Biology subjects

Abel, E. D.

Publications and source records attributed to Abel, E. D..

16 recordsLinked to original sources

bioRxiv2024

Lysosomal LRRC8 complex regulates lysosomal pH, morphology and systemic glucose metabolism

The lysosome integrates anabolic signalling and nutrient-sensing to regulate intracellular growth pathways. The leucine-rich repeat containing 8 (LRRC8) channel complex forms a lysosomal anion channel and regulates PI3K-AKT-mTOR signalling, skeletal muscle differentiation, growth, and systemic glucose metabolism. Here, we define the endogenous LRRC8 subunits localized to a subset of lysosomes in differentiated myotubes. We show LRRC8A regulates leucine-stimulated mTOR, lysosome size, number, pH, and expression of lysosomal proteins LAMP2, P62, LC3B, suggesting impaired autophagic flux. Mutating a LRRC8A lysosomal targeting dileucine motif sequence (LRRC8A-L706A;L707A) in myotubes recapitulates the abnormal AKT signalling and altered lysosomal morphology and pH observed in LRRC8A KO cells. In vivo, LRRC8A-L706A;L707A KI mice exhibit increased adiposity, impaired glucose tolerance and insulin resistance characterized by reduced skeletal muscle glucose-uptake, and impaired incorporation of glucose into glycogen. These data reveal a lysosomal LRRC8 mediated metabolic signalling function that regulates lysosomal activity, systemic glucose homeostasis and insulin-sensitivity.

cell biology↗

bioRxiv2023

Cardiomyocyte-specific deletion of PTP1B protects against HFD-induced cardiomyopathy through direct regulation of cardiac metabolic signaling

BackgroundHeart failure is the number one cause of death worldwide and mortality is directly correlated with the high incidence of obesity and diabetes. Indeed, the epidemic phenomenon of obesity was projected to reach 50% in the US by the year 2030. However, the mechanisms linking metabolic dysfunction with heart disease are not clear. Protein Tyrosine Phosphatase 1B (PTP1B), a negative regulator of insulin signaling, is considered to be an emerging therapeutic target against the development of obesity, insulin resistance, and diabetes. Increased PTP1B levels and activity have been observed in brain, muscle and adipose tissues isolated from obese and/or diabetic animals, as well as in human obese human patients. Its role, however, and the mechanisms by which it modulates metabolic processes in the heart remain unknown. Method and ResultsWe generated cardiomyocyte (CM)-specific PTP1B knock-out (PTP1Bfl/fl::MHCCre/+) mice to investigate the cardiomyocyte-specific role of PTP1B in response to high fat diet (HFD)-induced cardiac dysfunction. While we did not observe any physiological or functional cardiac differences at baseline, in response to HFD, we found that PTP1Bfl/fl::MHCCre/+ mice were protected against development of cardiac hypertrophy, mitochondrial dysfunction, and diminished cardiac steatosis. Metabolomics data revealed that hearts with CM-specific deletion of PTP1B had increased fatty acid oxidation and NAD+ metabolism, but reduced glucose metabolism; we further validated these findings by real-time qPCR analysis. Mechanistically, we identified a novel PTP1B PKM2-AMPK axis in the heart, which acts as a molecular switch to promote fatty acid oxidation. In this regard, we identified that hearts from PTP1Bfl/fl::MHCCre/+ mice had upregulated levels of nicotinamide adenine dinucleotide (NAD+) and NAD phosphate (NADPH), leading to higher levels of nicotinamide phosphoribosyl transferase (NAMPT), the rate-limiting step of the NAD+ salvage pathway and an enzyme associated with obesity and diabetes. ConclusionsTogether, these results suggest that CM-specific deletion of PTP1B mediates a substrate switch from glucose to fatty acid metabolism, protecting hearts against development of HFD-induced cardiac hypertrophy and dysfunction through mechanisms involving a novel PTP1B/PKM2/AMPK axis that is critical for the regulation of NAMPT and NAD+ biosynthesis.

molecular biology↗

bioRxiv2023

ATF4 Expression in Thermogenic Adipocytes is Required for Cold-Induced Thermogenesis in Mice via FGF21-Independent Mechanisms

In brown adipose tissue (BAT), short-term cold exposure induces the activating transcription factor 4 (ATF4), and its downstream target fibroblast growth factor 21 (FGF21). Induction of ATF4 in BAT in response to mitochondrial stress is required for thermoregulation, partially via upregulation of FGF21. In the present study, we tested the hypothesis that Atf4 and Fgf21 induction in BAT are both required for BAT thermogenesis by generating mice selectively lacking either Atf4 (ATF4 BKO) or Fgf21 (FGF21 BKO) in UCP1-expressing adipocytes. After 3 days of cold exposure, core body temperature was significantly reduced in ad-libitum-fed ATF4 BKO mice, which correlated with Fgf21 downregulation in brown and beige adipocytes, and impaired browning of white adipose tissue (WAT). Conversely, despite having reduced browning, FGF21 BKO mice had preserved core body temperature after cold exposure. Mechanistically, ATF4, but not FGF21, regulates amino acid import and metabolism in response to cold, likely contributing to BAT thermogenic capacity under ad libitum-fed conditions. Importantly, under fasting conditions, both ATF4 and FGF21 were required for thermogenesis in cold-exposed mice. Thus, ATF4 regulates BAT thermogenesis by activating amino acid metabolism in BAT in a FGF21-independent manner. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/531964v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1d2c3fdorg.highwire.dtl.DTLVardef@414f5forg.highwire.dtl.DTLVardef@1c1fa9eorg.highwire.dtl.DTLVardef@2cc815_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2022

GDF15 Is Required for Cold-Induced Thermogenesis and Contributes to Improve Systemic Metabolic Health Following Loss of OPA1 in Brown Adipocytes

Previously we showed that mice lacking the protein optic atrophy 1 (OPA1 BKO) in brown adipose tissue (BAT) have induction of the activating transcription factor 4 (ATF4), which promotes fibroblast growth factor 21 (FGF21) secretion as a batokine. FGF21 increases metabolic rates at baseline conditions but is dispensable for the resistance to diet-induced obesity (DIO) reported in OPA1 BKO mice [1]. To determine alternative mediators of this phenotype, we performed transcriptome analysis, which revealed increased levels of growth differentiation factor 15 (GDF15) in BAT. To determine if ATF4 induction was mediated by the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and to evaluate the contribution of GDF15 to the resistance to DIO, we selectively deleted PERK or GDF15 in OPA1 BKO mice. Mice lacking both OPA1 and PERK in BAT had preserved induction of ATF4. Importantly, simultaneous deletion of OPA1 and GDF15 partially reversed the resistance to diet-induced obesity and abrogated the improvements in glucose tolerance. Furthermore, GDF15 mediated cold-induced thermogenesis, likely via sympathetic activation of iWAT. Taken together, our data indicate that PERK is dispensable for ATF4 induction, but GDF15 contributes to the resistance to DIO, and is required for glucose homeostasis and thermoregulation in OPA1 BKO mice.

physiology↗

bioRxiv2022

Endosomal GLUT3 is essential for alternative macrophage signaling, polarization, and function

Macrophages play critical roles in both inflammation and tissue homeostasis. Classically activated (M1) macrophages promote antimicrobial and tumoricidal activity, while alternatively activated (M2) macrophages promote phagocytosis and tissue homeostasis. The facilitative GLUT1 and GLUT3 hexose transporters are expressed abundantly in different hematopoietic lineages, but their specific functions in macrophages is poorly understood. We discovered that GLUT3 expression was increased after M2-activation stimuli in macrophages. Notably, GLUT3 KO BMDM (bone marrow-derived macrophages) showed marked defects in M2, but not M1, polarization. Consistent with defects in M2 polarization, GLUT3 KO macrophages showed impaired wound healing and decreased inflammation in calcipotriol-induced, atopic dermatitislike inflammation. GLUT3 promoted IL-4/STAT6 signaling, the main signaling pathway for M2 polarization, in a glucose-transport independent manner. Unlike plasma membrane-localized GLUT1, GLUT3 and components of the IL-4 signaling pathway, localized primarily to endosomes. GLUT3, but not GLUT1, interacted with Ras through its intracytoplasmic loop, and Rac1-PAK-cofilin signaling and the endocytosis of IL4R subunits were impaired in the absence of GLUT3. Thus, GLUT3 is essential for alternative macrophage polarization and function and plays an unexpected role in the regulation of endosomal signaling.

immunology↗

bioRxiv2022

Cardiac MAO-A inhibition protects against catecholamine-induced ventricular arrhythmias via enhanced diastolic calcium control

BackgroundPeople with clinical depression exhibit increased risk for cardiac arrhythmias that could be related to differences in catecholamine metabolism. Emerging studies have implicated a pathophysiologic role for monoamine oxidase (MAO-A), which catalyzes catecholamine metabolism in the heart. MAO-A is the pharmacological target of some classes of anti-depressants. Here, we investigated the relationship between MAO-A activity and arrhythmogenesis. Methods & ResultsTriNetX database analysis of adult patients with depression (n=11,533) revealed that MAO inhibitor (MAOI) treatment is associated with significantly lower risk of arrhythmias compared with selective serotonin reuptake inhibitor (SSRI) treatment (16.7% vs 18.6%, p=0.0002). To determine a mechanistic link between MAO activity and arrhythmia, we utilized a genetically modified mouse model with cardiomyocyte-specific MAO-A deficiency (cMAO-Adef). Compared with wild-type (WT) mice, cMAO-Adef mice had a significant reduction in the incidence (38.9% vs. 77.8%, p=0.0409) and duration (55.33 {+/-} 26.21s vs.163.1 {+/-} 56.38s, p=0.0360) of catecholamine stress-induced ventricular tachyarrhythmias (VT). Reduced VT risk and duration were associated with altered cardiomyocyte Ca2+ handling in the cMAO-Adef hearts, including a marked increase in Ca2+ reuptake rate, decreased diastolic Ca2+ levels, decreased SR Ca2+ load and reduced Ca2+ spark activity following catecholamine stimulation relative to WT. Further analysis of molecular mechanisms revealed that altered Ca2+ handling in the cMAO-Adef hearts was related to decreased catecholamine-induced phosphorylation of Ca2+/calmodulin-dependent kinase II (CaMKII) and ryanodine receptor 2 (RyR2), and increased phosphorylation of phospholamban (PLB). ConclusionsThese findings suggest that MAO-A inhibition in cardiomyocytes mitigates arrhythmogenesis via enhanced Ca2+ reuptake that lowers diastolic Ca2+ levels thereby diminishing arrhythmic triggers following catecholamine stimulation. Thus, cardiac MAO-A represents a potential target for antiarrhythmic therapy.

physiology↗

bioRxiv2022

OPA1 Downregulation in Skeletal Muscle Induces MERC formation in an ATF4-Dependent Manner

Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are protein- and lipid-enriched hubs that mediate interorganellar communication by contributing to the dynamic transfer of Ca2+, lipid, and other metabolites between these organelles. Defective MERCs are associated with cellular oxidative stress, neurodegenerative disease, and cardiac and skeletal muscle pathology via mechanisms that are poorly understood. We previously demonstrated that skeletal muscle-specific knockdown (KD) of the mitochondrial fusion mediator optic atrophy 1 (OPA1) induced ER stress and correlated with an induction of Mitofusin-2, a known MERC protein. In the present study, we tested the hypothesis that Opa1 downregulation in skeletal muscle cells alters MERC formation by evaluating multiple myocyte systems, including from mice and Drosophila, and in primary myotubes. Our results revealed that OPA1 deficiency induced tighter and more frequent MERCs in concert with a greater abundance of MERC proteins involved in calcium exchange. Additionally, loss of OPA1 increased the expression of activating transcription factor 4 (ATF4), an integrated stress response (ISR) pathway effector. Reducing Atf4 expression prevented the OPA1-loss-induced tightening of MERC structures. OPA1 reduction was associated with decreased mitochondrial and sarcoplasmic reticulum, a specialized form of ER, calcium, which was reversed following ATF4 repression. These data suggest that mitochondrial stress, induced by OPA1 deficiency, regulates skeletal muscle MERC formation in an ATF4-dependent manner.

biophysics↗

bioRxiv2022

Gamma-interferon-inducible lysosomal thiol reductase maintains cardiac immuno-metabolic homeostasis in heart failure

BackgroundThe lysosome is a central player in maintaining immuno-metabolic homeostasis. However, mechanistic insights into the regulation of lysosome-dependent immuno-metabolism in the heart are lacking. Lysosomal reductase Gamma Interferon-Inducible Thiol Reductase (GILT) is the only identified lysosomal reductase that controls diverse sets of lysosomal enzymes and cargoes. MethodsThe role of cardiac GILT was assessed by generating a novel genetic mouse model and employing a multidisciplinary approach including surgical interventions, live in situ high resolution microscopy, whole-tissue respirometry analysis, unbiased transcriptomic and metabolomic analyses, and various cell biology and biochemical assays. ResultsWe found that expression and activity of GILT are reduced in hearts from humans and mice with heart failure (HF). Mice with cardiac specific loss of GILT develop late onset systolic HF at baseline. In the setting of nutrient-overload and experimental left ventricular pressure overload conditions, loss of GILT in cardiomyocytes accelerates the development of heart dysfunction. Transcriptomic and metabolic analyses further revealed that cardiac GILT deficiency alters adaptive immuno-metabolic signatures in the heart. Finally, at the cellular level, cardiac GILT deletion impaired mitochondrial respiration, which was in part due to NLR Family Pyrin Domain Containing 3 (NLRP3)-mediated elevation of mitochondrial oxidative stress. ConclusionsTogether, these findings identify a causal link between a lysosome-inflammation axis, mitochondrial function and heart failure. Elucidation of these mechanisms will identify novel therapeutic strategies for treating HF.

molecular biology↗

bioRxiv2022

Myelin lipids as nervous system energy reserves

Neuronal functions and impulse propagation depend on the continuous supply of glucose1,2. Surprisingly, the mammalian brain has no obvious energy stores, except for astroglial glycogen granules3. Oligodendrocytes make myelin for rapid axonal impulse conduction4 and also support axons metabolically with lactate5-7. Here, we show that myelin itself, a lipid-rich membrane compartment, becomes a local energy reserve when glucose is lacking. In the mouse optic nerve, a model white matter tract, oligodendrocytes survive glucose deprivation far better than astrocytes, by utilizing myelin lipids which requires oxygen and fatty acid beta-oxidation. Importantly, fatty acid oxidation also contributes to axonal ATP and basic conductivity. This metabolic support by fatty acids is an oligodendrocyte function, involving mitochondria and myelin-associated peroxisomes, as shown with mice lacking Mfp2. To study reduced glucose availability in vivo without physically starving mice, we deleted the Slc2a1 gene from mature oligodendrocytes. This caused a significant decline of the glucose transporter GLUT1 from the myelin compartment leading to myelin sheath thinning. We suggest a model in which myelin turnover under low glucose conditions can transiently buffer axonal energy metabolism. This model may explain the gradual loss of myelin in a range of neurodegenerative diseases8 with underlying hypometabolism9.

neuroscience↗

bioRxiv2022

Metabolic rescue ameliorates mitochondrial encephalo-cardiomyopathy in murine and human iPSC models of Leigh syndrome

Mice with deletion of complex I subunit Ndufs4 develop mitochondrial encephalomyopathy resembling Leigh syndrome (LS). We report that LS mice also develop severe cardiac bradyarrhythmia and diastolic dysfunction. Human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) with Ndufs4 deletion recapitulate LS cardiomyopathy. Mechanistically, we demonstrate a direct link between complex I deficiency, decreased intracellular NAD+/ NADH and bradyarrhythmia, mediated by hyperacetylation of the cardiac sodium channel NaV1.5, particularly at K1479 site. Neuronal apoptosis in the cerebellar and midbrain regions in LS mice was associated with hyperacetylation of p53 and activation of microglia. Targeted metabolomics revealed increases in several amino acids and citric acid cycle intermediates, likely due to impairment of NAD+-dependent dehydrogenases, and a substantial decrease in reduced Glutathione (GSH). Metabolic rescue by nicotinamide riboside (NR) supplementation increased intracellular NAD+/ NADH, restored metabolic derangement, reversed protein hyperacetylation through NAD+-dependent Sirtuin deacetylase, and ameliorated cardiomyopathic phenotypes, concomitant with improvement of NaV1.5 current and SERCA2a function measured by Ca2+- transients. NR also attenuated neuronal apoptosis and microglial activation in the LS brain and human iPS-derived neurons with Ndufs4 deletion. Our study reveals direct mechanistic explanations of the observed cardiac bradyarrhythmia, diastolic dysfunction and neuronal apoptosis in mouse and human iPSC models of LS.

pathology↗

bioRxiv2021

Practices for Measuring 3D Organelle Morphology and Generating Surfaces with Amira

Analysis of 3D structures is of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have remained staples for imaging cellular structures, they lack the ability to image in 3D. However, recent technological advances, such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM), have allowed researchers to observe cellular ultrastructure in 3D. Here, we propose a standardized protocol using the visualization software Amira to quantify organelle morphologies in 3D; this method allows researchers to produce accurate and reproducible measurements of cellular structure characteristics. We demonstrate this applicability by utilizing SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/461807v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@1c40041org.highwire.dtl.DTLVardef@ecde92org.highwire.dtl.DTLVardef@10870daorg.highwire.dtl.DTLVardef@1290c3b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Systematic Transmission Electron Microscopy-Based Identification of Cellular Degradation Machinery

Many interconnected degradation machineries including autophagosomes, lysosomes, and endosomes work in tandem to conduct autophagy, an intracellular degradation system that is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. Although many studies have investigated autophagy to elucidate disease pathogenesis, identification of specific components of the autophagy machinery has been challenging. The goal of this paper is to describe an approach to reproducibly identify and distinguish subcellular structures involved in macro autophagy. We provide methods that help avoid common pitfalls, including a detailed explanation for distinguishing lysosomes and lipid droplets and discuss differences between autophagosomes and inclusion bodies. These methods are based on using transmission electron microscopy (TEM), capable of generating nanometer-scale micrographs of cellular degradation components in a fixed sample. We also utilize serial block face-scanning electron microscopy (SBF-SEM) to offer a protocol for visualizing 3D morphology of degradation machinery. In addition to TEM and 3D reconstruction, we discuss other imaging techniques, such as immunofluorescence and immunogold labeling that can be utilized to reliably and accurately classify cellular organelles. Our results show how these methods may be used to accurately quantify the cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin-related protein 1.

biophysics↗

bioRxiv2021

A comprehensive approach for artifact-free sample preparation and assessment of mitochondrial morphology in tissue and cultured cells

Mitochondrial dynamics (fission, fusion, and the formation of nanotunnels) and morphology are very sensitive to the cellular environment. Mitochondria may be adversely affected by oxidative stress, changes in calcium levels, and hypoxia. Investigating the precise relationship between organelle structure and function requires methods that can adequately preserve mitochondria while providing accurate, quantitative measurements of morphological attributes. Here, we demonstrate a practical approach for preserving and measuring fine structural changes using two-dimensional, high-resolution electron micrographs. This approach is further applicable for three-dimensional volume renderings, obtained using serial block-face and focused ion beam-scanning electron microscopy, highlighting the specific advantages of these techniques. Additionally, this study defines a set of quantifiable metrics that can be applied to measure mitochondrial architecture and other organellar structures. Finally, we validated specimen preparation methods that avoid the introduction of morphological artifacts that may interfere with mitochondrial appearance and do not require whole-animal perfusion.

cell biology↗

bioRxiv2021

A Universal Approach to Analyzing Transmission Electron Microscopy with ImageJ

2Transmission electron microscopy (TEM) is a scientific research standard for producing nanometer-resolution ultrastructural images of subcellular components within cells and tissues. Mitochondria, endoplasmic reticulum (ER), lysosomes, and autophagosomes are organelles of particular interest to those investigating metabolic disorders. However, there is no clear consensus amongst regarding the best methods for quantifying the features of organelles in TEM images. In this protocol, we propose a standardized approach to accurately measure the morphology of these important subcellular structures using the free program ImageJ, developed by the National Institutes of Health (NIH). Specifically, we detail procedures for obtaining mitochondrial length, width, area, and circularity, in addition to assessing cristae morphology. We further provide methods for measuring interactions between the mitochondria and ER and measuring the length and width of lysosomes and autophagosomes. This standardized method can be used to quantify key features of organelle morphology, allowing investigators to produce accurate and reproducible measurements of organelle structures in their experimental samples. 1 SUMMARYWe discuss a standardized method for measuring and quantifying organelle features using transmission electron microscopy and accessing for interactions between subcellular structures; organelles of focus include mitochondria, endoplasmic reticulum, lysosomes, and autophagosomes.

biophysics↗

bioRxiv2021

OPA1 Deletion in Brown Adipose Tissue Improves Thermoregulation and Systemic Metabolism via FGF21

Adrenergic stimulation of brown adipocytes alters mitochondrial dynamics, including proteolytic processing of the mitochondrial fusion protein optic atrophy 1 (OPA1). However, direct mechanisms linking OPA1 to brown adipose tissue (BAT) physiology are incompletely understood. By deleting OPA1 selectively in BAT (OPA1 BAT KO), we demonstrate that OPA1 is required for cold-induced thermogenesis. Unexpectedly, OPA1 deficiency induced fibroblast growth factor 21 (FGF21) as a BATokine in an activating transcription factor 4 (ATF4)- dependent manner. BAT-derived FGF21 mediates an adaptive response in OPA1 BAT KO mice, by inducing browning of white adipose tissue (WAT), increasing resting metabolic rates, and improving thermoregulation. However, FGF21 does not mediate the resistance to diet-induced obesity observed in these animals. These findings reveal a requirement for OPA1 in BAT thermogenesis, and uncovers a homeostatic mechanism of BAT-mediated metabolic protection governed by an ATF4-FGF21 axis, that is activated independently of BAT thermogenic function.

physiology↗

bioRxiv2020

SWELL1-LRRC8 complex regulates skeletal muscle cell size, intracellular signalling, adiposity and glucose metabolism

Maintenance of skeletal muscle is beneficial in obesity and Type 2 diabetes. Mechanical stimulation can regulate skeletal muscle differentiation, growth and metabolism, however the molecular mechanosensor remains unknown. Here, we show that SWELL1 (LRRC8a) functionally encodes a swell-activated anion channel that regulates PI3K-AKT, ERK1/2, mTOR signaling, muscle differentiation, myoblast fusion, cellular oxygen consumption, and glycolysis in skeletal muscle cells. SWELL1 over-expression in SWELL1 KO myotubes boosts PI3K-AKT-mTOR signaling to supra-normal levels and fully rescues myotube formation. Skeletal muscle targeted SWELL1 KO mice have smaller myofibers, generate less force ex vivo, and exhibit reduced exercise endurance, associated with increased adiposity under basal conditions, and glucose intolerance and insulin resistance when raised on a high-fat diet, compared to WT mice. These results reveal that the SWELL1-LRRC8 complex regulates insulin-PI3K-AKT-mTOR signalling in skeletal muscle to influence skeletal muscle differentiation in vitro and skeletal myofiber size, muscle function, adiposity and systemic metabolism in vivo.

cell biology↗