Abstract 56: Platelet Specific Knockout of Glucose Transporter 3 Leads to Altered Metabolism and Decreased Platelet Activation

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Trevor P Fidler ◽  
Elizabeth Middleton ◽  
Jesse W Rowley ◽  
Luc Boudreau ◽  
Robert A Campbell ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Plasma Membrane ◽  
Glucose Uptake ◽  
Platelet Activation ◽  
Glucose Transporter ◽  
Platelet Reactivity ◽  
Thrombus Formation ◽  
Mitochondrial Uncoupling ◽  
Granule Release

Patients with diabetes display increased thrombosis and platelet activation. In these disorders, the systemic milieu is characterized by multiple metabolic changes including increased glucose concentrations. Preliminary metabolomics analysis of platelets from patients with type 2 diabetes revealed an accumulation of glycolytic and TCA intermediates relative to healthy controls. Therefore we hypothesized that decreasing platelet glucose uptake would limit glycolysis thereby decreasing energy production and platelet reactivity. Platelets import glucose via two glucose transporters GLUT1 and GLUT3. GLUT1 is expressed on the plasma membrane and GLUT3 is expressed predominantly on alpha granule membranes (85%) and to a lesser extent on the plasma membrane (15%). To better understand the consequences of glucose metabolism on platelet function we generated a platelet specific knockout (KO) of GLUT3 using a Pf4 Cre recombinase transgenic mouse crossed to mice that harbor floxed GLUT3 alleles. Platelet glycogen content and glycolytic intermediates were significantly reduced in GLUT3 KO platelets compared to controls, and following mitochondrial uncoupling exhibited reduced glycolysis rates. Interestingly, under these conditions, mitochondrial maximal respiration was increased two-fold, with no change in mitochondrial density, or citric acid cycle intermediates. In vitro , GLUT3 deficient platelets display a 90% reduction of spreading on fibrinogen and collagen matrixes and significant reductions in CD62p surface translocation and GPIIbIIIa activation following stimulation with multiple agonists. Additionally makers of alpha granule release were significantly reduced. In vivo analysis of GLUT3 KO mice using a 10% ferric chloride model of arterial thrombosis and a tail-bleed model indicated no alteration in thrombosis between littermate controls and knockouts. However in a KBx/N model of rheumatoid arthritis GLUT3 KO mice exhibited significantly reduced disease severity. Together, these data indicate that GLUT3-mediated glucose uptake is essential for platelet activation, spreading and alpha granule release. GLUT3 modulates mechanisms that promote rheumatoid arthritis but not those that regulate in vivo thrombus formation.

Glucose Transporter 3 in Platelets Facilitates Alpha-Granule Mediated Glucose Uptake, Driving Intragranular Glycolysis That Is Required for Platelet Degranulation and Activation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 417-417
Author(s):  
Fidler P.L Trevor ◽  
Elizabeth Middleton ◽  
Jesse W Rowley ◽  
Luc Boudreau ◽  
Robert A. Campbell ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Plasma Membrane ◽  
Glucose Metabolism ◽  
Glucose Uptake ◽  
Platelet Activation ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Conflicts Of Interest ◽  
Platelet Factor

Abstract Patients with diabetes display increased thrombosis and platelet activation. Preliminary metabolomics analysis of platelets from patients with type 2 diabetes revealed an accumulation of glycolytic and TCA intermediates relative to healthy controls. In vitro studies of platelets under hyperglycemic conditions suggest that glucose metabolism may lead to increased platelet activation. Platelets import glucose via two glucose transporters GLUT1, which is expressed on the plasma membrane, and GLUT3, which is expressed on the plasma membrane (15%) and the remaining 85% on α-granule membranes. Following stimulation, platelet α-granules translocate to the plasma membrane and release their cargo. To better understand the consequences of glucose metabolism on platelet function we generated a platelet specific knockout of GLUT3 using a Pf4 Cre recombinase transgenic mouse crossed to mice that harbor floxed GLUT3 alleles. GLUT3 KO platelets displayed a 23% reduction in basal glucose uptake compared to littermate controls. Control platelets stimulated with thrombin displayed a significant increase in glucose uptake whereas KO platelets failed to show any change. Additionally, platelet glycogen content and glycolysis intermediates were significantly reduced in KO platelets, which exhibited reduced glycolytic rates following metabolic stress (mitochondrial uncoupling). Because GLUT3 KO platelets had only a minor decrease in glucose uptake under basal conditions, but platelet glucose metabolism was dramatically altered when stimulated, we hypothesized that under basal conditions, GLUT3 facilitates glucose uptake into α-granules, to generate glycogen and fuel intragranular glycolysis that generates the energy required for α-granule degranulation. To test this hypothesis we permeabilized platelet plasma membranes, but not α-granule membranes using saponin and incubated the platelets with C13-glucose. Under these conditions, control platelets produced 2.5-fold more C13 -lactic acid than KO platelets. In vitro, GLUT3 knockout platelets display a 90% reduction in spreading on fibrinogen and collagen matrices and significant reductions in α-granule degranulation as marked by CD62p surface translocation, platelet factor 4 release, and the persistence of α-granules observed in electron micrographs of stimulated platelets. In vivo in a KBx/N model of rheumatoid arthritis, which is dependent in part on platelet activation, GLUT3 KO mice exhibited significantly reduced severity of disease. Analysis of GLUT3 mice in models of arterial thrombosis, deep vein thrombosis and tail-bleeding indicated no alteration in thrombosis between littermate controls and KO mice. Together these data indicate that GLUT3 mediated α-granule glucose uptake is essential for platelet activation and degranulation. Moreover reducing platelet GLUT3 may ameliorate the course of rheumatoid arthritis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Alterations in Glucose Metabolism During the Transition to Heart Failure: The Contribution of UCP-2

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 552 ◽  
Author(s):  
Hanna Sarah Kutsche ◽  
Rolf Schreckenberg ◽  
Martin Weber ◽  
Christine Hirschhäuser ◽  
Susanne Rohrbach ◽  
...  
Keyword(s):  
Heart Failure ◽  
Glucose Metabolism ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Contractile Function ◽  
Uncoupling Protein ◽  
Left Ventricular ◽  
Mitochondrial Uncoupling ◽  
Glut 4

The cardiac expression of the mitochondrial uncoupling protein (UCP)-2 is increased in patients with heart failure. However, the underlying causes as well as the possible consequences of these alterations during the transition from hypertrophy to heart failure are still unclear. To investigate the role of UCP-2 mechanistically, expression of UCP-2 was silenced by small interfering RNA in adult rat ventricular cardiomyocytes. We demonstrate that a downregulation of UCP-2 by siRNA in cardiomyocytes preserves contractile function in the presence of angiotensin II. Furthermore, silencing of UCP-2 was associated with an upregulation of glucose transporter type (Glut)-4, increased glucose uptake, and reduced intracellular lactate levels, indicating improvement of the oxidative glucose metabolism. To study this adaptation in vivo, spontaneously hypertensive rats served as a model for cardiac hypertrophy due to pressure overload. During compensatory hypertrophy, we found low UCP-2 levels with an upregulation of Glut-4, while the decompensatory state with impaired function was associated with an increase of UCP-2 and reduced Glut-4 expression. By blocking the aldosterone receptor with spironolactone, both cardiac function as well as UCP-2 and Glut-4 expression levels of the compensated phase could be preserved. Furthermore, we were able to confirm this by left ventricular (LV) biopsies of patients with end-stage heart failure. The results of this study show that UCP-2 seems to impact the cardiac glucose metabolism during the transition from hypertrophy to failure by affecting glucose uptake through Glut-4. We suggest that the failing heart could benefit from low UCP-2 levels by improving the efficiency of glucose oxidation. For this reason, UCP-2 inhibition might be a promising therapeutic strategy to prevent the development of heart failure.

scholarly journals Specialized sorting of GLUT4 and its recruitment to the cell surface are independently regulated by distinct Rabs

2013 ◽  
Vol 24 (16) ◽  
pp. 2544-2557 ◽  
Author(s):  
L. Amanda Sadacca ◽  
Joanne Bruno ◽  
Jennifer Wen ◽  
Wenyong Xiong ◽  
Timothy E. McGraw
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Receptor Activation ◽  
Glut4 Translocation ◽  
Insulin Stimulation ◽  
Transport Vesicles ◽  
Glucose Transporter Glut4

Adipocyte glucose uptake in response to insulin is essential for physiological glucose homeostasis: stimulation of adipocytes with insulin results in insertion of the glucose transporter GLUT4 into the plasma membrane and subsequent glucose uptake. Here we establish that RAB10 and RAB14 are key regulators of GLUT4 trafficking that function at independent, sequential steps of GLUT4 translocation. RAB14 functions upstream of RAB10 in the sorting of GLUT4 to the specialized transport vesicles that ferry GLUT4 to the plasma membrane. RAB10 and its GTPase-activating protein (GAP) AS160 comprise the principal signaling module downstream of insulin receptor activation that regulates the accumulation of GLUT4 transport vesicles at the plasma membrane. Although both RAB10 and RAB14 are regulated by the GAP activity of AS160 in vitro, only RAB10 is under the control of AS160 in vivo. Insulin regulation of the pool of RAB10 required for GLUT4 translocation occurs through regulation of AS160, since activation of RAB10 by DENND4C, its GTP exchange factor, does not require insulin stimulation.

Deoxyandrographolide promotes glucose uptake through glucose transporter-4 translocation to plasma membrane in L6 myotubes and exerts antihyperglycemic effect in vivo

2015 ◽  
Vol 768 ◽  
pp. 207-216 ◽  
Author(s):  
Deepti Arha ◽  
Sukanya Pandeti ◽  
Akansha Mishra ◽  
Swayam Prakash Srivastava ◽  
Arvind Kumar Srivastava ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Glucose Transporter 4 ◽  
Antihyperglycemic Effect ◽  
L6 Myotubes

scholarly journals VAS2870 and VAS3947 attenuate platelet activation and thrombus formation via a NOX-independent pathway downstream of PKC

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Wan Jung Lu ◽  
Jiun Yi Li ◽  
Ray Jade Chen ◽  
Li Ting Huang ◽  
Tzu Yin Lee ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Therapeutic Agents ◽  
Independent Manner ◽  
Downstream Signaling ◽  
Nox Inhibitors ◽  
Normal Hemostasis ◽  
Granule Release

AbstractNADPH oxidase (NOX) enzymes are involved in a various physiological and pathological processes such as platelet activation and inflammation. Interestingly, we found that the pan-NOX inhibitors VAS compounds (VAS2870 and its analog VAS3947) exerted a highly potent antiplatelet effect. Unlike VAS compounds, concurrent inhibition of NOX1, 2, and 4 by treatment with ML171, GSK2795039, and GKT136901/GKT137831 did not affect thrombin and U46619-induced platelet aggregation. These findings suggest that VAS compounds may inhibit platelet aggregation via a NOX-independent manner. Thus, we aimed to investigate the detailed antiplatelet mechanisms of VAS compounds. The data revealed that VAS compounds blocked various agonist-induced platelet aggregation, possibly via blocking PKC downstream signaling, including IKKβ and p38 MAPK, eventually reducing platelet granule release, calcium mobilization, and GPIIbIIIa activation. In addition, VAS compounds inhibited mouse platelet aggregation-induced by collagen and thrombin. The in vivo study also showed that VAS compounds delayed thrombus formation without affecting normal hemostasis. This study is the first to demonstrate that, in addition to inhibiting NOX activity, VAS compounds reduced platelet activation and thrombus formation through a NOX-independent pathway downstream of PKC. These findings also indicate that VAS compounds may be safe and potentially therapeutic agents for treating patients with cardiovascular diseases.

scholarly journals Cathepsin G Cleavage of PAR4 Generates a Novel Tethered Ligand That Induces Platelet Activation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 2-2
Author(s):  
Michelle L. Stoller ◽  
Indranil Basak ◽  
James Alsobrooks ◽  
Paul F. Bray ◽  
Robert A. Campbell
Keyword(s):  
Platelet Activation ◽  
Cleavage Site ◽  
Conflicts Of Interest ◽  
Platelet Reactivity ◽  
Thrombus Formation ◽  
Cathepsin G ◽  
Cleavage Sites ◽  
Tethered Ligand ◽  
Tethered Ligands

Atherosclerotic vessel injury induces recruitment of both platelets and neutrophils where multiple proteases induce platelet activation and aggregation. Platelets contain two protease activated receptors, PAR1 and PAR4, the cleavage of which results in exposure of a new amino terminus to serve as a tethered ligand. Released neutrophil cathepsin G (CatG) has been shown to be a physiologic modulator of platelet thrombus formation in mice. CatG activates PAR4 and not PAR1, presumably because CatG cleaves PAR1 by removing its tethered ligand. However, neither the CatG biochemical cleavage of PAR4 nor the resulting tethered ligands have been reported. The goals of the current study are to (1) identify the CatG-PAR4 cleavage sites and resulting tethered ligands and (2) determine how CatG-stimulated PAR4 signaling is altered by the PAR4 Ala120Thr variant. We synthesized two portions of the PAR4 extracellular N-terminus: amino acids Asp38-Ser58 (PAR4-B) and Asp57-Arg78 (PAR4-C) and exposed each peptide to purified CatG. Mass spectrometry identified two major cleavage sites for PAR4-B: the previously documented CatG and thrombin site Arg47-Gly48 and a novel Cys54-Ala55. Analysis of PAR4-C digestion yielded an additional three novel CatG cleavage sites, two major: Arg68-Ala69 and Leu71-Leu72, and one minor: Leu70-Leu71. Neither concentration or time of exposure appeared to alter the CatG cleavage sites. To assess functionality, we generated peptides based on the novel cleavage sites produced by CatG cleavage. Human washed platelets were treated with each peptide, and platelet activation was assessed by PAC-1 binding. As expected, the known tethered ligand sequence GYPGQV showed a statistically significant increase in PAC-1 binding (p=.02) compared to resting platelets. Three of the remaining four novel peptides generated no significant change in PAC-1 binding compared to baseline. However, peptide 3, representing novel tethered ligand ALLLGW, induced a substantial increase (462%) in PAC-1 binding compared to resting platelets. To assess the effect of the PAR4Ala120Thr variant on CatG-stimulated platelet reactivity, human washed platelets were collected from donors homozygous for Ala120 or Thr120 and stimulated with CatG. Platelets expressing the Thr120 variant displayed a significant increase in PAC-1 compared to Ala120 platelets (54%; p=.004). Addition of a CatG inhibitor caused a significant decrease in platelet activation triggered by CatG for both groups (Thr: -81%, p=.000001; Ala: -78%, p=.00017), and abrogated the significant increase in platelet activation displayed by Thr120 platelets (p=.99). To further examine the relationship between the PAR4 Ala120Thr variant and CatG, platelets from each variant were subjected to increasing amounts of CatG. Platelet activation was measured by PAC-1 binding and P-selectin expression. Both PAC-1 binding and P-selectin expression were significantly increased in platelets from Thr120 donors compared to Ala120 platelets (PAC-1, p=.026; P-selectin, p=.025). Overall, our study identified a previously unidentified CatG cleavage site in PAR4, which produced a novel tethered ligand capable of activating platelets. Because cleavage resulting in the ALLLGW ligand is downstream of the thrombin cleavage site, CatG may modulate in vivo thrombin-induced signaling in platelets or other cell types. Our findings also indicate the hyperactive response of 120Thr platelets is not dependent on a specific PAR4 protease. These new insights into PAR4 biology may provide targets for future antithrombotic therapies. Disclosures No relevant conflicts of interest to declare.

Investigation of the Interaction of Blood Platelets with the Coagulation System at the Site of Plug Formation In Vivo in Man - Effect of Low-Dose Aspirin

1987 ◽  
Vol 57 (01) ◽  
pp. 062-066 ◽  
Author(s):  
P A Kyrle ◽  
J Westwick ◽  
M F Scully ◽  
V V Kakkar ◽  
G P Lewis
Keyword(s):  
Platelet Activation ◽  
Thrombin Generation ◽  
Low Dose ◽  
Bleeding Time ◽  
Blood Platelets ◽  
Dose Aspirin ◽  
Low Dose Aspirin ◽  
Plug Formation ◽  
Granule Release

SummaryIn 7 healthy volunteers, formation of thrombin (represented by fibrinopeptide A (FPA) generation, α-granule release (represented by β-thromboglobulin [βTG] release) and the generation of thromboxane B2 (TxB2) were measured in vivo in blood emerging from a template bleeding time incision. At the site of plug formation, considerable platelet activation and thrombin generation were seen within the first minute, as indicated by a 110-fold, 50-fold and 30-fold increase of FPA, TxB2 and PTG over the corresponding plasma values. After a further increase of the markers in the subsequent 3 minutes, they reached a plateau during the fourth and fifth minute. A low-dose aspirin regimen (0.42 mg.kg-1.day-1 for 7 days) caused >90% inhibition of TxB2formation in both bleeding time blood and clotted blood. At the site of plug formation, a-granule release was substantially reduced within the first three minutes and thrombin generation was similarly inhibited. We conclude that (a) marked platelet activation and considerable thrombin generation occur in the early stages.of haemostasis, (b) α-granule release in vivo is partially dependent upon cyclo-oxygenase-controlled mechanisms and (c) thrombin generation at the site of plug formation is promoted by the activation of platelets.

ALY688 elicits adiponectin-mimetic signaling and improves insulin action in skeletal muscle cells

2021 ◽  
Author(s):  
Hye Kyoung Sung ◽  
Patricia L. Mitchell ◽  
Sean Gross ◽  
Andre Marette ◽  
Gary Sweeney
Keyword(s):  
Skeletal Muscle ◽  
Insulin Sensitivity ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Muscle Cells ◽  
Temporal Analysis ◽  
Skeletal Muscle Cells ◽  
Adiponectin Receptor ◽  
Metabolic Effects

Adiponectin is well established to mediate many beneficial metabolic effects, and this has stimulated great interest in development and validation of adiponectin receptor agonists as pharmaceutical tools. This study investigated the effects of ALY688, a peptide-based adiponectin receptor agonist, in rat L6 skeletal muscle cells. ALY688 significantly increased phosphorylation of several adiponectin downstream effectors, including AMPK, ACC and p38MAPK, assessed by immunoblotting and immunofluorescence microscopy. Temporal analysis using cells expressing an Akt biosensor demonstrated that ALY688 enhanced insulin sensitivity. This effect was associated with increased insulin-stimulated Akt and IRS-1 phosphorylation. The functional metabolic significance of these signaling effects was examined by measuring glucose uptake in myoblasts stably overexpressing the glucose transporter GLUT4. ALY688 treatment both increased glucose uptake itself and enhanced insulin-stimulated glucose uptake. In the model of high glucose/high insulin (HGHI)-induced insulin resistant cells, both temporal studies using the Akt biosensor as well as immunoblotting assessing Akt and IRS-1 phosphorylation indicated that ALY688 significantly reduced insulin resistance. Importantly, we observed that ALY688 administration to high-fat high sucrose fed mice also improve glucose handling, validating its efficacy in vivo. In summary, these data indicate that ALY688 activates adiponectin signaling pathways in skeletal muscle, leading to improved insulin sensitivity and beneficial metabolic effects.

FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

2018 ◽  
Vol 115 (11) ◽  
pp. 1672-1679 ◽  
Author(s):  
Qi Ma ◽  
Weilin Zhang ◽  
Chongzhuo Zhu ◽  
Junling Liu ◽  
Quan Chen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Mitochondrial Protein ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Akt Phosphorylation ◽  
Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

Genetic impairment of AMPKα2 signaling does not reduce muscle glucose uptake during treadmill exercise in mice

2009 ◽  
Vol 297 (4) ◽  
pp. E924-E934 ◽  
Author(s):  
Stine J. Maarbjerg ◽  
Sebastian B. Jørgensen ◽  
Adam J. Rose ◽  
Jacob Jeppesen ◽  
Thomas E. Jensen ◽  
...  
Keyword(s):  
Glucose Uptake ◽  
Glucose Transporter ◽  
Treadmill Exercise ◽  
Surface Membrane ◽  
Absolute Intensity ◽  
Wild Type ◽  
Running Speed ◽  
Mouse Muscle ◽  
Glucose Clearance

Some studies suggest that the 5′-AMP-activated protein kinase (AMPK) is important in regulating muscle glucose uptake in response to intense electrically stimulated contractions. However, it is unknown whether AMPK regulates muscle glucose uptake during in vivo exercise. We studied this in male and female mice overexpressing kinase-dead AMPKα2 (AMPK-KD) in skeletal and heart muscles. Wild-type and AMPK-KD mice were exercised at the same absolute intensity and the same relative intensity (30 and 70% of individual maximal running speed) to correct for reduced exercise capacity of the AMPK-KD mouse. Muscle glucose clearance was measured using 2-deoxy-[3H]glucose as tracer. In wild-type mice, glucose clearance was increased at 30 and 70% of maximal running speed by 40 and 350% in the quadriceps muscle and by 120 and 380% in gastrocnemius muscle, respectively. Glucose clearance was not lower in AMPK-KD muscles compared with wild-type regardless of whether animals were exercised at the same relative or the same absolute intensity. In agreement, surface membrane content of the glucose transporter GLUT4 was increased similarly in AMPK-KD and wild-type muscle in response to running. We also measured signaling of alternative exercise-sensitive pathways that might be compensatorily increased in AMPK-KD muscles. However, increases in phosphorylation of CaMKII, Trisk95, p38 MAPK, and ERK1/2 were not higher in AMPK-KD than in WT muscle. Collectively, these findings suggest that AMPKα2 signaling is not essential in regulating glucose uptake in mouse skeletal muscle during treadmill exercise and that other mechanisms play a central role.

Sign in / Sign up

Export Citation Format

Share Document