bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of obesity suppression by adipose tissue creatine energetics
Authors: Kazak Lawrence , Lu Gina Z. , Spiegelman Bruce M.
Published: June 2021

The increase in obesity worldwide has led to a surge in the prevalence of type 2 diabetes, cardiovascular disease and many cancers. The capacity of thermogenic (brown and beige) fat to dissipate chemical energy as heat offers tremendous potential to combat obesity. Exploiting thermogenesis to regulate body weight requires a precise molecular understanding of its control at the level of mitochondria.

We have previously demonstrated that creatine drives energy expenditure in adipose tissue by stimulating a futile cycle of mitochondrial ATP turnover. Our recent findings show that genetic deletion of the rate‐limiting enzyme of creatine synthesis (Gatm) results in rapid‐onset obesity due to the suppression of elevated energy expenditure that occurs in response to high calorie feeding. This is paralleled by a blunted capacity for β3‐adrenergic activation of metabolic rate. Moreover, abolishing adipocyte creatine uptake, by deleting the creatine transporter (CrT), similarly impairs adrenergic thermogenesis and causes obesity. These results provide strong in vivo genetic support for a role of adipose tissue creatine metabolism in the activation of energy expenditure and the defense against diet‐induced obesity. However, the mechanism underlying creatine‐dependent thermogenesis is not fully understood.

We have now identified the primary Creatine Kinase (CK) isoform that controls thermogenic respiration in brown fat. Using quantitative proteomics from purified brown adipocytes and brown adipocyte mitochondria, we find brain‐type CK (B‐CK) to be the most abundant CK in murine brown fat. Surprisingly a subset of B‐CK is targeted to mitochondria. Mitochondria from primary brown adipocytes have robust capacity for creatine‐dependent thermogenic respiration, and knockdown of B‐CK impairs this process. Notably, B‐CK is the most abundance CK isoform in purified human subcutaneous adipocytes and its expression is significantly lower in insulin‐resistant compared to insulin‐sensitive patients. Therefore, B‐CK is the first molecular handle that should result in the identification of additional components regulating creatine‐dependent thermogenesis. In addition, adipose tissue creatine energetics is correlated with healthy glucose homeostasis in humans.

Support or Funding Information

Canadian Institutes of Health Research postdoctoral fellowship and NIDDK, NIH Pathway to Independence (K99/R00), K99DK114528 (to L.K.)

NIH grant (DK31405) and the JPB Foundation (to B.M.S.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Approach to Characterize Size Variance In Third Instar Larvae Following Multigenerational Dietary Modification
Authors: Arnal Gustavo , Rogers Ryan Patricia
Published: June 2021

Mutant InR female Drosophila melanogaster flies are considered dwarfs due to reduced growth and experienced up to an 85% increase in longevity (Tatar et al. 2001) compared to yw D. melanogaster flies. For this reason, it was of interest to investigate if this growth deviance occurred in the developmental stages of the flies, specifically in the third instar larvae stage. As part of this experiment, yw and InR mutant flies were placed in vials containing food of two different sucrose concentrations, 0% and 10%. Multigenerational exposure to elevated metabolic substrate was studied for both types of flies. Size measurements were performed at regular time intervals using a self‐developed method which involves image processing with ImageJ. This approach begins by placing larvae on a high contrast surface, next to an object of known size for scaling purposes. Then a picture is taken and ran through the ImageJ macro, which prompts the user to follow three steps meant to achieve a clean outline of the larvae. ImageJ calculates the area of each outline and records it, and these are used as relative measurements of size between larvae. This method can be more efficient in time and cost than current sizing techniques which require expensive equipment, or meticulous set ups. Preliminary results show a significant growth of larvae across generations, but no significant changes in size between yw and InR mutant fly larvae, or between metabolic substrate concentrations. Results suggest that dwarfs InR female flies begin exhibiting variation in their size post metamorphosis, and a 10% increase of metabolic substrate does not impact their physical growth during development.

Support or Funding Information

This work was supported by funds from the Department of Sciences at Wentworth Institute of Technology

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Organ Reserve, Excess Metabolic Capacity, and Aging
Authors: Atamna Hani , Dhahbi Joseph
Published: June 2021

“Organ reserve” refers to the ability of an organ to successfully recover its original metabolic homeostatic set‐points following repeated episodes of stress. Clinical evidence shows that organ reserve correlates with the ability of older adults to cope with an added workload or stress, suggesting a role in the process of aging. Although organ reserve is well documented clinically, it is not clearly defined at the molecular level. Interestingly, several metabolic pathways exhibit excess metabolic capacities (e.g., bioenergetics pathway, antioxidants system, neuroplasticity). These pathways comprise molecular components that have an excess of quantity and/or activity than that required for normal demand (e.g., mitochondrial complex IV or glycolytic enzymes). We propose that the excess in mtDNA copy number and tandem DNA repeats of telomeres are additional examples of intrinsically embedded structural components that could comprise excess capacity. These excess capacities may grant intermediary metabolism the ability to instantly cope with, or manage, added workload or stress. Therefore, excess metabolic capacities could be viewed as an innate mechanism of adaptability that substantiates organ reserve and contributes to the cellular defense systems. If metabolic excess capacities or organ reserves are impaired or exhausted, the ability of the cell to cope with stress is reduced. Under these circumstances cell senescence, transformation, or death occurs. In this presentation, we will discuss excess metabolic and structural capacities as integrated metabolic pathways in relation to organ reserve and cellular aging.

Support or Funding Information

NIHAFAR

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Reevaluation of the Role of Phosphatidylinositol Transfer Protein a in Growth Factor Signaling
Authors: McDermott Mark I. , Diz Ramiro , Hur Seong , Lete Marta G. , Applebee Christopher J. , Grabon Aby , Tripathi Ashutosh , Wakelam Michael J. O. , Larijani Banafshe , Bankaitis Vytas A.
Published: June 2021

Despite current dogma stating that phosphatidylinositol transfer protein a (PITPa) is required for growth factor signaling, little evidence has emerged supporting these claims. Data from our laboratory, gathered from multiple experimental systems, strongly dispute the current doctrine and indicate that in contrast to previous reports PITPa is dispensable for epidermal growth factor (EGF), and netrin signaling. Mouse and human cell lines lacking PITPa sense and chemotax towards EGF. Furthermore, signaling responses in these lines, including phosphatidylinositol (3,4,5)P3 generation and phosphorylation of protein kinase B (Akt), downstream of EGF‐receptor (EGFR) stimulation are unimpaired. Surprisingly, human cells lacking PITPa displayed elevated Akt (Thr308) phosphorylation following EGF‐stimulation, indicating that not only is PITPa dispensable for Akt phosphorylation, but contrary to current thinking it may actually repress this pathway. PITPa dispensability for EGF‐signaling explains the striking absence of EGF‐signaling related phenotypes in PITPa nullizygous mice. We have previously reported that these animals display a complex phenotype including spinocerebellar neurodegeneration characterized by neuronal apoptosis and gliosis of cerebellum, and hindbrain. Netrin signaling through the netrin receptor (DCC) is required for axon guidance and mouse forebrain development, and is reported to require PITPa. We demonstrate that absence of PITPa does not result in structural brain derangements associated with compromise of netrin‐signaling. Moreover, the previously reported interaction between the netrin receptor (DCC) and PITPa could not be reproduced. Clearly the ‘essential’ role of PITPa in growth factor signaling must be reevaluated.

Support or Funding Information

This research was funded by NIH grant R01‐GM112591 and grant BE‐0017 from the Robert A. Welch Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel biosensors for an enigmatic phosphoinositide
Authors: Goulden Brady , Zewe James , Wills Rachel , Hammond Gerry
Published: June 2021

Phosphatidylinositol (3,4)‐bisphosphate (PtdIns(3,4)P2) functions at the nexus of plasma membrane (PM) signaling and trafficking. Specifically PtdIns(3,4)P2 is produced from PtdIns4P during endocytosis or from PtdInsP3 during signaling. Elucidating the relative roles of these pathways requires the capacity to image this lipid's production with real‐time live‐cell microscopy. However, the biosensors currently employed either recognize both PtdIns(3,4)P2 and PtdInsP3, or have insufficient affinity to image these low abundance lipids. We therefore set out to identify PtdIns(3,4)P2 and PtdInsP3 probes that have high sensitivity and fidelity. Pleckstrin homology (PH) domains from Akt, Btk, ARNO, and GRP1 have been shown to recognize and bind PtdInsP3, while the C‐terminal PH domain from TAPP1 has been reported to interact with PtdIns(3,4)P2. To increase their efficacy as probes, we cloned a fluorescent protein in tandem with these PH domains as monomers, dimers, or trimers. Using TIRF and confocal microscopy, we demonstrate that our trimer TAPP1‐PH and dimer ARNO‐2G‐PH constructs are specific and sufficient for detecting PtdIns(3,4)P2 and PtdInsP3, respectively. In addition, we developed several chemical‐ and opto‐genetic tools to specifically manipulate these lipids in living cells. A rapamycin inducible system was used by conjugating FKBP to PI(3)K‐C2a and INPP4B (PtdIns(3,4)P2‐specific 4‐phosphatase), and a photoactivatable PtdIns3P 4‐kinase derived from Legionella pneumophila (LepB) was also used to manipulate PtdIns(3,4)P2. Collectively, the tools produced in this study provide powerful new means of understanding the spatiotemporal control of PtdIns(3,4)P2 and PtdInsP3 in signaling and membrane traffic.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Subcellular Distribution of Cholesterol and Sphingolipids in Rat Hepatocytes
Authors: Stieger Bruno , Steiger Julia , Jiménez Noemi , Riezman Isabelle , Riezman Howard
Published: June 2021

Hepatocytes are central in energy and lipid homeostasis of mammals and as such are key players in maintaining and regulating serum cholesterol levels. In addition, hepatocytes produce bile, which has detergent properties and is the main product of cholesterol metabolism. The canalicular membrane is the site of bile formation and needs protective mechanisms against the detergent properties of bile. One such mechanism could be a specific lipid composition. We therefore aimed to use a targeted lipidomics approach to determine simultaneously the cholesterol and sphingolipid composition of the organelles involved in lipid synthesis in hepatocytes. Subcellular organelles were isolated from rat liver with standard methodologies and lipids were analyzed by either electrospray ionization mass spectrometry (glycerophospholipids and sphingolipids) or gas chromatography coupled mass spectrometry (sterols). Our data indicate that there is a distinct difference in cholesterol and sphingolipid content in the organelle fractions. Highest cholesterol content is found in basolateral plasma membranes and in the Golgi fraction, while endoplasmic reticulum displays a low cholesterol content. A high proportion of sphingomyelin is present in basolateral plasma membranes, smooth endoplasmic reticulum and in the Golgi apparatus. Ceramide content is higher in basolateral membranes compared to organelles and the canalicular plasma membrane. Glucosylceramide levels are very low and are mostly found in Golgi fractions. Our preliminary data so far show a parallel distribution of cholesterol and sphingolipids in subcellular fractions of rat hepatocytes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Brewster Angle Microscopy and Langmuir Monolayer Films: Construction of an Instrument and Basic Software Development for Visualization of Lipid Domains and Lipid Raft Formation
Authors: Allen Brandon C. , Sturgeon Bradley E. , Sostarecz Audra Goach
Published: June 2021

Brewster Angle Microscopy is a useful technique for the analysis of surface characteristics of films at the air‐water interface in a Langmuir monolayer trough due to its ability to obtain images while simultaneously collecting isothermal data. It provides an effective way of visualizing lipid domains, as well as lipid aggregation to form structures such as lipid rafts. The goal of this research was to construct a relatively low‐cost Brewster Angle Microscope (BAM) using LEGO™ Mindstorms, 3D printed components, a laser, a 10x microscope objective, and a CCD camera. This BAM was then used to obtain images of dipalmitoylphosphatidylcholine (DPPC) films and DPPC/Cholesterol films during a compression analysis. At the moment, we are working on programmatically quantifying the phase differences in lipid domains, as well as the surface density and thickness of films for analysis of lipid monolayers for drug delivery applications.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deciphering TRAPP complex function in yeast
Authors: Joiner Aaron , Fromme Chris
Published: June 2021

All eukaryotes contain organelles that perform specialized functions essential for life. The Golgi complex, an organelle that is important for transport of cellular material, serves as the “Grand Central Station” of the cell by receiving, then sorting proteins and membranes to various locations. Many steps of vesicular trafficking at the Golgi complex are regulated by several families of small GTPases, which modulate between an active and inactive state. One particularly interesting GTPase is Rab1, which also has an established role in autophagy. Rab1 is activated by a guanine nucleotide exchange factor (GEF), a process that is conserved from mammals to yeast. Discordantly, mammals contain two protein complexes named TRAPPII and TRAPPIII (transport protein particle) which activate Rab1; while in yeast, four (TRAPPI ‐ TRAPPIV) have been implicated as GEFs in Ypt1 (Rab1 homolog) activation. Yeast TRAPP complexes share a core set of six subunits. Additional subunits are thought to regulate the complex by adding substrate and compartmental specificity. TRAPPII contains four more subunits, while TRAPPIII and TRAPPIV include one additional subunit. Importantly, the precise functions of the accessory subunits remain unclear. We demonstrate that TRAPPII and TRAPPIII are the only two complexes present in yeast cells, which is consistent with findings in mammalian cells, by quantifying isolated TRAPP complexes and simultaneously monitoring the localization of all TRAPP complexes in vivo. We also find that, using an in vitro fluorescent nucleotide exchange assay, TRAPPIII activates Ypt1 more effectively than the other complexes. Concurrently, we notice that Trs85 localizes to the Golgi complex and that Ypt1 is mislocalized in vivo in the absence of the TRAPPIII‐specific subunit, Trs85. Additionally, using an in vitro membrane binding assay, we find that Trs85 has an affinity for membranes. These findings suggest that there are only two TRAPP complexes in yeast, and that TRAPPIII is involved in trafficking at the Golgi complex in addition to its established role in autophagy. Taken together, our model suggests that the main role of Trs85 in the TRAPPIII complex is to bring the catalytic core close to a membrane surface, localizing the active site to the right compartment in an orientation that facilitates localization and activation of Ypt1.

Support or Funding Information

This material is based upon work supported by the NSF GRFP under Grant DGE‐1144153, by NIH/NIGMS Grant R01GM116942, and by the Alfred P. Sloan Foundation. Any opinions, conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the NSF, NIH, the Sloan Foundation or their trustees, officers, or staff.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PML‐Nuclear Bodies Regulate the Stability of the Fusion Protein Dendra2‐Nrf2 in the Nucleus of Single Live Cells
Authors: Burroughs Andrea Flores , Eluhu Sylvia , Whalen Diva , Goodwin J. Shawn , Sakwe Amos M. , Arinze Ifeanyi J.
Published: June 2021

Nuclear factor erythroid 2‐related factor 2 (Nrf2) is a basic leucine‐zipper transcription factor essential for cellular responses to oxidative stress. Degradation of Nrf2 in the cytoplasm, mediated by Keap1‐Cullin3/RING box 1 E3 ubiquitin ligase and the proteasome, is considered the primary pathway controlling the cellular abundance of Nrf2. Although the nucleus has been implicated in the degradation of Nrf2, little information is available on how this compartment participates in degrading Nrf2. Here, we fused the photoconvertible fluorescent protein Dendra2 to Nrf2 and capitalized on the irreversible change in color (green to red) that occurs when Dendra2 undergoes photoconversion to study degradation of Dendra2‐Nrf2 in single live cells. Using this approach, we show that the half‐life (t1/2) of Dendra2‐Nrf2 in the whole cell, under homeostatic conditions, is 35 min. Inhibition of the proteasome with MG‐132 or induction of oxidative stress with tert‐butylhydroquinone (tBHQ) extended the half‐life of Dendra2‐Nrf2 by 6‐ and 28‐fold, respectively. By inhibiting nuclear export using leptomycin B, we provide direct evidence that degradation of Nrf2 also occurs in the nucleus and involves PML‐NBs. We further demonstrate that co‐expression of Dendra2‐Nrf2 and Crimson‐PML‐I lacking two PML‐I sumoylation sites (K65R and K490R) changed the decay rate of Dendra2‐Nrf2 in the nucleus and stabilized the nuclear derived Nrf2 levels in whole cells. Altogether, these data suggest that functional PML‐NBs alter the rate of degradation of nuclear Nrf2, which underscores the importance of these structures in Nrf2‐mediated response to oxidative stress.

Support or Funding Information

HHS |National Institutes of Health (NIH):, SC1CA143985; HHS |National Institutes of Health (NIH):, SC1CA211030; HHS |National Institutes of Health (NIH):, UL1TR000445; HHS |National Institutes of Health (NIH):, U54MD007593; HHS |National Institutes of Health (NIH):, G12MD007586; HHS |National Institutes of Health (NIH):, U54CA163069; HHS |National Institutes of Health (NIH):, R24DA036420; HHS |National Institutes of Health (NIH):, S10RR0254970; HHS |National Institutes of Health (NIH):, 5R25GM059994; HHS |National Institutes of Health (NIH):, 5T32GM007628‐37

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of GIV‐GRP78 Interaction During Endoplasmic Reticulum Stress
Authors: Limso Clariss , Ngo Jordan , Nguyen Peter , Bhandari Deepali
Published: June 2021

Endoplasmic Reticulum (ER) stress occurs when misfolded proteins accumulate in the ER. Cells facing ER stress initially attempt to restore cellular homeostasis, but if the stress becomes chronic and homeostasis is not achieved within a reasonable timeframe, cells initiate programmed cell death. Cancer cells are able to withstand and survive ER stress better than regular cells. Overexpression of the chaperone Glucose Regulated Protein 78 kDa (GRP78) is one of the key factors that promote cancer cell survival during ER stress. Although it is an ER resident protein, recent studies have shown that when overexpressed, GRP78 can translocate to other cellular locations including the cell surface from where it can activate cytoprotective signals; however, the mechanism by which it does so remains elusive. Our laboratory has recently identified Gα‐Interacting Vesicle associated protein (GIV) ‐ a known enhancer of the pro‐survival Akt signaling ‐ as a novel binding partner of GRP78. We hypothesize that this interaction may be the missing link in understanding the mechanism by which cell surface GRP78 increases cell viability. Because GIV and GRP78 are normally localized in different cellular compartments, we characterized the spatiotemporal dynamics of this interaction by analyzing the sub‐cellular localization of these proteins in HeLa cells treated with Tunicamycin (an ER stress inducer) through differential centrifugation and immunofluorescence microscopy. Our results showed that GIV is present in both membrane and cytosolic fractions during normal as well as ER stress conditions. GRP78, on the other hand, is in the membrane fraction under normal cellular conditions but appears in the cytosolic fraction upon ER stress. GIV and GRP78 co‐localized in a perinuclear region as well as at the cell surface of the ER stressed cells. Finally, we also mapped the interaction between GIV and GRP78 to the carboxyl terminus domains on both proteins by performing pull‐down assays. Taken together, this work has characterized GIV‐GRP78 interaction in the face of ER stress. Our current and future goals include studying the role of this novel interaction in mediating cell survival during chronic ER stress.

Support or Funding Information

This project is supported by the NIH‐NIGMS grant # SC2GM121246 (DB). JN is supported by the NIH MARC program award number: T34GM008074.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial Dysfunction in Type 2 Diabetic Skeletal Muscle Cells
Authors: Castaneda Diana L. , Yamazaki Katrina
Published: June 2021

Type 2 diabetes (T2D) is an epidemic disease characterized by high blood glucose levels and insulin resistance and is usually associated with obesity. Inadequate glucose utilization by the cell leads to an increase in high fat utilization to make cellular energy, which can lead to mitochondrial dysfunction. Typically, autophagy maintains homeostasis by removing damaged portions of the mitochondria and recycling the functional components to form new efficient mitochondria. The main focus of this project is to analyze if there is an impairment of the autophagy process in T2D that leads to an accumulation of dysfunctional mitochondria. We also evaluated mitochondrial dynamics to determine if the rate of mitochondrial fission exceeds that of fusion, as this may also explain the accumulation. Using an in vitro model of T2D, fluorescent microscopy and biochemical analysis of cells treated with high fats were evaluated to look at mitochondrial dynamics and number. Microscopic analysis showed a decrease in mitochondrial number and a decrease in mitochondrial membrane potential in T2D cells versus control. Furthermore, results revealed that in a diabetic environment there was an increase in mitochondrial fragmentation as seen with an increase in DRP‐1 expression. Lastly, results showed that Beclin‐1 increases under diabetic conditions signifying that autophagy is still being activated. In conclusion, our results demonstrate that there is an increase in mitochondrial fission and activation of autophagy in T2D. More work is needed to determine if fission occurs at a rate faster than the normal autophagy response can handle to explain the consequences of T2D.

Support or Funding Information

California State University, Los Angeles Louis Stokes Alliance for Minority Participation (CSULA‐ LSAMP)andCalifornia State University, Los Angeles Minority Opportunities in Research Programs‐ Minority Biomedical Research Support Program‐Research Initiative for Scientific Enhancement (CSULA MORE‐ RISE Undergraduate)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Potential mitochondrial dysfunction in skeletal muscle of mouse models of Osteogenesis imperfecta.
Authors: Gremminger Victoria , Jeong Youngjae , Cunningham Rory , Meers Grace , Rector R. Scott , Phillips Charlotte
Published: June 2021

Osteogenesis imperfecta (OI), also referred to as brittle bone disease, is a heritable connective tissue disorder characterized by short stature, fragile bones, craniofacial deformity and muscle weakness. OI is most often the result of autosomal dominant gene defects in the type I collagen genes: COL1A1 and COL1A2. The remaining rare autosomal recessive forms result from defects in gene products responsible for modification and packaging of type I collagen. Muscle weakness not only impacts a patient's quality of life, but likely negatively affects bone health via altered bone‐muscle crosstalk.

The oim/oim mouse models severe human type III OI with reduced body mass, skeletal fragility, and reduced muscle mass and function compared to their wildtype (WT) counterparts. Whereas, the G610C OI mouse models the less severe human type I/IV OI with skeletal weakness, but near normal muscle function. The differential presence of inherent muscle pathology between oim/oim and G610C mice suggest mutation specific mechanisms.

To investigate the mechanisms underlying the muscle pathology, we examined citrate synthase activity, a known marker of mitochondrial activity and determined, in the mixed fiber type gastrocnemius muscle, that citrate synthase activity was significantly increased only in oim/oim mice relative to WT mice, despite evidence of reduced muscle function; whereas G610C mice did not have altered citrate synthase activity. The unexpected increase in citrate synthase activity led to the question of whether or not mitochondria play a role in the muscle weakness observed in OI. To assess mitochondrial health, we examined biomarkers of mitogenesis and mitophagy, and found increased mitogenesis in oim/oim mice relative to WT as determined by PGC1α and TFAM protein content in the mixed gastrocnemius muscle. Additionally, measurement of mitochondrial electron transport complex proteins also suggest altered mitochondria in the oim/oim mice. These data suggest that oim/oim mice have increased mitogenesis and altered mitochondrial function accompanying decreased muscle function.

Support or Funding Information

Supported by March of Dimes, National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (RO1 AR055907), the Leda J. Sears Trust, the Kansas City Area Life Sciences Institute Patton Trust Research, Inc., University of Missouri Research Board Grant, and in part by the Department of Veterans Affairs (VA‐Merit Grant I01BX003271‐01). A portion of this work was also supported by resources and the use of facilities at the Harry S. Truman Memorial Veterans Hospital in Columbia, MO.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dual Perturbation of Electron Transport Chain (ETC) Complex and ATP Synthase Triggers PINK1/Parkin‐dependent Mitophagy
Authors: Ramirez Adrian Thomas , Liu Xuedong
Published: June 2021

Neurodegenerative disorders like Parkinson's Disease arise in part due to accumulation of unhealthy mitochondria. PINK1 and Parkin, found to be deficient in some Familial Parkinson's Disease patients, are known to orchestrate mitochondrial quality control responses like mitophagy. Research into PINK1 and Parkin function has been focused primarily on downstream signaling events, but the exact type of damage signals originating from defected mitochondria to trigger PINK1 stabilization and subsequent Parkin recruitment to the outer membrane of damaged mitochondria remains elusive. We propose PINK1 and Parkin act in tandem to monitor the integrity and activity of ETC complexes and ATP synthase to ensure robust energy production. To test this hypothesis, we profiled several small molecule inhibitors with well‐documented inhibitory activities towards ETC complexes for their effects on PINK1 and Parkin activation. We found that while dual inhibition of complex III and V results in robust Parkin recruitment to damaged mitochondria and mitophagy responses, dual inhibition of complex I and V has only modest effect and other combinations have no effect. PINK1 and Parkin activation as a result of dual assault of ETC complexes can be phenocopied with perturbations of these complexes with siRNA. Thus, our study supports the idea that PINK1 and Parkin are guardians of ETC quality and status. Damaged mitochondria as a result of specific defects in oxidative phosphorylation can be cleared by PINK1 and Parkin dependent mitophagy. This mechanism could be relevant in understanding their dysregulation in Parkinson's Disease.

Support or Funding Information

National Institute of General Medical Sciences R01 GM113141, National Institute of General Medical Sciences T32GM08759, S10 RR026680

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding Specificity of Glycosaminoglycan Interactions with Proteins
Authors: Desai Umesh R. , Patel Bhaumik B.
Published: June 2021

Glycosaminoglycans (GAGs) play key roles in virtually all biologic responses through their interaction with proteins. Generally, GAGs are believed to interact non‐specifically with proteins. The primary reason for this belief is the highly electrostatic nature of their interactions and the fact that only one homogeneous GAG sequence has reached the clinic – the anticoagulant heparin pentasaccharide sequence. Are highly specific GAG–protein interactions so rare? Does nature rely primarily on non‐specificity to mediate the biological roles of GAGs? These questions are of major significance and insights into specificity of GAG–protein interactions may lead to many more GAG sequences reaching the clinic. Yet, a major challenge in understanding these interactions is the massive structural complexity of GAGs. Computational approaches are extremely useful in navigating this challenge and, in some cases, the only avenue to gain comprehensive insight. Computationally, most basic and many advanced questions on GAG–protein interactions can be answered including 1) does my protein bind to GAGs?, 2) where does the GAG bind?, 3) does the protein preferentially recognize a particular GAG type?, 4) what is the most optimal GAG chain length?, 5) what is the structure of the most favored GAG sequence?, 6) is the GAG–protein system ‘specific’, ‘non‐specific’, or a combination of both?, etc. Recent advances in the areas of thrombosis and cancer from our laboratories have led to discoveries of GAG sequences that are highly specific for their protein targets. The results raise the promise that many proteins may recognize GAGs with high specificity and clinically relevant GAG sequences are waiting to be identified.

Support or Funding Information

P01 HL107152R01 HL090586R25 HL128639

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biochemical Characterization of Missense Mutations in O‐GlcNAc Transferase Found in Patients With X‐Linked Intellectual Disability
Authors: George Stephan Nicholas , Selvan Nithya , Stephen Hannah , Wells Lance
Published: June 2021

O‐GlcNAc is an abundant post‐translational modification of nucleocytoplasmic proteins that has been implicated in biological processes such as transcription, cell‐cycle regulation, and nutrient‐sensing. O‐GlcNAc transferase (OGT) catalyzes the reversible transfer of the monosaccharide N‐acetylglucosamine (GlcNAc) from the donor substrate UDP‐GlcNAc to serine and threonine residues on nuclear, cytosolic, and mitochondrial substrates, while the enzyme O‐GlcNAcase (OGA) reverses this process. OGT consists of a tetratricopeptide repeat (TPR) region and catalytic domains, both of which are required for substrate glycosylation. Through exome sequencing of multiple patient samples, 5 individual missense mutations in the TPR region of OGT appear causal for X‐linked intellectual disability (XLID). However, the effect of each of these mutations on enzyme characteristics is poorly understood. To elucidate the mechanism through which each mutation causes development of XLID, the recombinant, purified OGT variants are being biochemically characterized. We are currently in the process of defining the differential interactome/O‐GlcNAc proteome of OGT variants using quantitative glycoproteomic approaches and performing functional assays to identify processes linking OGT to XLID.

Support or Funding Information

This work is funded by grants from the Keck Foundation and the NIH (P41GM103490) to Dr. Lance Wells.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mapping astrocytes to understand development and function of the hindbrain
Authors: Mostafa Hasnaa R. , Czeisler Catherine , Otero Jose J. , Aljuhani Mona
Published: June 2021

Astrocytes, the most abundant glial cells in the mammalian brain, are critical regulators of brain development and physiology through dynamic and often bidirectional interactions with neuronal synapses. The embryonic origin of astrocytes is known to play a major role in defining the functional diversity of astrocytes. Astrogliogenesis is known to temporally follow neurogenesis and neural migration in the forebrain. However, recent studies have demonstrated that the differentiation and distribution of astrocytes may vary between different brain regions. The molecular mechanisms by which astrocytes are specified, interact, and grow in order to shape their complex morphologies are still unknown. A first step in understanding this process is to address how astrocytes develop using lineage markers, including PHOX2B, NKX2.2, OLIG3, and ATOH1, in the murine hindbrain. Characterizing the development of astrocytes will significantly improve the understanding astrocytic contributions to neural function. Furthermore, given their multifaceted quality, it is imperative to attribute astrocytic dysfunction to a wide range of disease states, such as neurodegenerative disorders.

Support or Funding Information

3R01hl132355‐01a1s1

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex‐specific neurobiological differences in addiction neuropathology: An educational pilot program for next generation STEM workforce
Authors: Vieira Philip
Published: June 2021

AIM

California State University Dominguez Hills (CSUDH) is located in central Los Angeles county, servicing a diverse community. This region is considered a hotspot for drug abuse due to a variety of factors, including a close proximity with the Mexico border, an abundance of transportation facilities to support trafficking, and a substantial population of several at‐risk groups. While graduates from CSUDH often go on to work in the community, these students do not yet receive the education in the factors leading to substance addiction. The aim of this program is to provide a set of courses which will train students on the latest addiction prevention and treatment research. The initial pilot course focused on sex‐specific and neurobiological corollaries to drug dependence.

METHODS

During the 2016–2018 academic years, 6 classes of senior undergraduate students were targeted for this program. Students worked individually and in small groups to prepare discussions on the neurobiology of addiction, following several original and secondary literature sources, including Michael Kuhar's The Addicted Brain (2011), peer‐reviewed publications and published abstracts from the several conferences (including Experimental Biology) from 2014–2017. Discussions with addiction researchers were also included. Students submitted research proposals to address potential gaps in the neuropharmacology literature, focusing on sex‐specific studies. Survey data were collected before and after the course to assess learning outcomes and career trajectories for these next generation STEM workforce students.

RESULTS

This pilot program has been well‐received by the participating students. While it was clear that some students struggled with the content, all of them were personally engaged in the material. Indeed, many students openly shared their own experiences with drug dependence.

CONCLUSION

Students need earlier exposure to the neurobiology of drug abuse to support their success in this course. We will therefore offer a psychopharmacology course to students prior to taking this course.

Support or Funding Information

California State University, Dominguez Hills Faculty Research Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effect of myotonic dystrophy on intracellular calcium handling and mitochondrial function.
Authors: Rios Gregory Herrera , Yamazaki Katrina
Published: June 2021

Myotonic dystrophy (DM) is a multisystemic genetic disorder that leads to skeletal muscle, cardiac and neurological impairment. DM is characterized by multiple CUG repeats along the DMPK gene, with the severity of the disease increasing with the number of repeats. DM patients exhibit extended muscle contractions due to misregulated ion channels. As a result, normal ion balance, notably calcium, is disrupted leading to cellular damage and eventual death. While research has been aimed at identifying how the CUG repeats regulate gene expression, little is known regarding the effects of DM on organelle function. This study works to understand how DM affects calcium handling between the mitochondria and smooth endoplasmic reticulum to cause extended muscle contractions. An in vitro model of DM will be created by transfecting C2C12 cells with a plasmid containing 950 CUG repeats. This will allow for testing of overall calcium distribution and organelle functions in the control versus diseased state. We will run a mixture of fluorescent microscopy experiments to evaluate calcium distribution and organelle structure, as well as biochemical and functional assays to assess the difference in organelle function between these two groups. I hypothesize that DM cells will exhibit a misregulation of calcium handling leading to calcium overload in the mitochondria, thereby decreasing mitochondrial function. Results should indicate calcium mishandling and inhibited ATP production as shown by the results of the various assays. Therefore, if results should hold to be true, DM will indeed induce calcium mishandling through misregulation of calcium handling protein channels and a decrease in ATP synthesis due to mitochondrial dysfunction. Currently, there are limited DM treatments available and the identification of a new possible cell target may lead to the development of new pharmacological agents to combat the disease.

Support or Funding Information

The project will be supported and funded by the project advisor, Dr. Katrina Yamazaki.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An overview of minority‐based research on Diabetes type II in the US between 2012–2017
Authors: Tajkarimi Ali
Published: June 2021

In 2015, diabetes killed more people than breast cancer and AIDs combined. According to the American Diabetes Association, diabetes affects nearly 29 million Americans each year. The purpose of this research was to explore possible pattern with diabetes with respect to racial disparities in the United States. The method used to find available research articles based on the medical research search engine called PubMed within the years of 2012 until 2017 and 2010 bureau of census data. According to the results found in this study, there is a consistent quantity of race‐based study reports each year. The data demonstrated that percentage of research data on Diabetes in minorities are African American(20.02%), Native Americans (42.3%), Asian (22.0%)and Hispanics(15.7%). The percentages were calculated with the ratio between the number of articles made for each minority for the last 5 years. In light of findings of this study, there is a need for diabetes research in the African American and Hispanic groups.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Egg consumption and risk of cardiovascular diseases, evidence from China Health and Nutrition Survey 2000–2011
Authors: Zhang Ji , Du Wenwen , Huang Feifei , Su Chang , Zhang Jiguo , Jiang Hongru , Jia Xiaofang , Ouyang Yifei , Wang Yun , Li Li , Wang Huijun
Published: June 2021

Objective

The morbidity and hospitalization of CVDs in China have kept increasing since 2000, outpacing the GDP growth. CVD may bring heavier burden in the coming decade to Chinese people, economically and physically. There are evidence indicating the strong relationship between high plasma concentration of cholesterol and CVDs, but the association between CVDs and dietary cholesterol intake remains unclear. We therefore conducted a longitudinal analysis with multifactor adjusting models to assess the nonfatal myocardial infarction (MI) and stroke risks of egg consumption in a sample of Chinese adults.

Method

Five rounds of China Health and Nutrition Survey from 2000 to 2011 were used in this study. 5289 non‐myocardial infarction or non‐stroke subjects >30y in 2000 and followed at least once in the subsequence rounds were included in the final analysis. Three consecutive 24h dietary recall was used to collect egg consumption data, and self‐reported nonfatal MI and stroke history at each follow‐up was used for the CVD diagnosis. Cox proportional hazards regression models with time‐dependent explanatory variables was conducted to compute relative risks and 95% CIs.

Result

The follow‐up time of the myocardial infarction(MI) patients was averagely 9.62 years, while that of the stroke patients was 9.59 years. In 2000, the average daily egg consumption of the whole population was 50.01±38.39g/day, while the figure slightly decreased in the year of 2011, at 46.52 ±34.18g/day. Adjusted for age, gender, education level, household income, urbanized level, geographic location, and individual behavioral factors (including drinking, smoking, physical activity level, weight status, and chronic disease history ), multivariate Cox regression analysis revealed that egg consumption was not a significant risk factor of the CVDs in this study(MI and stroke).

Conclusion

Our study shows no significant relation between egg consumption and CVD risks in this population of which egg consumption was mostly below 100g/day.

Support or Funding Information

The authors thank the team at National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention, and the Carolina Population Center, University of North Carolina at Chapel Hill. The China Health and Nutrition Survey data collection was in part funded by research grants from the NIH (R01‐HD30880, DK056350, R24 HD050924, and R01‐HD38700) and the NIH Fogarty International Center and by the Chinese Center for Disease Control and Prevention. The content of the paper is solely the responsibility of the authors and does not necessarily represent the official views of the funders.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of Field Measures Versus Imaging and Total Versus Regional Body Composition in Predicting Inflammation in Preadolescent Hispanic Girls
Authors: Bea Jennifer W. , Funk Janet , Hetherington‐Rauth Megan , Wertheim Betsy , Mosqueira Lucia , Thuraisingam Ravina , Lee Vinson , Blew Robert , Lohman Timothy , Going Scott B.
Published: June 2021

Importance

C‐reactive protein (CRP) is an inflammatory marker linked with obesity, particularly abdominal obesity, and cardiovascular disease (CVD) in adults. The optimal measure of adiposity to predict CRP in children is not known.

Objective

To compare total and regional estimates of body composition, by imaging and field techniques, for the prediction of CRP among Hispanic girls.

Design

This observational, cross‐sectional study examined participants from the STAR Study between October 2013 and November 2016.

Setting

A population‐based study performed at a research laboratory, with subject recruitment performed in schools and pediatric clinics in the greater area of Tucson, Arizona USA.

Participants

Healthy girls aged 9 to 12 years, with no known metabolic disorders, were recruited. Of the 239 Hispanic subjects who provided informed consent to participate, 232 had complete data and were included in the current analysis.

Main Outcomes and Measures

Standard anthropometric techniques were used to measure height, weight, and waist circumference. Densitometric measures of total and regional lean and fat depots by dual energy X‐ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT) were completed. Fasting serum CRP was measured by the Beckman Coulter AU5812 Clinical Chemistry Analyzer. Associations between each body composition parameter and CRP were tested using linear regression (log‐transformed, continuous CRP) and ordinal logistic regression (CRP <1, >1<3, ≥3 mg/L), controlling for maturation, dietary energy, physical activity, and medications.

Results

All measures of total and regional body fat were positively associated with CRP (P<0.0001), except for intermuscular fat by pQCT. There were no clinically relevant differences between anthropometric (BMI; waist circumference) and DXA‐derived (total fat and regional fat: trunk, gynoid, android fat, leg) measures of fat in their associations with CRP. Total lean mass by DXA was significantly, inversely associated with CRP (P<0.0001).

Support or Funding Information

Funding was provided by the National Institute of Child Health and Human Development (HD074565) and the National Cancer Institute (P30CA023074).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.