Glucose Metabolism Is Required for PlateletHyperactivation in a Murine Model of Type 1 DiabetesTrevor P. Fidler,1,2 Alex Marti,1 Katelyn Gerth,1 Elizabeth A. Middleton,3 Robert A. Campbell,3

Matthew T. Rondina,3,4 Andrew S. Weyrich,3 and E. Dale Abel1,3

Diabetes 2019;68:932–938 | https://doi.org/10.2337/db18-0981

Patients with type 1 diabetes mellitus (T1DM) have in-creased thrombosis and platelet activation. The mech-anisms for platelet hyperactivation in diabetes areincompletely understood. T1DM is accompanied by hy-perglycemia, dyslipidemia, and increased inflammationin addition to an altered hormonal milieu. In vitro analysisof platelets revealed that normal glucose reduces plate-let activation whereas hyperglycemic conditions in-crease platelet activation. We therefore hypothesizedthat hyperglycemia increases platelet glucose utilization,which increases platelet activation to promote thrombo-sis. Glucose uptake and glycolysis were increased inplatelets isolated from mice given streptozotocin (STZ)to induce T1DM in concert with induction of GLUT3.Platelets from STZ-induced diabetic mice exhibited in-creased activation after administration of protease-activated receptor 4 peptide and convulxin. In contrast,platelets isolated fromGLUT1andGLUT3double-knockout(DKO) mice, which lack the ability to use glucose,failed to increase activation in hyperglycemic mice.Diabetic mice displayed decreased survival in acollagen/epinephrine-induced pulmonary embolismmodel of in vivo platelet activation relative to nondiabeticcontrols. Survival after pulmonary embolism was in-creased in diabetic DKO mice relative to nondiabeticcontrols. These data reveal that increased platelet glu-cosemetabolism in vivo contributes to increased plateletactivation and thrombosis in a model of T1DM.

Patients with type 1 diabetes mellitus (T1DM) displayincreased platelet activation and thrombosis (1–3). The

mechanisms underlying this dysfunction are incompletelyunderstood. T1DM is accompanied by multiple systemicabnormalities, including hyperglycemia, dyslipidemia, in-creased inflammation, and an altered hormonal milieu, anyof which could contribute to platelet dysfunction. Plateletsfrom diabetic rats display an enrichment of glycolyticintermediates (4); however, whether this is due to in-creased glycolytic flux or an impaired ability to metabolizeglucose is unclear. Furthermore, platelet activation corre-lates with glucose metabolism. Platelets incubated in hy-perglycemic conditions demonstrate increased plateletactivation (5), and platelets lacking the ability to takeup glucose display decreased platelet activation andin vivo thrombosis (6). In T1DM, the specific contributionof glucose uptake to platelet dysfunction is unknown.

To determine the direct consequence of altered glucoseutilization on platelet function, we induced insulin-deficient diabetes (T1DM) in mice lacking the ability tometabolize glucose in platelets. These mice with platelet-specific double-knockout (DKO) of GLUT1 and GLUT3are unable to take glucose up from the extracellularcompartment (6). Using these mice, we evaluated thehypothesis that increased glucose metabolism in plateletsfrom T1DM mice promoted platelet hyperactivation andthrombosis. Here we show that platelets from mice givenstreptozotocin (STZ) to model T1DM exhibited increasedglucose uptake and glycolysis that correlated with in-creased GLUT3 protein expression. Furthermore, plateletsfrom STZ-induced diabetic mice exhibited increased plate-let activation and thrombosis, which was prevented whenplatelet glucose metabolism was abrogated.

1Fraternal Order of Eagles Diabetes Research Center and Division of Endocrinologyand Metabolism, Carver College of Medicine, University of Iowa, Iowa City, IA2Division of Molecular Medicine, Department of Medicine, Columbia UniversityMedical Center, New York, NY3Molecular Medicine Program, Department of Internal Medicine, University of Utah,Salt Lake City, UT4Geriatric Research, Education, and Clinical Center, Department of InternalMedicine, George E. Wahlen Department of Veterans Affairs Medical Center, SaltLake City, UT

Corresponding author: E. Dale Abel, drcadmin@uiowa.edu

Received 10 September 2018 and accepted 7 February 2019

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0981/-/DC1.

© 2019 by the American Diabetes Association. Readers may use this articleas long as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

932 Diabetes Volume 68, May 2019

METABOLISM

RESEARCH DESIGN AND METHODS

AnimalsAll animals were generated on a C57Bl6 background andhoused under normal light and temperature conditions.DKO mice were generated as previously described (6) bycrossing mice expressing a platelet factor 4 driven Crerecombinase to mice harboring homozygous GLUT1 andGLUT3 floxed alleles.

For STZ administration, 8- to 12-week-old mice wereinjected i.p. with 50 mg/kg STZ or citrate buffer daily for5 days (7). All studies were conducted on mice between7 and 12 weeks after STZ administration. The institutionalanimal care and use committee at the University of Iowaand the University of Utah approved all animal studies.

Platelet IsolationsBlood was isolated into 1:20 acid citrate dextrose throughcarotid artery cannulation as previously described (8). ForGLUT1 protein analysis and Seahorse analysis, platelets werenegatively depleted of leukocytes and red blood cells byincubation with CD45 and Terr119 microbeads (MiltenyBiotec, Cambridge, MA). Platelet counts were determined byCellometer AutoM10 (NexcelomBioscience, Lawrence, MA).

Platelet CountsPlatelet counts were obtained using the Advia 120 whole-blood analyzer (Siemens, Berlin, Germany). For circulatinghalf-life and depletion assays, platelet concentrations weredetermined as CD41-APC+ events normalized to fluo-Spheres (BD Biosciences, San Jose, CA). For circulatinghalf-life, mice were injected i.v. with anti-GPIbb-FITCantibody (Emfret Analytics, Eibelstadt, Germany), andblood was obtained via cheek bleed. For depletion studies,DKO and littermate control mice were injected i.v. with2 mg/g anti-GPIba antibody (Emfret Analytics), and plate-let counts were obtained every 24 h.

Metabolic CharacterizationPlasma glucose concentrations were determined witha Contour clinical glucometer (Bayer, Leverkusen, Ger-many). Serum free fatty acids (FFA) were measured usingan FFA assay kit (MAK044; MilliporeSigma, St. Louis, MO).

Bone Marrow–Derived MegakaryocytesProplatelet formation assays were determined as previ-ously described with some modifications (6,9). Bone mar-row fromDKO or control mice was cultured in DMEMwith5 mmol/L glucose, glutamate, and recombinant thrombo-poietin for 5 days. At day 6, megakaryocytes were enrichedusing a gravity BSA gradient and plated into fibrinogen-coated chamber slides. Megakaryocytes were then treatedwith 42 mmol/L BSA (vehicle), 250 mmol/L palmitatecomplexed with BSA (Cat #102720-100; Agilent, SantaClara, CA), 250 mmol/L oleic acid, or 250 mmol/L myristicacid (Nu-Chek Prep, Elysian, MN) for 24 h. Oleic andmyristic acid were conjugated to BSA in the presenceof NaOH, then neutralized to pH 7.2. The fraction

of proplatelet-forming megakaryocytes to total megakar-yocytes was then quantified in a blinded manner examin-ing .100 megakaryocytes/biological replicates.

Glucose MetabolismTo determine glucose uptake, washed platelets in 1mmol/L glucose DMEM were incubated with 10 mmol/L[3H]2-deoxy-D-glucose for 10 min with or without 0.5units/mL thrombin. Platelets were washed then lysed in1 mol/L NaOH. Glucose uptake was normalized to proteincontent using bicinchoninic acid analysis.

Seahorse analysis was conducted as previously de-scribed (6). Briefly, platelets were negatively depleted ofleukocytes and red blood cells. Platelets bioenergetics wereevaluated by Seahorse XF96 Analyzer (Agilent Technolo-gies). Data were normalized to platelet counts.

ImmunoblotsPlatelets were lysed in radioimmunoprecipitation assaybuffer and analyzed via Western blot. For GLUT1 proteinanalysis, platelets were depleted of leukocytes and redblood cells. Primary antibodies to GLUT1 (07-1401) andGLUT3 (AB1344) were purchased from MilliporeSigma.ImageJ software (National Institutes of Health) was usedfor densitometry quantification.

Platelet ActivationWashed platelets were incubated in HEPES Tyrode’s bufferfor 1 h, then incubated in the presence of the indicatedagonist with JonA-PE, CD62p-FITC (Emfret Analytics),and CD41-APC (eBioscience) for 10 min at 37°C. Reactionswere stopped by the addition of FACS lysis buffer (BectonDickinson, Franklin Lakes, NJ) and analyzed using flowcytometry LSR II (Becton Dickinson).

Collagen/Epinephrine-Induced Pulmonary EmbolismCollagen/epinephrine-induced pulmonary embolism wasconducted as previously described (6,10). Briefly, micewere injected i.v. with 430 mg/kg collagen (Chrono-Log,Columbia, MD) and 20 mg/kg epinephrine (Millipore-Sigma) in PBS. Survival was determined when spontaneousrespiratory chest expansions ceased for 1 min.

StatisticsStatistical analyses were performed using GraphPad 7 orMicrosoft Excel 2011 software. Data are presented asmean 6 SD. The statistical significance threshold ofP , 0.05 was determined.

RESULTS

Diabetes Does Not Impair Platelet Survival orBiogenesisTo evaluate platelet glucose metabolism in a model ofT1DM, we gave mice lacking both GLUT1 and GLUT3specifically in platelets (DKO) and littermate controls STZor citrate buffer. Mice subjected to STZ (control-STZ andDKO-STZ) displayed an approximately twofold increase in

diabetes.diabetesjournals.org Fidler and Associates 933

circulating glucose concentrations at 4 weeks, which wassustained for up to 12 weeks (Fig. 1A). STZ administrationdid not alter circulating platelet counts in control mice(Fig. 1B). We previously reported decreased circulatingplatelets in DKO mice (6). Interestingly, 6 and 8 weeksafter STZ administration, circulating platelet counts wereincreased in DKO-STZ mice to levels similar to controls(Supplementary Table 1 and Fig. 1B). To determinewhether increased platelet counts were due to decreasedclearance, we monitored the platelet circulating half-life.DKO mice exhibited decreased circulating half-life; how-ever, STZ administration did not increase survival (Fig.1C). We therefore investigated platelet biogenesis. To dothis we depleted platelets by injecting mice with anti-GPIba antibodies and monitored the time of plateletregeneration. No change in platelet regeneration wasobserved in control-STZ mice. DKO mice demonstrateda significantly longer time to platelet regeneration (Fig.

1D). Platelet regeneration was increased in DKO-STZ miceto levels similar to controls. These data indicate thatincreased platelet counts in DKO-STZ mice are due toincreased platelet production relative to nondiabetic DKOmice.

We sought to determine mechanisms responsible fornormalizing platelet production in diabetic DKO mice.Because we have previously demonstrated that DKO pla-telets and megakaryocytes have very little glucose uptakeand metabolism (6), we thought it unlikely that hypergly-cemia was directly influencing megakaryocyte function.Wetherefore considered the possibility that increased avail-ability of FFA in the diabetic state enhanced megakaryo-cyte FFA metabolism, which compensated for the absenceof glucose uptake. Circulating FFA were increased in di-abetic mice (Fig. 2A). Incubation of bone marrow–derivedmegakaryocyte cultures with BSA-conjugated palmitic acid,oleic acid, and myristic acid increased proplatelet

Figure 1—Platelet counts, circulating half-life, and regeneration in diabetic mice. A: Blood glucose concentrations were measured at theindicated times after STZ or citrate buffer administration (n. 6) in wild-typemice (control), diabetic wild-typemice (control-STZ), nondiabeticplatelet GLUT1/3 DKOmice (DKO), and diabetic DKOmice (DKO-STZ). B: Platelet counts in whole blood (n = 10). C: Platelet circulating half-life was determined after administration of GPIbb antibodies (n = 6). D: Platelet regeneration was determined by monitoring platelets countsafter depletion of platelets following administration of GPIba antibodies (black arrow) (n = 6). Error bars are SD. ****P , 0.0001 relative tocontrol genotype. B: One-way ANOVA followed by Tukey post hoc test. A, C, and D: Two-way ANOVA followed by Bonferroni multiplecomparison post hoc test.

934 Glucose Regulates Platelet Function in Diabetes Diabetes Volume 68, May 2019

formation in DKO megakaryocytes (Fig. 2B and C); how-ever, no FFA-mediated increase was observed in controls.These data suggest that in the absence of glucose metab-olism, increased FFA availability and presumably increasedutilization can promote proplatelet formation.

Diabetes Increases Platelet Glucose UptakePlatelets from diabetic wild-typemice exhibited significantlyincreased basal glucose uptake relative to nondiabetic con-trols (Fig. 3A). After thrombin administration, glucoseuptake increased approximately threefold and twofold, re-spectively, in nondiabetic and STZ-administered wild-typemice, such that absolute levels of glucose uptake wereequivalent (Fig. 3A). Similar to our previous findings,

DKO platelets displayed negligible glucose uptake (6), whichwas not altered by diabetes (Fig. 3A). We next evaluatedglycolysis rates using Seahorse flux analysis. DKO plateletsdemonstrated significantly impaired glycolysis (Fig. 3B).Under basal conditions, hyperglycemia did not alter glycol-ysis in platelets isolated from wild-type mice (Fig. 3B);however, after administration of mitochondrial inhibitors,platelets from diabetic wild-type mice displayed increasedglycolysis relative to nondiabetic controls (Fig. 3B). Thesedata support the conclusion that platelet glucose utilizationis increased in a murine model of T1DM.

GLUT1 and GLUT3 are the physiologically relevantGLUTs in platelets (6). Because glucose uptake wasincreased, we examined GLUT expression. GLUT1 is

Figure 2—FFA potentiate megakaryocyte-mediated proplatelet formation. A: Plasma FFA (n = 5). B: Representative images of bone marrow–

derived megakaryocytes, proplatelet-forming megakaryocytes (white arrowheads), and nonproplatelet-forming megakaryocytes (red arrow-heads) treated for 24 h with 42 mmol/L BSA or 250 mmol/L BSA-conjugated palmitate, 250 mmol/L BSA-conjugated oleic acid, or 250 mmol/LBSA-conjugated myristic acid. C: Quantification of proplatelet-producing megakaryocytes (n = 4). Error bars are SD. ***P, 0.001 relative tocontrol genotype; #P, 0.05, ##P, 0.01 relative to control or BSA, same genotype. A: One-way ANOVA followed by Tukey post hoc test. C:Two-way ANOVA followed by Tukey post hoc test.

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expressed in red blood cells and leukocytes; therefore, toexamine GLUT1 protein expression, platelet preparationswere depleted of cells positive for Ter119 and CD45.GLUT1 protein content was unchanged in platelets iso-lated from diabetic wild-type mice relative to nondiabeticcontrols (Fig. 3C and D). However, GLUT3 platelet proteincontent was significantly increased by diabetes (Fig. 3C andE). Diabetes increases GLUT3 protein content in theplacenta (11) and neuronal tissue (12), suggesting glucose-dependent regulation of GLUT3. Together these findingsreveal that platelets from mice given STZ increase GLUT3

protein expression that may contribute to increased glu-cose uptake and glycolysis.

Diabetes Increases Platelet Activation and Thrombosis,Which Is Prevented by Deletion of GLUT1 and GLUT3Platelets from patients with T1DM and platelets incubatedunder hyperglycemic conditions display increased plateletactivation (1–3,5). Therefore, we hypothesized that in-creased platelet glucose metabolism results in increasedactivation. Under basal conditions, no differences in plate-let activation marked by GPIIbIIIa activation (relative

Figure 3—Glucose utilization is increased in platelets fromSTZ-diabetic mice.A: Uptake of [3H]2-deoxy-D-glucose in washed platelets underbasal and thrombin-stimulated conditions (n $ 4). B: Analysis of glycolysis as measured by the extracellular acidification rate (ECAR) bySeahorse flux analysis of CD45- and Ter119-depleted platelets. Platelets were sequentially treated with oligomycin (1 mmol/L), carbonylcyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 0.5mmol/L), and rotenone (1mmol/L) (n = 3).C: Western blot analysis of protein lysates.Densitometric analysis of GLUT1 (n. 4) (D) and GLUT3 (E ) normalized to Ponceau S staining (n. 10). Error bars are SD. *P, 0.05, **P,0.01, ****P , 0.0001 relative to control genotype; #P , 0.05, ###P , 0.001 relative to equivalently treated platelets of the samegenotype. D and E: One-way ANOVA followed by Tukey post hoc test. A and B: Two-way ANOVA followed by Bonferroni multiplecomparison post hoc test.

936 Glucose Regulates Platelet Function in Diabetes Diabetes Volume 68, May 2019

JonA geometric mean fluorescence intensity) or a-granulerelease (CD62p geometric mean fluorescence intensity)were observed (Fig. 4A and B). In response to submaximalconcentrations of protease-activated receptor 4 (PAR4)peptide and convulxin, nondiabetic and diabetic wild-type platelets both revealed increased activation. However,platelet activation was significantly higher in diabetic micerelative to similarly treated controls (Fig. 4A and B).Consistent with our previous reports, DKO platelets dem-onstrated blunted activation (6). Importantly, diabeticDKO platelets did not display an additional increase inagonist-mediated activation relative to DKO platelets (Fig.4A and B). This insensitivity was not due to STZ-inducedtoxicity or suppression of platelet activation, because inresponse to maximal doses of PAR4 peptide, DKO andDKO-STZ platelets exhibit equivalent degrees of activationrelative to submaximal agonist concentrations (Fig. 4A andB). Together, these data indicate that glucose metabolismis a critical mediator of the hyperglycemia-associated in-crease in agonist-mediated activation.

To determine whether diabetes increases in vivo throm-bosis, we subjected mice to collagen/epinephrine-inducedpulmonary embolism. This in vivo assay is believed to belargely driven by platelet activation versus endothelialchanges that could also develop in diabetes. Control-STZmice demonstrated significantly reduced length of survivalrelative to controls (Fig. 4C). In contrast, DKO and DKO-STZ mice both exhibited prolonged survival relative tocontrol groups; however, no significant change in survivalwas observed between DKO and DKO-STZ mice (Fig. 4C).Thus, reducing platelet glucose utilization protects againstdiabetes-associated platelet hyperactivation.

DISCUSSION

This study identifies an important role for glucose utili-zation in the increased platelet activation that character-izes diabetes and also identifies a potential role for FFA inmaintaining proplatelet biogenesis when platelet glucoseutilization is impaired. Diabetes reversed thrombocyto-penia in mice lacking the ability to import glucose intoplatelets and megakaryocytes, likely through increasedFFA utilization. Thus, hyperglycemia and increased FFAavailability exhibit distinct effects on platelet biogenesisversus activation, with glucose or FFA being interchange-able for proplatelet biogenesis from megakaryocytes,whereas glucose is required for platelet activation.

Here we demonstrate that in a model of T1DM, plate-lets increase glucose uptake and glycolysis in concert withincreased GLUT3 expression. Importantly, when glucoseutilization is blocked, the hyperglycemia-associated in-crease in platelet function and thrombosis is abolished,indicating that glucose metabolism in the T1DM modeldrives platelet hyperactivation. These findings indicatethat inhibition of glucose metabolism in platelets in indi-viduals with diabetes could ameliorate platelet hyperfunc-tion, providing a potential therapeutic opportunity to

reduce the associated increased risk of thrombosis. Met-abolic inhibitors have become a promising target in cancertherapeutics, that some of these glycolytic inhibitors couldbe repurposed to decrease glucose metabolism in diabetic

Figure 4—In vitro platelet activation and in vivo thrombosis isincreased in STZ-diabetic mice. Washed platelets stimulated inthe presence of the indicated agonist and monitored for GPIIbIIIaactivation marked by relative JonA geometric mean fluorescenceintensity (Geo. MFI) (A) and a-granule release marked by CD62pGeo. MFI (B) (n = 4 PAR4, n = 3 Convulxin) (C). Survival curves ofmice subjected to collagen/epinephrine-induced pulmonary embo-lism (n$ 6). Error bars are SD. *P, 0.05, **P, 0.01, ****P, 0.0001relative to control genotype; #P , 0.05 relative to equivalent treat-ment, same genotype. A and B: Two-way ANOVA followed byBonferroni multiple comparison post hoc test. C: Log-rank (Mantel-Cox) test.

diabetes.diabetesjournals.org Fidler and Associates 937

platelets may be possible. Future work to elucidate themechanisms responsible for GLUT3 protein induction inT1DM may also reveal additional therapeutic targets.

Acknowledgments. The data presented herein were obtained at the FlowCytometry Facility, which is a Carver College of Medicine core research facility atthe University of Iowa.Funding. This work was supported by National Institutes of Health grants TL1-TR-001875 to T.P.F., R01-AG-048022 to M.T.R., R01-HL-126547-01 to A.S.W.,and U54-HL-112311 to A.S.W. and E.D.A., who are both established investigatorsof the American Heart Association.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. T.P.F. contributed to writing the original draft.T.P.F., A.M., K.G., E.A.M., and R.A.C. contributed to the investigation. T.P.F.,R.A.C., M.T.R., A.S.W., and E.D.A. conceptualized the study and contributed tostudy methodology. T.P.F., A.S.W., and E.D.A. contributed to writing, reviewing, andediting the manuscript. R.A.C., M.T.R., A.S.W., and E.D.A. supervised the study.A.S.W. and E.D.A. acquired funding and contributed to resources. T.P.F. andE.D.A. are the guarantors of this work and, as such, had full access to all the datain the study and take responsibility for the integrity of the data and the accuracyof the data analysis.Prior Presentation. Parts of this study were presented in abstract form atthe American Heart Association’s Vascular Discovery: From Genes to MedicineScientific Sessions 2018, San Francisco, CA, 10–12 May 2018.

References1. Davì G, Ciabattoni G, Consoli A, et al. In vivo formation of 8-iso-prostaglandinf2alpha and platelet activation in diabetes mellitus: effects of improved metaboliccontrol and vitamin E supplementation. Circulation 1999;99:224–229

2. Stolla MC, Li D, Lu L, Woulfe DS. Enhanced platelet activity and thrombosis ina murine model of type I diabetes are partially insulin-like growth factor 1-dependent and phosphoinositide 3-kinase-dependent. J Thromb Haemost 2013;11:919–9293. Bern MM. Platelet functions in diabetes mellitus. Diabetes 1978;27:342–3504. Paterson RA, Heath H, Cranfield T. The effect of O-(beta-hydroxyethyl) ru-toside on platelet intermediary metabolism in normal and streptozotocin diabeticrats. Biochem Pharmacol 1974;23:1591–15975. Yamagishi SI, Edelstein D, Du XL, Brownlee M. Hyperglycemia potentiatescollagen-induced platelet activation through mitochondrial superoxide over-production. Diabetes 2001;50:1491–14946. Fidler TP, Campbell RA, Funari T, et al. Deletion of GLUT1 and GLUT3 revealsmultiple roles for glucose metabolism in platelet and megakaryocyte function. CellReports 2017;20:881–8947. Ni R, Zheng D, Xiong S, et al. Mitochondrial calpain-1 disrupts ATPsynthase and induces superoxide generation in type 1 diabetic hearts: a novelmechanism contributing to diabetic cardiomyopathy. Diabetes 2016;65:255–2688. Fidler TP, Rowley JW, Araujo C, et al. Superoxide dismutase 2 is dispensablefor platelet function. Thromb Haemost 2017;117:1859–18679. Shi DS, Smith MC, Campbell RA, et al. Proteasome function is required forplatelet production. J Clin Invest 2014;124:3757–376610. Fidler TP, Middleton EA, Rowley JW, et al. Glucose transporter 3 potentiatesdegranulation and is required for platelet activation. Arterioscler Thromb Vasc Biol2017;37:1628–163911. Boileau P, Mrejen C, Girard J, Hauguel-de Mouzon S. Overexpression ofGLUT3 placental glucose transporter in diabetic rats. J Clin Invest 1995;96:309–31712. Reagan LP, Magariños AM, Lucas LR, van Bueren A, McCall AL, McEwen BS.Regulation of GLUT-3 glucose transporter in the hippocampus of diabetic ratssubjected to stress. Am J Physiol 1999;276:E879–E886

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