RESEARCH ARTICLE

Altered glucose metabolism and hypoxic
response in alloxan-induced diabetic
atherosclerosis in rabbits
Yunosuke Matsuura1,2, Atsushi Yamashita1*, Yan Zhao3, Takashi Iwakiri1,2,
Kazuaki Yamasaki3, Chihiro Sugita1,4, Chihiro Koshimoto5, Kazuo Kitamura2,
Keiichi Kawai6,7, Nagara Tamaki8, Songji Zhao3, Yuji Kuge9,10, Yujiro Asada1

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1 Department of Pathology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan, 2 Department ofInternal Medicine, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan, 3 Department of Tracer
Kinetics and Bioanalysis, Graduate School of Medicine, Hokkaido University, Sapporo, Japan, 4 Departmentof Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Kyusyu University of Health andWelfare, Nobeoka, Japan, 5 Frontier Science Research Center, University of Miyazaki, Miyazaki, Japan,
6 Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa
University, Kanazawa, Japan, 7 Biomedical Imaging Research Center, University of Fukui, Fukui, Japan,
8 Department of Nuclear Medicine, Graduate School of Medicine, Hokkaido University, Sapporo, Japan,
9 Department of Integrated Molecular Imaging, Graduate School of Medicine, Hokkaido University, Sapporo,
Japan, 10 Central Institute of Isotope Science, Hokkaido University, Sapporo, Japan* atsushi_yamashita@med.miyazaki-u.ac.jp

OPEN ACCESS
Citation: Matsuura Y, Yamashita A, Zhao Y, Iwakiri
T, Yamasaki K, Sugita C, et al. (2017) Alteredglucose metabolism and hypoxic response inalloxan-induced diabetic atherosclerosis in rabbits.
PLoS ONE 12(4): e0175976. https://doi.org/
10.1371/journal.pone.0175976
Editor: Toshiyuki Miyata, National Cerebral andCardiovascular Center, JAPANReceived: January 14, 2017
Accepted: April 3, 2017
Published: April 14, 2017
Copyright: © 2017 Matsuura et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which
permits unrestricted use, distribution, andreproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information
files.
Funding: This study was supported in part by
Grants-in-Aid for Scientific Research in Japan(Nos. 25460440, 23390084, 16K08670, and
16H05163) from the Ministry of Education,
Science, Sports and Culture of Japan, http://www.
jsps.go.jp, Clinical Research from Miyazaki
University Hospital, http://www.med.miyazaki-u.ac.
jp/home/ Mitsui Life Welfare Foundation,

Abstract
Diabetes mellitus accelerates atherosclerosis that causes most cardiovascular events.
Several metabolic pathways are considered to contribute to the development of atherosclerosis, but comprehensive metabolic alterations to atherosclerotic arterial cells remain
unknown. The present study investigated metabolic changes and their relationship tovascular histopathological changes in the atherosclerotic arteries of rabbits with alloxaninduced diabetes. Diabetic atherosclerosis was induced in rabbit ilio-femoral arteries byinjecting alloxan (100 mg/kg), injuring the arteries using a balloon, and feeding with a
0.5% cholesterol diet. We histologically assessed the atherosclerotic lesion development,
cellular content, pimonidazole positive-hypoxic area, the nuclear localization of hypoxiainducible factor-1α, and apoptosis. We evaluated comprehensive arterial metabolism byperforming metabolomic analyses using capillary electrophoresis-time of flight mass
spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using
18
F-fluorodeoxyglucose and pimonidazole. Plaque burden, macrophage content, andhypoxic areas were more prevalent in arteries with diabetic, than non-diabetic atherosclerosis. Metabolomic analyses highlighted 12 metabolites that were significantly altered
between diabetic and non-diabetic atherosclerosis. A half of them were associated withglycolysis metabolites, and their levels were decreased in diabetic atherosclerosis.
The uptake of glucose evaluated as 18F-fluorodeoxyglucose in atherosclerotic lesions
increased according to increased macrophage content or hypoxic areas in non-diabetic,
but not diabetic rabbits. Despite profound hypoxic areas, the nuclear localization of hypoxia-inducible factor-1α decreased and the number of apoptotic cells increased in diabetic atherosclerotic lesions. Altered glycolysis metabolism and an impaired response to

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Vascular metabolism in diabetic atherosclerosis

http://www.kousei-zigyodan.or.jp/igakukenkyu_
zyosei.html, SENSHIN Medical Research
Foundation, https://www.smrf.or.jp, Intramural
Research Fund (25-4-3) for Cardiovascular
Diseases of the National Cerebral andCardiovascular Center, http://www.ncvc.go.jp, andthe Bayer Scholarship for Cardiovascular Research,
http://www.jcvrf.jp/research/baieru.html.

hypoxia in atherosclerotic lesions under conditions of insulin-dependent diabetes might
be involved in the development of diabetic atherosclerosis.

Competing interests: The authors have declaredthat no competing interests exist.

Atherosclerosis is the cause of most cardiovascular diseases (CVD), and diabetes mellitus(DM) accelerates progression of atherosclerotic disease and the subsequent occurrence ofatherothrombosis [1–4]. In general, the quantity and quality of atherosclerotic plaques aremajor determinants of the incidence of cardiovascular events (CVE). A recent series of imaging and pathological studies has confirmed that plaque burden is increased in patients withDM, compared with those without DM [5,6]. On the other hand, whether or not diabetic andnon-diabetic atherosclerotic lesions histologically differ is unclear [7]. These lines of evidence
indicate that the underlying mechanisms of plaque progression and the subsequent onset of
CVE in patients with DM remains unknown. Medical intervention and efforts to lower glucose
levels have failed to prevent the development and progression of diabetes associated withmacroangiopathy in the clinical setting [8,9]. Therefore, innovative approaches are urgently
required to prevent atherosclerosis in patients with DM.
Persistent hyperglycemia seems to be responsible for the development of atherosclerotic
lesions. Increased glucose flux through various metabolic pathways is thought to generate
toxic precursors that contribute to vascular cell damage. Harmful metabolic pathways include
the formation of advanced glycation end products, protein kinase C activation and disrupted
polyol and hexosamine pathways [10]. Dyslipidemia due to increased lipolysis might also contribute to the progression of atherosclerosis. Therefore, DM is considered to largely affect vascular cell metabolism, which leads to the development of atherosclerosis. Identifying changes
in metabolic pathways and metabolites in atherosclerotic lesions under the conditions imposed
by DM would be critical to understanding the underlying mechanisms of atherogenesis inpatients with DM and might provide the means to develop novel therapeutic targets.
Metabolomics can simultaneously identify levels of endogenous metabolites in biologicalsamples, such as blood, urine and tissues, and analytical datasets provide comprehensive information in a snapshot of metabolic status during dynamic disease processes. Recent studies
using metabolomics profiling have revealed serum levels of branched-chain and aromatic
amino acids, including leucine, isoleucine, valine, phenylalanine, and tyrosine, are closely associated with the risk of future diabetes [11, 12]. However, metabolic alterations in diabetic atherosclerosis have not been reported.
We assessed metabolomics and the effects of hypoxia on atherosclerotic lesions in rabbitswith alloxan-induced diabetes to identify metabolic changes in diabetic atherosclerosis and its
relationship to vascular pathology.

Introduction

Materials and methods
Diabetic rabbit atherosclerotic model
The animal research protocol of the present study conformed to the Guide for the Care and Useof Laboratory Animals published by the US National Institutes of Health, and was approved by
the Animal Care Committee of Miyazaki University (2010–541). Experiments proceeded underaseptic conditions and general anaesthesia induced via subcutaneous administration of medetomidine (0.16mg/kg) and butorphanol tartrate (0.4mg/kg) and intravenous infusion of midazolam (0.6 mg/kg). Diabetic rabbits were generated from Japanese white rabbits initially weighing

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Vascular metabolism in diabetic atherosclerosis

2.5–3.5 kg using a single dose of alloxan [13]. Atherosclerotic lesions were produced by feeding
the rabbits with a high-cholesterol diet and denuding the endothelium of the femoral arteries indiabetic and non-diabetic rabbits. In detail, a single 100-mg/kg dose of alloxan monohydrate(Sigma, St Louis, MO, USA) dissolved in 10 mL of sterile saline was injected to induce diabetes.
Non-diabetic rabbits were injected with saline. Initial hypoglycemia after alloxan injection wasprevented by an immediate injection of 10 mL of 20% glucose and the provision of 5% glucose
in the drinking water ad libitum for 24 hours. Thereafter, blood glucose levels were monitored
weekly and rabbits with fasting plasma glucose levels > 200 mg/dL during the entire experimental period were defined as diabetic and included in this study. One week after injection
with alloxan or saline, the rabbits were started on the 0.5% cholesterol diet for four weeks. One
week after starting the cholesterol diet, the endothelium of the ilio-femoral arteries was denuded
using an angioplasty balloon catheter as described [14]. At baseline and at five weeks after
alloxan or saline injection, the rabbits were weighed and levels of insulin, total cholesterol, triglyceride and free fatty acid were measured after an overnight fast.

Metabolomic profiling of vascular tissues using Capillary Electrophoresis
Time-Of-Flight Mass Spectrometry (CE-TOFMS)
Tissue preparation. The rabbits were sacrificed with intravenous infusion of pentobarbital (60 mg/kg). Soft tissues surrounding isolated rabbit arteries were removed and the arteries
were immediately frozen in liquid nitrogen and stored at -80˚C. Arteries were weighed andthen the tissues (30mg) were added to 500 μL of methanol containing 50 μM of internal standard and homogenized using a tissue disrupter. Homogenates were mixed with 200 μL of
Milli-Q water and 500 μL of chloroform and then separated by centrifugation at 2300 × g for 5
min at 4˚C. The upper aqueous layer (400 μL) was passed through a filter (5-kDa cutoff; Millipore) by centrifugation at 9,100 g for 120 min at 4˚C to remove proteins. The filtrates werelyophilized and suspended in 50 μL of Milli-Q water for metabolome analysis.
Instrumentation. Metabolomes were profiled by CE-TOFMS using an Agilent CE Capillary Electrophoresis System equipped with a 6210 Time-of-Flight mass spectrometer, an 1100
isocratic HPLC pump, a G1603A CE-MS adapter kit and a G1607A CE-ESI-MS sprayer kit(all from Agilent Technologies, Waldbronn, Germany), and this system was controlled by
G2201AA ChemStation software version B.03.01 for CE (Agilent Technologies).
CE-TOFMS conditions. Cationic metabolites were analyzed using a fused silica capillary(50 μm i.d. × 80 cm length), with H3301-1001 cation electrophoresis buffer (Human Metabolome Technologies, Tsuruoka, Yamagata, Japan) as the electrolyte. Samples were injected at apressure of 50 mbar for 10 sec. The applied voltage was set at 27 kV. Electrospray ionizationmass spectrometry proceeded in positive-ion mode, and the capillary voltage was set at 4,000
V. Samples were scanned by the spectrometer between 50 and 1,000 m/z [15].
Anionic metabolites were analyzed using a fused silica capillary (50 μm i.d. × 80 cm length),with H3302-1021 anion electrophoresis buffer (Human Metabolome Technologies) as the electrolyte. Samples were injected at a pressure of 50 mbar for 25 sec. The applied voltage was set
at 30 kV. Electrospray ionization-mass spectrometry proceeded in negative-ion mode, and thecapillary voltage was set at 3,500 V. Samples were scanned by the spectrometer between 50 and
1,000 m/z [15].
Processing and analysis of metabolites data. Raw data derived by CE-TOFMS wereautomatically processed by the proprietary software MasterHands [11]. Signal peaks corresponding to isotopomers, adduct ions and other product ions of known metabolites were
excluded. All signal peaks potentially corresponding to authentic compounds were extracted
and their migration times (MT) were normalized to internal standards. Thereafter, peaks were

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Vascular metabolism in diabetic atherosclerosis

aligned in accordance with m/z values and normalized MT. Finally, peak areas were normalized against those of the respective internal standards for cations, anions, methionine sulfone
and D-camphol-10-sulfonic acid. Values for relative areas were further normalized by sample
weight. Annotation tables were produced from values of standard compounds and alignedwith datasets according to similar m/z values and normalized MT.

Hypoxia and 18F-fluorodeoxyglucose (FDG) uptake
We investigated 18F-FDG uptake as described with minor modifications [15]. Diabetic andnon-diabetic rabbits (n = 6 in each) were fasted for four hours and then infused with 18F-FDG(average, 207 MBq/rabbit) and 60 mg/kg of the hypoxia marker, pimonidazole (Hypoxyprobe1; Hypoxyprobe, Inc., Burlington, MA, USA). Two hours later, amounts of radioactivity in thefemoral arteries, and blood were measured using a 1480 WIZARD 3 well-type γ-counter (Wallac Co. Ltd., Turku, Finland). The results were calculated as (%ID/g) × kg.
Atherosclerotic and control ilio-femoral arteries were excised, cut into 8 to 12 segments,
embedded in Tissue-Tek (Sakura, Tokyo, Japan) and frozen. Ten and 5-μm consecutive slices
were prepared for autoradiography and histological analysis, respectively, and then 10-μm
cryostat sections were exposed on phosphor imaging plates (Fuji Imaging Plate BAS-SR 2025,
Fuji Photo Film Co. Ltd., Tokyo, Japan) for 12 h together with a set of calibrated standards.
The plates were then scanned using a Fuji Bio-imaging Analyzer BAS-5000 (Fuji Photo Film)with an internal resolution of 25-μm and the images were assessed using Multi Gauge Ver.
3.0 image analysis software (Fuji Photo Film). The amount of radioactivity in each image isexpressed as photo-stimulated luminescence (PSL) per unit area (PSL = a × D × t, where a is aconstant, D is the amount of radioactivity exposed on the imaging plate, and t is exposure
duration). All PSL values/mm2 from the arterial tissue were converted to ratios (%) of theactivity of the standard injected dose/mm2 of lesion area (% ID/m2). Data were normalizedaccording to the weight of each rabbit (% ID/m2 × kg).

Histopathology and immunohistochemistry
Serial frozen sections (5-μm thick) were stained with hematoxylin and eosin (HE) and Victoria
Blue (VB). Sections were immunohistochemically assessed using mouse monoclonal anti-αsmooth-muscle-cell actin for smooth muscle cells (SMC) (Dako, Glostrup, Denmark), RAM11 mouse monoclonal anti-rabbit macrophages (Dako), mouse monoclonal anti-pimonidazole(Hypoxyprobe), and mouse monoclonal hypoxia inducible factor (HIF)-1α (Abcam, Cambridge, UK) as primary antibodies. Horseradish peroxidase activity was visualized by staining
with 3, 3’-diaminobenzidine tetrahydrochloride and counterstaining with Meyer’s hematoxylin. The negative control was stained with universal negative control mouse immunoglobulins(Dako) instead of primary antibodies.

Morphometric analysis
Vessel areas and positive immunostained parts of whole cross-sectional areas of vascular tissues were quantified using a color-imaging morphometry system (Win Roof, Mitani, Fukui,Japan). Immunopositive areas are expressed as ratios (%) of vessel areas. Two investigators(T. I. and C. S.) accomplished all quantitative analyses in a blinded manner.

Western blotting
Nuclear proteins of rabbit diabetic and non-diabetic atherosclerotic arteries were extractedusing a mammalian nuclear protein extraction reagent (Thermo Fisher Scientific Inc.,

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Vascular metabolism in diabetic atherosclerosis

Waltham, MA, USA) with 1% protease inhibitor cocktail (Thermo Fisher). Protein concentrations were determined using DC protein assay kits (Biorad, Hercules, CA, USA).
The nuclear translocation of HIF-1α in diabetic and non-diabetic atherosclerosis was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10%
gels (Technical Frontier Co., Tokyo, Japan). Samples were incubated with sample buffer containing SDS and 2-mercaptoethanol for three minutes at 98˚C before SDS-PAGE. Separated
proteins were electrophoretically transferred to Immobilon membranes (Millipore, Billerica,
MA, USA). Non-specific binding was blocked overnight with 5% skimmed milk, and then theseparated proteins were incubated with anti-HIF-1α antibody (Abcam) followed by horseradish peroxidase conjugated anti-mouse IgG (Nacalai Tesque Inc., Kyoto, Japan). After stripping
the antibodies, the membrane was reprobed with anti-lamin A/C (Sigma) and the secondary
antibody. The proteins of interest were detected by chemiluminescence using a LAS-4000
Lumino image analyzer (Fuji).

TUNEL assay
The numbers of apoptotic cells in the ilio-femoral arteries of diabetic and non-diabetic rabbits
were determined using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assays. Apoptotic cells in arterial samples that had been
fixed in 4% paraformaldehyde and embedded in paraffin were visualized using in situ apoptosis detection kits (Takara Bio Inc., Kusatsu, Japan) and data are expressed as numbers of positive nuclei per 10,000 μm2. Double immunofluorescence emission by apoptotic cells andhypoxia was determined using TUNEL assays, fluorescein isothiocyanate-labeled anti-BrdU
antibody (Apo-BrdU in situ DNA fragmentation assay kits [BioVison Inc., Milpitas, CA,USA]), and Dylight™549 fluorophore-labeled anti-pimonidazole antibody (Hypoxyprobe).

Statistical analysis
Data were analyzed using SampleStat (Human Metabolome Technologies Inc., Tsuruoka,
Japan) for Principal Component Analysis (PCA), PeakStat (Human Metabolome Technologies) for Hierarchical Cluster Analysis (HCA), JMP 7.0 (SAS Institute Inc., Cary, NC, USA)and Graph Pad Prism 5.01 (GraphPad Software Inc., San Diego, CA). Values are expressed asmeans with standard deviation (SD). Variables between or among groups were comparedusing Mann-Whitney U test or One-way ANOVA with Bonferroni’s multiple comparisonstest. Relationships between factors were evaluated using Spearman’s test. P values < 0.05 wereconsidered statistically significant.

Results
Body weight and blood parameters in diabetic and non-diabetic rabbits
fed with diet containing 0.5% cholesterol
Diabetic rabbits were generated using a single 100-mg/kg dose of alloxan monohydrate. S1Table shows body weight (BW) and serum levels of glucose, insulin, total cholesterol and triglyceride in blood samples obtained after an overnight fast at baseline and after five weeks
from non-diabetic (n = 10) and diabetic (n = 10) rabbits fed with the cholesterol diet. None ofthe parameters differed between the two groups at baseline. At five weeks later, however, BW
and insulin levels were significantly lower, and mean values of glucose, total cholesterol andtriglyceride were ~ 3.5-, 2.0- or 8.0-fold higher, respectively, after five weeks in diabetic, thannon-diabetic rabbits.

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Vascular metabolism profiling using CE-TOFMS
Metabolomic analysis proceeded using CE-TOFMS in; non-diabetic, non-atheroscleroticarteries (n = 5; control), non-diabetic and diabetic atherosclerotic arteries (n = 5 each). Atherosclerotic lesions were produced by feeding the rabbits with a high-cholesterol diet anddenuding the endothelium of the ileo-femoral arteries in diabetic and non-diabetic rabbits.
Overall, 211 peaks (122 cations and 89 anions) were identified based on m/z values and migration times, in the arterial tissues. In detail, 155, 184 and 205 peaks were detected in control,and in non-diabetic and diabetic atherosclerotic samples, respectively. We also absolutely
quantified 110 metabolites including glycolytic and citric acid cycle intermediates, amino acids
and purine nucleoside phosphates.
Fig 1A shows the results of principal component analysis (PCA). The difference between
control and atherosclerotic arteries was distinctly separated along with the first principal
components (PC1) axis, whereas, non-diabetic and diabetic atherosclerotic arteries
were obviously discriminated along with the second principle components (PC2) axis. S2Table shows factor loading values for PC1 and PC2 in score plots. Metabolites with highest
absolute factor loading values in PC1 comprised 2-hydroxyglutaric acid, Ala, Ser, Thr, Ile,Asn, Asp and Gly-Asp, which are mainly involved in amino acid metabolism. On the other
hand, those in PC2 were phosphoenolpyruvic acid, dihydroxyacetone phosphate, betaine
aldehyde+H2O, glucose 6-phosphate, fructose 6-phosphate, 2-deoxyglucose 6-phosphate,o-hydroxybenzoic acid, glyceraldehyde 3-phosphate, and UMP, GDP-mannose, CMP
and GMP, which are mainly involved in glycolysis/glycogenesis and purine/pyrimidine
metabolism.
The results of the hierarchical clustering analysis (HCA) are represented as a heat map(Fig 1B, S3 Table). The results of HCA combined with those of PCA in the three types ofarteries confirmed that UMP, GDP-mannose, CMP and GMP belonged to a metabolite
cluster with specifically increased levels in diabetic atherosclerotic lesions. In contrast,
phosphoenolpyruvic acid, dihydroxyacetone phosphate, betaine aldehyde + H2O, glucose
6-phosphate, fructose 6-phosphate, 2-deoxyglucose 6-phosphate, o-hydroxybenzoic acid,glyceraldehyde 3-phosphate belonged to a metabolite cluster with specifically reduced levels
in diabetic atherosclerotic lesions (S2, S3 and S4 Tables, Fig 2). Levels of glycolytic metabolites did not significantly differ in control arteries from rabbits with or without diabetes(Fig 2, S4 Table).

Glucose uptake in diabetic and non-diabetic atherosclerosis in rabbits
The metabolomic analysis confirmed that the glycolytic pathway is altered in diabetic atherosclerosis. Since we previously found that glucose uptake in atherosclerotic lesions is enhanced
by hypoxia [15], we examined glucose uptake and hypoxia in diabetic and non-diabetic atherosclerotic lesions using 18F- FDG and pimonidazole, respectively.
Fig 3A shows autoradiographic and corresponding histological images of atherosclerotic
lesions from diabetic and non-diabetic rabbits. Immunohistochemical staining shows hypoxic
areas (pimonidazole positive) within macrophage-rich areas located deep in atherosclerotic
lesions. The immunopositive areas of hypoxia and macrophage accumulation were larger indiabetic, than non-diabetic atherosclerotic lesions. The SMC area was significantly smaller indiabetic atherosclerotic, than non-diabetic atherosclerotic arteries (Table 1). The 18F-FDG
uptake in atherosclerotic arteries was significantly higher and larger than those in control
arteries from both non-diabetic and diabetic rabbits, but significantly lower in diabetic, thannon-diabetic rabbits (Fig 3A and 3B, S4 Table).

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Fig 1. Metabolomic analysis of arterial metabolites in rabbits with and without alloxan-induced
diabetes. A. Principal Component Analysis (PCA) discriminates metabolic features among non-diabetic,
control arteries (1–5, blue), non-diabetic atherosclerotic arteries (6–10, red), and diabetic atheroscleroticarteries (11–15, green). S2 Table shows factor-loading values for PC1 and PC2 on score plots. B.
Representative heat map assessed using hierarchical clustering analysis shows metabolic differences amonggroups. S3 Table shows original data for each metabolite.
https://doi.org/10.1371/journal.pone.0175976.g001

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Vascular metabolism in diabetic atherosclerosis

Fig 2. Levels of metabolites associated with glycolysis pathway in arteries of rabbits with and without alloxan-induced diabetes. Metabolite
levels are expressed as nmol/g. Control arteries, n = 4; atherosclerotic arteries, n = 5 (Mann Whitney U-test). DHAP, dihydroxyacetone phosphate;
F6P, fructose 6-phosphate; Glc, glucose; G3P, glyceraldehyde 3-phosphate; G6P, glucose 6-phosphate; PEP, phosphoenolpyruvic acid.
https://doi.org/10.1371/journal.pone.0175976.g002

Glucose uptake does not correlate with areas of hypoxia andmacrophage infiltration in diabetic atherosclerosis
We examined whether or not glucose uptake correlates with macrophage infiltration or hypoxic areas in diabetic atherosclerosis. The uptake of 18F-FDG correlated positively with both

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Fig 3. Uptake of 18F-FDG in arteries of rabbits with and without alloxan-induced diabetes. A. Representative
immunohistochemically stained macrophages, pimonidazole stained with hematoxylin eosin/Victoria blue (HE/VB)
and autoradiography of sections of atherosclerotic and control femoral arteries from diabetic and non-diabetic rabbits,
respectively. B. Uptake of 18F-FDG in control or atherosclerotic arterial sections of diabetic and non-diabetic rabbits(One-way ANOVA with Bonferroni’s multiple comparisons test).
https://doi.org/10.1371/journal.pone.0175976.g003

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Table 1. Histological characteristics of control and atherosclerotic arteries in non-diabetic and diabetic rabbits, respectively, fed with 0.5% cholesterol diet.
Histologicalvariables

Non-DM control (n = 64), Atherosclerotic (n = 76) arterial
sections

DM control (n = 57), Atherosclerotic (n = 64) arterial
sections

P value

Control

0.67 ± 0.17

0.63 ± 0.21

0.25

Atherosclerotic

1.50 ± 0.38

1.96 ± 0.50

< 0.0001

Control

90.1 ± 4.6

90.0 ± 5.3

0.95

Atherosclerotic

70.2 ± 15.0

56.7 ± 18.9

< 0.0001

Vessel area (mm2)

SMC area (%)

Macrophage area (%)
Control00-Atherosclerotic

23.5 ± 1.8

40.6 ± 1.9< 0.0001

Pimonidazole area(%)
Control00-Atherosclerotic

11.7 ± 10.3

28.4 ± 15.3

< 0.01

Values are shown as means ± SD and compared between groups using Mann-Whitney u test.
DM, diabetes mellitus; SMC, smooth muscle cells.
https://doi.org/10.1371/journal.pone.0175976.t001

macrophage accumulation and hypoxic areas in non-diabetic atherosclerotic lesions, which
was similar to our previous findings [15]. On the other hand, 18F-FDG uptake was significantly
lower and did not correlate with either of these factors in diabetic rabbits (Fig 4, S4 Table).

Reduced nuclear translocation of HIF-1α in diabetic atherosclerosis
We assessed the nuclear translocation of HIF-1α to evaluate cellular responses to hypoxia in
rabbit arteries. Both the HIF-1α content in nuclear extracts and the number of immunopositive nuclei for HIF-1α were significantly reduced in diabetic, compared with non-diabetic atherosclerotic arteries (Fig 5A–5C, S4 Table). The number of HIF-1α positive nuclei correlated
positively with hypoxic areas in non-diabetic, but not in diabetic atherosclerotic arteries (Fig
5D). There were no immunopositive nulcei for HIF-1α in non-atherosclerotic control arteries
in diabetic or non-diabetic rabbits.

Enhanced apoptosis in hypoxic areas of diabetic atherosclerotic lesions
We determined numbers of apoptotic cells in atherosclerotic lesions using TUNEL assays.
Double immunofluorescence emission revealed significantly more apoptotic cells in hypoxic(pimonidazole-positive) areas of atherosclerotic lesions from diabetic, than non-diabetic rabbits (Fig 6A and 6B, S4 Table). The number of apoptotic cells significantly correlated withthe vascular area (r = 0.64, p<0.001) and macrophage area (r = 0.46, p<0.05) in non-diabetic
atherosclerotic arteries, but not in diabetic atherosclerotic arteries (vascular area, r = 0.004,
p = 0.98, macrophage area, r = -0.75, p = 0.70). The result suggests the difference in contributing factors for apoptotic cell death in diabetic and non-diabetic atherosclerotic arteries.

Discussion
We found altered levels of metabolites associated with the glycolysis/glycogenesis and nucleotide intermediate pathways, reduced 18F-FDG uptake, impaired HIF-1α regulation of hypoxia

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Fig 4. Relationship between 18F-FDG uptake and hypoxia in arteries of rabbits with and without alloxan-induced diabetes. Uptakeof 18F-FDG relative to macrophage or hypoxic areas (Mann-Whitney u test), and correlations between 18F-FDG uptake and macrophage
infiltration or hypoxic areas (Spearman’s correlation coefficient) in atherosclerotic arterial sections of diabetic and non-diabetic rabbits.
https://doi.org/10.1371/journal.pone.0175976.g004

and increased numbers of apoptotic cells in atherosclerotic lesions from rabbits with alloxaninduced DM.
Atherosclerosis is the principle cause of mortality among patients with types 1 and type 2
DM. Although epidemiological data have established that DM accelerates atherosclerosis andCVD, the underlying mechanisms remain obscure [16]. Therefore, we developed a rabbit
model of diabetic atherosclerosis by combining balloon-induced injury, alloxan injection anda high-cholesterol diet. The lesions were larger and more macrophage-rich in the rabbits with,than without DM. Since alloxan damages pancreatic β cells, the developed DM condition wasinsulin-dependent type 1 DM [17]. Its condition has been also observed in end-stage type 2
DM [18]. Our model developed hyperglycemia along with obviously increased plasma levels of
T-cholesterol and triglyceride. Plasma levels of lipoprotein and plaque formation are increased
in several mouse models of type 1 DM [16,19]. Since patients with type 1 DM are frequently
associated with dyslipidemia, our animal model simulates atherosclerosis in type 1 DM withdyslipidemia.
The histological features of human diabetic vascular tissue have been previously investigated. A post-mortem study found a larger plaque burden, increased macrophage infiltration

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Fig 5. Expression of HIF-1α in atherosclerotic arteries of rabbits with and without alloxan-induced diabetes. A. Western
blots of HIF-1α in nuclear extract of atherosclerotic arteries of diabetic and non-diabetic rabbits (Mann-Whitney U-test). Data areshown as fold change relative to non-DM and expressed as mean ± SD. B. Representative immunohistochemical images ofmacrophages, pimonidazole and HIF-1α in atherosclerotic arteries from diabetic and non-diabetic rabbits. C. Numbers of HIF-1α
immunopositive nuclei in atherosclerotic arterial sections of diabetic and non-diabetic rabbits (Mann-Whitney u test). D.

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Correlations between pimonidazole-immunopositive areas and numbers of HIF-1α immunopositive nuclei in sections ofatherosclerotic arteries in diabetic and non-diabetic rabbits (Spearman’s correlation coefficient).
https://doi.org/10.1371/journal.pone.0175976.g005

and a necrotic core in coronary arteries of patients with types 1 and 2 DM [5], and anotherstudy of coronary atherectomy specimens also found more lipid-rich atheroma and macrophage infiltration in patients with DM [20]. The histological features of our animal model
were compatible with these findings in patients with DM.
Our metabolomic analysis highlighted 12 metabolites that differed between diabetic andnon-diabetic atherosclerosis. These comprised six glycolysis/glycogenesis metabolites, four
nucleotides, betaine aldehyde and o-hydroxybenzoic acid. Levels of metabolites associatedwith glycolysis/glycogenesis were notably decreased in rabbits with diabetic, compared with
non-diabetic atherosclerosis. Although few studies have examined metabolic pathways in diabetic vascular tissue, Filla et al. [21] found increases in all metabolites associated with glycolysis
and in half of those associated with the tricarboxylic acid cycle, as well as methylglyoxal andamino acids in the non-atherosclerotic aorta in diabetic mouse (db/db). The results are notcomparable of those of the present study. The inflammatory and hypoxic microenvironmentsin the atherosclerotic lesions affect metabolic status of these cells [15]. The metabolic differences might have been due to differences in diabetic models and the presence of atherosclerotic lesions in our model. Peiró et al. [22] reported that high glucose enhances activities of
glucose-6-phosphate dehydrogenase, a pentose phosphate pathway enzyme, and nicotinamide
adenine dinucleotide phosphate oxidase by interleukin 1β in cultured SMCs. Although metabolite levels of pentose phosphate pathway increased in the diabetic and non-diabetic atherosclerotic arteries compared with non-atherosclerotic arteries, the diabetic condition did not
potentiate the metabolic change in our in vivo study (S3 Table).
We assessed metabolic changes in glycolysis by measuring 18F-FDG uptake and determining its relationship to vascular hypoxia. The uptake of 18F-FDG was reduced in diabetic,
compared with non-diabetic atherosclerotic lesions. These findings are compatible with thepresent results of the metabolome analysis. We previously found that increased 18F-FDG
uptake in macrophage-rich atherosclerotic lesions was positively associated with thrombogenicity [23]. Hyperglycemia itself impairs 18F-FDG uptake, reportedly via competition withendogenous blood glucose [24,25], whereas contradictory findings have also been published[26,27]. We found here that 18F-FDG uptake in non-injured arteries did not significantly differ
between diabetic and non-diabetic rabbits, indicating that reduced 18F-FDG uptake in diabetic
atherosclerotic lesions is due to metabolic changes in vascular cells rather than competition
with endogenous blood glucose. Hellberg et al. [28] found lower absolute 18F-FDG uptake anda lower aorta-to-myocardial 18F-FDG uptake ratio determined by autoradiography in diabetic,
than in non-diabetic mice. The uptake of 18F-FDG positively correlated with macrophage content and hypoxic (pimonidazole-positive) areas of atherosclerotic lesions in rabbits withoutDM, but a correlation was not found in diabetic atherosclerosis. The lack of relationshipbetween 18F-FDG uptake and macrophage content in diabetic atherosclerosis suggests alteration of macrophage glucose metabolism in alloxan-induced diabetic rabbits.
The promotion of glucose uptake and the glycolytic pathway by hypoxia is generally HIF1-dependent in many types of cells including macrophages [15]. We found reduced HIF-1
expression in diabetic atherosclerotic lesions, and this did not increase as the size of hypoxic
areas increased. Since HIF-1 signaling protects against cell death and apoptosis by reducing
oxidative stress, mitochondrial injury and metabolic derangement [29], the inappropriate
response of HIF-1 to hypoxia associated with diabetes would induce cell death and increase
the necrotic core. A study of wound healing in diabetic mice model found reduced levels of

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Vascular metabolism in diabetic atherosclerosis

Fig 6. Apoptosis and hypoxic areas in diabetic and non-diabetic atherosclerosis. A. Representative
double immunofluorescence staining of hypoxic areas and apoptotic cells in diabetic and non-diabetic
atherosclerosis. Images are stained with fluorescein isothiocyanate-labeled anti-BrdU antibody (green),
Dylight™549 fluorophore-labeled anti-pimonidazole antibody (red), and merged. B. Numbers of apoptotic cells
in diabetic and non-diabetic atherosclerosis (Mann-Whitney U-test).
https://doi.org/10.1371/journal.pone.0175976.g006

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Vascular metabolism in diabetic atherosclerosis

HIF-1α and HIF-1 target genes in lesions despite hypoxia [30]. Taken together, these and thepresent findings suggest that hypoxic responses are impaired in diabetic atherosclerosis, andthat reduced HIF-1α nuclear translocation partly contributes to altered glucose metabolism,
macrophage accumulation and enhanced apoptosis in atherosclerotic lesions.
Betaine aldehyde, intermediate metabolite of choline and betaine, belonged to the metabolite cluster with reduced levels in diabetic atherosclerosis. Betaine is an essential osmolyte andmethyl group donor [31]. Its metabolism links several metabolites, including choline, homocystine, and methionine. Clinical studies showed that plasma betaine levels were negatively
associated with triglyceride and non-high-density lipoprotein cholesterol, and that lowest
quintile of plasma betaine was associated with secondary acute myocardial infarction [32,33].
Although exact mechanisms are unclear, the evidence suggests that alteration of betaine
metabolism is associated with diabetic atherosclerotis and atherothrombotic complication.
This study has several limitations. The metabolites investigated herein represent only a portion of the total vascular metabolites. Further analysis by CE-TOFMS combined with liquid
chromatography-mass spectrometry might unveil further comprehensive metabolic changes.
We analyzed specimens at only five weeks after inducing diabetes and thus time-coursechanges in vascular metabolism during atherogenesis were not investigated. Alloxan-induced
diabetes is a model of type 1 DM, but type 2 DM is a more common risk factor for CVD. Studies of type 2 DM models should provide further understanding of the underlying mechanismsof diabetic atherosclerosis.
In conclusion, our results suggest that glycolytic metabolism and glucose uptake are alteredin diabetic atherosclerosis. An altered hypoxic response might partly contribute to metabolicchanges and enhanced apoptosis in diabetic atherosclerosis.

Supporting information
S1 Table. Body weight and serum parameters in non-diabetic and diabetic rabbits fed with
0.5% cholesterol diet at baseline and five weeks later.
(DOCX)S2 Table. Factor loading value for principal component 1 and 2 in arterial metabolites.
(XLSX)S3 Table. Hierarchical clustering analysis of arterial metabolites.
(XLSX)S4 Table. Original data of Figs 2, 3B, 4, 5 and 6B.
(XLSX)

Acknowledgments
We thank Ritsuko Sotomura and Kyoko Ohashi for excellent technical assistance.

Author Contributions
Conceptualization: YM AY SZ CK K. Kitamura K. Kawai YK NT YA.
Data curation: YM AY.
Formal analysis: YM AY TI CS.
Funding acquisition: AY YA.
Investigation: YM AY YZ SZ KY TI CS.

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Vascular metabolism in diabetic atherosclerosis

Methodology: YM AY TI CK.
Project administration: YM AY CK K. Kawai YK YA.
Supervision: K. Kitamura K. Kawai YK NT YA.
Visualization: YM AY.
Writing – original draft: YM AY.
Writing – review & editing: K. Kitamura K. Kawai YK NT YA.

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