challenging to study due to their solitary nature and intricate cytoskeleton. Current imaging modalities are limited in their ability to visualize structural details that are important for understanding primary cilia function. Ultrastructure expansion microscopy (U-ExM) is a recent superresolution imaging technique that physically expands biological specimens using a swellable hydrogel, allowing structural interrogation of small cellular components such as cilia. In this study, we apply U-ExM to mouse and human pancreatic islets to visualize the axonemal cytoskeleton and associated proteins in primary cilia. Our study reveals the expression of axonemal dynein in islet primary cilia and centrioles, with DNAI1 being a principal subunit which we validate using targeted shRNA knockdown. We conclude that U-ExM is suitable for localizing protein expression in pancreatic islet cilia which contain axonemal dynein.

cell biology↗

bioRxiv2024

Intravital imaging reveals glucose-dependent cilia movement in pancreatic islets in vivo

Pancreatic islet cells harbor primary cilia, small sensory organelles that detect environmental changes to regulate hormone secretion and intercellular communication. While the sensory and signaling capacity of primary cilia are well-appreciated, it is less recognized that these organelles also possess active motility, including in dense multicellular tissues such as the pancreatic islet. In this manuscript, we use transgenic cilia reporter mice and an intravital imaging approach to quantitate primary cilia dynamics as it occurs in live mouse pancreatic islets. We validate this imaging workflow as suitable for studying islet cilia motion in real time in vivo and demonstrate that glucose stimulation corresponds to a change in cilia motility, which may be a physiologic measure of nutrient-dependent fluxes in islet cell function.

cell biology↗

bioRxiv2024

Where is the boundary of the human pseudoautosomal region?

A recent publication describing the assembly of the Y chromosomes of 43 males was remarkable not only for its ambitious technical scope, but also for the startling suggestion that the boundary of the pseudoautosomal region 1 (PAR1), where the human X and Y chromosomes engage in crossing-over during male meiosis, lies 500 kb distal to its previously reported location1. Where is the boundary of the human PAR1? We first review the evidence that mapped the PAR boundary, or PAB, before the human genome draft sequence was produced, then examine post-genomic datasets for evidence of crossing-over between the X and Y, and lastly re-examine the sequence assemblies presented by Hallast and colleagues to see whether they support a more distal PAB. We find ample evidence of X-Y crossovers throughout the 500 kb in question, some as close as 246 bp to the previously reported PAB. Our new analyses, combined with previous studies over the past 40 years, provide overwhelming evidence to support the original position, and narrow the probable location of the PAB to a 201-bp window.

genetics↗

bioRxiv2024

Immuno-Scanning Electron Microscopy of Islet Primary Cilia

The definitive demonstration of protein localization on primary cilia has been a challenge for cilia biologists. Primary cilia are solitary thread-like projections that contain specialized protein composition, but as the ciliary structure overlays the cell membrane and other cell parts, the identity of ciliary proteins are difficult to ascertain by conventional imaging approaches like immunofluorescence microscopy. Surface scanning electron microscopy combined with immuno-labeling (immuno-SEM) bypasses some of these indeterminacies by unambiguously showing protein expression in the context of the 3D ultrastructure of the cilium. Here we apply immuno-SEM to specifically identify proteins on the primary cilia of mouse and human pancreatic islets, including post-translationally modified tubulin, intraflagellar transport (IFT) 88, the small GTPase Arl13b, as well as subunits of axonemal dynein. Key parameters in sample preparation, immuno-labeling, and imaging acquisition are discussed to facilitate similar studies by others in the cilia research community.

cell biology↗

bioRxiv2021

Epitome: Predicting epigenetic events in novel cell types with multi-cell deep ensemble learning

The accumulation of large epigenomics data consortiums provides us with the opportunity to extrapolate existing knowledge to new cell types and conditions. We propose Epitome, a deep neural network that learns similarities of chromatin accessibility between well characterized reference cell types and a query cellular context, and copies over signal of transcription factor binding and modification of histones from reference cell types when chromatin profiles are similar to the query. Epitome achieves state-of-the-art accuracy when predicting transcription factor binding sites on novel cellular contexts, and can further improve predictions as more epigenetic signals are collected from both reference cell types and the query cellular context of interest.

bioinformatics↗

bioRxiv2020

Spontaneous restoration of functional β-cell mass in obese SM/J mice

Maintenance of functional {beta}-cell mass is critical to preventing diabetes, but the physiological mechanisms that cause {beta}-cell populations to thrive or fail in the context of obesity are unknown. High fat-fed SM/J mice spontaneously transition from hyperglycemic-obese to normoglycemic-obese with age, providing a unique opportunity to study {beta}-cell adaptation. Here, we characterize insulin homeostasis, islet morphology, and {beta}-cell function during SM/Js diabetic remission. As they resolve hyperglycemia, obese SM/J mice dramatically increase circulating and pancreatic insulin levels while improving insulin sensitivity. Immunostaining of pancreatic sections reveals that obese SM/J mice selectively increase {beta}-cell mass but not -cell mass. Obese SM/J mice do not show elevated {beta}-cell mitotic index, but rather elevated -cell mitotic index. Functional assessment of isolated islets reveals that obese SM/J mice increase glucose stimulated insulin secretion, decrease basal insulin secretion, and increase islet insulin content. These results establish that {beta}-cell mass expansion and improved {beta}-cell function underlie the resolution of hyperglycemia, indicating that obese SM/J mice are a valuable tool for exploring how functional {beta}-cell mass can be recovered in the context of obesity.

physiology↗

bioRxiv2020

Spontaneous activity changes in large-scale cortical networks in older adults couple to distinct hemodynamic morphology

Neurovascular coupling is a dynamic core mechanism supporting brain energy demand. Therefore, even spontaneous changes in neural activity are expected to evoke a vascular hemodynamic response (HDR). Here, we developed a novel procedure for estimating transient states in intrinsic activity of neural networks based on source-localized electroencephalogram in combination with HDR estimation based on simultaneous rapid-acquisition functional magnetic resonance imaging. We demonstrate a readily apparent spatiotemporal correspondence between electrophysiological and HDR signals, describing for the first time how features of neurovascular coupling may differ among large-scale brain networks. In the default mode network, the HDR pattern in our older adult participants was associated with a surrogate marker of cerebrovascular deterioration and predicted alterations in temporal structure of fast intrinsic electrophysiological activity linked to memory decline. These results show the potential of our technique for making inferences about neural and vascular processes in higher-level cognitive networks in healthy and at-risk populations.

neuroscience↗

bioRxiv2019

Transcranial Near-Infrared Light: Dose-Dependent Effects on EEG Oscillations but not Cerebral Blood Flow

ObjectiveOur objective was to assess whether transcranial photobiomodulation (tPBM) with near-infrared light (NIR) shows modulatory effects on cerebral electrical activity through electroencephalogram (EEG) and cerebral blood flow (CBF). BackgroundtPBM has emerged as a novel intervention for several neuropsychiatric conditions due to its neuroprotective and neuroenhancement effects. MethodsWe conducted a single-blind, sham-controlled pilot study to test the effect of continuous (c-tPBM), pulse (p-tPBM) and sham (s-tPBM) transcranial photobiomodulation on EEG oscillations and CBT using diffuse correlation spectroscopy (DCS) in a sample of ten healthy subjects [6 F/ 4 M; mean age 28.6 {+/-} 12.9 (SD) years]. c-tPBM NIR (830 nm; 54.8 mW/cm{superscript 2}; 65.8 J/cm{superscript 2}; 2.3 kJ) and p-tPBM (830 nm; 10Hz; 54.8 mW/cm{superscript 2}; 33%; 21.7 J/cm{superscript 2}; 0.8 kJ) were delivered concurrently to the frontal areas (F4, Fp2, Fp1, F3 - total surface 35.8 cm{superscript 2}) by four LED clusters (TPBM-1000 Litecure). EEG and DCS recordings were performed weekly before, during, and after each tPBM session (in sequence: c-tPBM, s-tPBM and p-tPBM). ResultsOnly c-tPBM significantly boosted gamma (t = 3.02, df = 7, p < .02) and beta (t = 2.91, df = 7, p < .03) EEG spectral powers in eyes-open recordings and gamma power (t = 3.61, df = 6, p < .015) in eyes-closed recordings, with the largest effects in the posterior regions. There was no significant effect of NIR-tPBM on CBF compared to sham. ConclusionsOur data suggest a dose-dependent effect of tPBM with NIR (c-tPBM) on cerebral gamma and beta neuronal activity. Altogether, our findings support the neuromodulatory effect of transcranial NIR.

neuroscience↗