COMMENTARIES

Accelerated Atherosclerosis in Diabetic Mice Mediated by High Glucose Levels and Anti-Atherosclerotic Effects of Insulin

Tony Hayek1 and Marielle Kaplan2, 1Lipid Research Laboratory and 2Clinical Biochemistry Laboratory
Rambam Medical Center
Haifa
Israel

Diabetes mellitus (DM) is associated with premature and accelerated atherosclerosis, and patients with diabetes are at 2-4 times increased risk for coronary artery diseases, which accounts for the major cause of death in these patients [1,2]. The precise mechanisms underlying the acceleration of atherosclerosis in DM is poorly understood, but it was suggested that hyperglycemia accelerates atherosclerosis by induction of vascular dysfunction, increased inflammatory burden, increased lipid peroxidation of lipoproteins, and arterial cells which lead to enhanced macrophage foam cell formation, the hallmark of early atherosclerosis [3,4]. Moreover, the role of insulin in atherosclerosis progression is uncertain. Hyperinsulinemia is considered to be an independent risk factor for atherosclerosis development, but there are some lines of evidence suggesting a protective role for insulin [5-7].

          The aim of the present study was to identify mechanisms involved in diabetes-induced macrophage cholesterol accumulation and foam cell formation. For this purpose, we first analyzed macrophage oxidative stress and cellular cholesterol metabolism in diabetic mice, followed by analysis of the direct effects of glucose on macrophage oxidative stress and cellular cholesterol accumulation. Furthermore, we wished to analyze whether insulin treatment can reverse the pro-atherosclerotic effects of diabetes mediated by glucose on macrophage foam cell formation.

Accelerated Atherosclerosis and Foam Cell Formation in Diabetic Mice

Induction of diabetes in atherosclerotic mice led to an increase in the aortic atherosclerotic lesion area after 3 months of diabetes by 106%, compared to the lesion area in age-matched untreated E0 mice as well as the appearance of more advanced lesions compared to untreated E0 mice. Peritoneal macrophages from E0 diabetic mice exhibited increased atherogenic properties as illustrated by increased macrophages lipid peroxidation and increased cellular cholesterol accumulation. MPM isolated from diabetic E0 mice exhibited a significant increase, by up to 140% in their lipid peroxide content and superoxide production respectively, as well as increased Ox-LDL uptake and cholesterol content by up to 50%, compared to MPM harvested from untreated E0 mice.

          These results were further assessed by inducing diabetes in normocholesterolemic non-atherosclerotic Balb C mice. Macrophage total peroxides, and lipid peroxides test were increased by 290% and by 61%, respectively, in comparison to control age-matched mice. Moreover, MPM from diabetic non-atherosclerotic mice exhibited higher cholesterol content by 4.2 fold, in comparison to MPM isolated from control mice. This could be related to the fact that MPM from diabetic mice demonstrated a significant increase in their ability to synthesize cholesterol, as well as to take up oxidized LDL by 5.9 fold and by 31%, respectively, in comparison to control mice macrophages.

Effect of High Glucose Concentrations on Macrophages Lipid Peroxidation and Cellular Cholesterol Accumulation

To analyze whether increased macrophages atherogenicity demonstrated in MPM from diabetic mice, was related to a direct effect of glucose on macrophages, J-774 A.1 macrophages were incubated with increasing concentrations of glucose and analyzed for their cellular lipid peroxidation and lipoproteins uptake. Furthermore, to determine mechanisms involved in glucose-induced macrophage foam cell formation, we compared the effect of the two glucose enantiomers, D-glucose and L-glucose. As L-glucose, unlike D-glucose, does not cross cell membranes, the use of both isomers enables us to specifically determine the effect of glucose uptake by macrophages on their oxidative status.

Cellular Lipid Peroxidation

Macrophages peroxides content increased by cell incubations with high D-glucose levels (30 mM), by 120%, in comparison to cells incubated with low D-glucose concentration, whereas L-glucose had no significant effect. Since regulation of the NADPH oxidase complex is involved in macrophage oxidative stress [8], we analyzed the effect of glucose on macrophage NADPH p47 mRNA expression, which is needed for NADPH activation. p47 mRNA expression was significantly up-regulated by 61% following macrophage incubations with high D-glucose concentration. To determine whether NADPH oxidase activation was requested in the glucose-induced macrophage lipid peroxidation, we used the p-47 knockout mice, where cellular NADPH oxidase is inactive. Incubation of 30 mM D-glucose with macrophages isolated from p-47 knockout failed to induce superoxide release in contrast to the results obtained with control Balb C mice.

Cholesterol Accumulation

Incubations of J-774 A.1 macrophage with high glucose concentrations of D-glucose (30 mM) led to an increase in macrophage cholesterol content by 418%, in comparison to macrophages incubated with low concentrations of glucose (5mM).

          Incubations of J774 A.1 macrophages with high D-glucose concentration increased the cell’s ability to take up oxidized LDL by 56%, paralleled by an up-regulation of the Ox-LDL receptor, CD36 mRNA expression, by 21%, in comparison to cells incubated with low D-glucose concentration.

          As cholesterol biosynthesis rate limiting step involves activation of the enzyme HMG-CoA reductase, we next questioned whether the increased macrophage cholesterol biosynthesis shown in MPM from diabetic mice is present also in macrophage incubated with high glucose concentration and if such an effect is mediated by a stimulatory effect on HMG-CoA reductase expression. Incubation of J774A.1 macrophages with high D-glucose concentration increased the cell’s ability to synthesize cholesterol, by 7.2 fold, paralleled by an up-regulation of HMG-CoA reductase mRNA expression, by 190%.

Effect of Insulin on Diabetes and Glucose-Induced Foam Cell Formation

In the next part of the study, we investigated the effect of insulin on diabetes induction of foam cell formation by analyses of macrophage lipids peroxidation, macrophage uptake of oxidized LDL by the cells, and macrophage cholesterol biosynthesis.

          Macrophage total peroxide and lipid peroxide levels in diabetic mice treated with insulin were decreased by 41% and by 40%, respectively, compared to age-matched diabetic mice. Similarly, macrophage Ox-LDL uptake and CD36 mRNA levels in diabetic mice treated with insulin were also decreased by 29% and by 41%, respectively, compared to diabetic mice. We next turned to an in vitro system, using J-774 A.1 macrophages that were incubated with high levels of glucose (30 mM) in the presence or absence of insulin. Addition of insulin to glucose-enriched cells led to a significant decrease in cellular lipid peroxidation, by 43%, compared to cells incubated with the high concentrations of glucose, but with no insulin. p47 mRNA expression was significantly down-regulated, by 26%, in glucose-enriched macrophages following their incubation with insulin, compared to macrophages incubated with high glucose, but without insulin. Incubations of glucose-enriched macrophages with insulin led to a significant up-regulation of the Ox-LDL receptor, CD36 mRNA expression, by 37%, compared to cells incubated only with glucose.

          Most important, macrophage cholesterol accumulation was significantly affected since total cholesterol content of macrophages incubated with insulin with high concentration of glucose was lower by 41% compared to macrophages incubated only with glucose. Incubations of macrophage with high glucose concentrations led to an increase in macrophage cholesterol content by 418% compared to macrophages incubated with low concentrations of glucose.

Conclusions

Diabetes induction in mice led to a dose-dependent increase in their atherosclerotic lesion area. This was associated with an increase in their macrophage foam cell formation as measured by the cell lipid peroxidation and ability to take up oxidized LDL (Ox-LDL). Glucose enrichment of macrophages was directly involved in induction of macrophage-derived foam cell formation since in an in vitro system macrophage enrichment with glucose was shown to induce lipid peroxidation Ox-LDL uptake and expression of the scavenger receptor CD36 at the mRNA level. In vitro studies in cultured macrophages showed that high glucose concentration can induce the above atherogenic effects and they required glucose uptake by the cells as D-glucose, but not L-glucose exhibited most of those effects [9,10]. Moreover, insulin treatment to diabetic mice or insulin incubation to macrophages was able to counteract both diabetes and glucose cellular enrichment, regarding the macrophages atherogenic properties.

The present study presents important novel insights on the events connecting diabetes and glucose stimulation of macrophages foam cell formation leading to atherosclerosis. Hyperglycemia directly affected the pathophysiology of accelerated atherosclerosis in diabetes, by leading to macrophage glucose accumulation and macrophages-derived foam cell formation. Most important, insulin exhibited inhibitory effects of on diabetes-mediated (and high glucose-induced) increased macrophage foam cell formation.

References

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