Altered glucose metabolism and hypoxic response in alloxan-induced diabetic atherosclerosis in rabbits

<div><p>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 to vascular histopathological changes in the atherosclerotic arteries of rabbits with alloxan-induced diabetes. Diabetic atherosclerosis was induced in rabbit ilio-femoral arteries by injecting 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 hypoxia-inducible factor-1α, and apoptosis. We evaluated comprehensive arterial metabolism by performing metabolomic analyses using capillary electrophoresis-time of flight mass spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using ¹⁸F-fluorodeoxyglucose and pimonidazole. Plaque burden, macrophage content, and hypoxic 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 with glycolysis 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 hypoxia in atherosclerotic lesions under conditions of insulin-dependent diabetes might be involved in the development of diabetic atherosclerosis.</p></div

Apoptosis and hypoxic areas in diabetic and non-diabetic atherosclerosis.

<p>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).</p

Metabolomic analysis of arterial metabolites in rabbits with and without alloxan-induced diabetes.

<p>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 atherosclerotic arteries (11–15, green). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175976#pone.0175976.s002" target="_blank">S2 Table</a> shows factor-loading values for PC1 and PC2 on score plots. B. Representative heat map assessed using hierarchical clustering analysis shows metabolic differences among groups. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175976#pone.0175976.s003" target="_blank">S3 Table</a> shows original data for each metabolite.</p

Expression of HIF-1α in atherosclerotic arteries of rabbits with and without alloxan-induced diabetes.

<p>A. Western blots of HIF-1α in nuclear extract of atherosclerotic arteries of diabetic and non-diabetic rabbits (Mann-Whitney U-test). Data are shown as fold change relative to non-DM and expressed as mean ± SD. B. Representative immunohistochemical images of macrophages, 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. Correlations between pimonidazole-immunopositive areas and numbers of HIF-1α immunopositive nuclei in sections of atherosclerotic arteries in diabetic and non-diabetic rabbits (Spearman’s correlation coefficient).</p

Relationship between ¹⁸F-FDG uptake and hypoxia in arteries of rabbits with and without alloxan-induced diabetes.

<p>Uptake of 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.</p

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

<p>Histological characteristics of control and atherosclerotic arteries in non-diabetic and diabetic rabbits, respectively, fed with 0.5% cholesterol diet.</p

Increased Metabolite Levels of Glycolysis and Pentose Phosphate Pathway in Rabbit Atherosclerotic Arteries and Hypoxic Macrophage

<div><p>Aims</p><p>Inflammation and possibly hypoxia largely affect glucose utilization in atherosclerotic arteries, which could alter many metabolic systems. However, metabolic changes in atherosclerotic plaques remain unknown. The present study aims to identify changes in metabolic systems relative to glucose uptake and hypoxia in rabbit atherosclerotic arteries and cultured macrophages.</p><p>Methods</p><p>Macrophage-rich or smooth muscle cell (SMC)-rich neointima was created by balloon injury in the iliac-femoral arteries of rabbits fed with a 0.5% cholesterol diet or a conventional diet. THP-1 macrophages stimulated with lipopolysaccharides (LPS) and interferon-γ (INFγ) were cultured under normoxic and hypoxic conditions. We evaluated comprehensive arterial and macrophage metabolism by performing metabolomic analyses using capillary electrophoresis-time of flight mass spectrometry. We evaluated glucose uptake and its relationship to vascular hypoxia using ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) and pimonidazole, a marker of hypoxia.</p><p>Results</p><p>The levels of many metabolites increased in the iliac-femoral arteries with macrophage-rich neointima, compared with those that were not injured and those with SMC-rich neointima (glycolysis, 4 of 9; pentose phosphate pathway, 4 of 6; tricarboxylic acid cycle, 4 of 6; nucleotides, 10 of 20). The uptake of ¹⁸F-FDG in arterial walls measured by autoradiography positively correlated with macrophage- and pimonidazole-immunopositive areas (r = 0.76, and r = 0.59 respectively; n = 69 for both; p<0.0001). Pimonidazole immunoreactivity was closely localized with the nuclear translocation of hypoxia inducible factor-1α and hexokinase II expression in macrophage-rich neointima. The levels of glycolytic (8 of 8) and pentose phosphate pathway (4 of 6) metabolites increased in LPS and INFγ stimulated macrophages under hypoxic but not normoxic condition. Plasminogen activator inhibitor-1 protein levels in the supernatant were closely associated with metabolic pathways in the macrophages.</p><p>Conclusion</p><p>Infiltrative macrophages in atherosclerotic arteries might affect metabolic systems, and hypoxia but not classical activation might augment glycolytic and pentose phosphate pathways in macrophages.</p></div

Uptake of ¹⁸F-FDG in arteries of rabbits with and without alloxan-induced diabetes.

<p>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).</p

Levels of metabolites of glycolysis, the pentose phosphate pathway, tricarboxylic acid cycle and glyconeogenesis/glycogenolysis in rabbit iliac-femoral arteries.

<p>Gray, blue and red bars: iliac-femoral arteries that were not injured (conventional diet), and those with SMC-rich (conventional diet) and macrophage-rich (0.5% cholesterol diet) neointima, respectively (n = 3 for all). Metabolite levels are expressed as nmol/g. *p<0.05 vs. other groups, ^†p<0.05 vs. non-injured femoral artery. 1,3BPG, 1,3-bisphosphoglycerate; 2PG, 2-phosphoglyceric acid; 3PG, 3-phosphoglyceric acid; 2OG, 2-oxoglutaric acid; 6PG, 6-phosphogluconic acid; DHAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; F1-6P, fructose 1,6-diphosphate; F6P, fructose 6-phosphate; FFA, free fatty acid; G1P, glucose 1-phosphate; G3P, glyceraldehyde 3-phosphate; G6P, glucose 6-phosphate; Glu, glucose; Gly, glycogen; PEP, phosphoenolpyruvic acid; R5P, ribose 5-phosphate; Ru5P, ribulose 5-phosphate; S7P, sedoheptulose 7-phosphate; Xu5P, xylulose 5-phosphate.</p

Histological findings of iliac-femoral arteries that were not injured or those at three weeks after balloon injury in rabbits fed with conventional or 0.5% cholesterol diets.

<p>A. Representative histological images of arterial sections stained with hematoxylin eosin (HE), and antibodies for muscle actin or rabbit macrophages. N, neointima; BI, balloon injury. B. Vascular areas, as well as areas that are immunopositive for SMCs and macrophages, and numbers of Ki-67-immunopositive nuclei in arteries. White and black bars, conventional and 0.5% cholesterol diet, respectively; n = 12 each). BI, balloon injury. Arteries at three weeks after balloon injury shows neointimal formation rich in SMCs (conventional diet) or macrophages (0.5% cholesterol diet), compared with non-injured arteries. C. Representative double immunofluorescent images for Ki-67 and macrophage or SMC in arteries 3 weeks after balloon injury in rabbits fed with 0.5% cholesterol diet. Images stained with CF488A labeled anti-Ki-67 antibody (green), CF495-labeled anti-macrophage antibody (red) or anti-SMC antibody (red), and merged immunofluorescent images. There are Ki-67 immunopositive nuclei in macrophages (upper row). The cells with Ki-67-immunopositive nuclei don't have SMC-positive cytoplasm (arrows).</p