Diabetes and atherosclerosis: is there a role for hyperglycemia?

Introduction

Development of atherosclerosis

Diabetes and development of arteriosclerosis

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Patients with insulin resistance and early type 2 diabetes exhibit an increased propensity to develop a diffuse and extensive pattern of arteriosclerosis. Typically, these diabetes and development of arteriosclerosis show elevated serum levels of the proinsulin cleavage product C-peptide and immunohistochemical data from our group revealed C-peptide deposition in early lesions of these individuals.

Moreover, in vitro studies suggest that C-peptide could promote atherogenesis. This study examined whether C-peptide promotes vascular inflammation and lesion development in a mouse model of arteriosclerosis.

ApoE-deficient mice on a high fat diet were treated with C-peptide or control injections for 12 weeks and the effect on lesion size and plaque composition was analysed. C-peptide treatment significantly increased C-peptide blood levels by 4. In these mice, C-peptide deposition in atherosclerotic plaques was significantly increased compared with controls. Moreover, lesions of C-peptide葉reated mice contained significantly more macrophages 1.

Finally, lipid deposition measured by Oil-red-O staining in the aortic arch was significantly higher in the C-peptide group compared with controls. Our results demonstrate that elevated C-peptide levels promote inflammatory cell infiltration and lesion development in ApoE-deficient mice without having metabolic effects, diabetes and development of arteriosclerosis. These data obtained in a mouse model of arteriosclerosis support the hypothesis that C-peptide may have an active role in atherogenesis in patients with diabetes and insulin resistance.

Patients with insulin resistance and early type 2 diabetes typically develop a diffuse and extensive pattern of arteriosclerosis [ 12 ]. Temporarily, diabetes and development of arteriosclerosis, these patients demonstrate elevated levels of the proinsulin cleavage product C-peptide.

For years, C-peptide has been considered to be biologically inert, until recent work has demonstrated that C-peptide can activate intracellular signalling pathways in various cell types [ 3 - 6 ]. Previous data from our group raise the hypothesis that C-peptide might play a causal role in atherogenesis in patients with diabetes [ 7 ]: In vitro migration assays, employing a modified Boyden chamber, demonstrated that C-peptide induces chemotaxis of both monocytes and CD4-positive lymphocytes [ 789 ].

These data raise the hypothesis that C-peptide may deposit in the subendothelial space in early lesions of patients with insulin resistance and diabetes subsequently promoting the recruitment of inflammatory cells into the vessel wall through its chemotactic action. Such mechanisms may contribute to lesion development and potentially explain why patients with diabetes develop a diffuse and extensive pattern of arteriosclerosis at diabetes and development of arteriosclerosis very early time-point.

In addition to its chemotactic effect on diabetes and development of arteriosclerosis and CD4-positive lymphocytes, C-peptide also colocalized with medial vascular smooth muscle cells VSMCs in some diabetic patients and enhanced VSMC proliferation in vitro [ 10 ], thus potentially promoting both the development of arteriosclerotic lesions as well as neointima formation after coronary intervention, diabetes and development of arteriosclerosis.

Still, these results only refer to in vitro data and observational studies and to date the causal role of C-peptide in lesions development in experimental in vivo models of arteriosclerosis remains unexplored, diabetes and development of arteriosclerosis.

Therefore, this study examined the effect of elevated C-peptide levels on lesion development in ApoE-deficient mice. A non-diabetic mouse model was employed to dissect direct effects of C-peptide on diabetes and development of arteriosclerosis development from potential confounding effects of insulin resistance and diabetes. Two groups served as a controls. Mice in the chow diet control group and high fat diet control group were fed with standard chow diet and Western diet, respectively, for 13 weeks.

At the beginning of the study, blood was obtained from the tail vein from each mouse to determine basal C-peptide levels in serum in each group.

After 12 weeks of treatment, blood was drawn again four hrs after last injectionto determine serum Dogs and upset stomach and pepcid levels after the treatment. On the next day the animals have been euthanized. This model using non-immunogenic rat C-peptide in a diabetic diabetes and acupuncture has previously been established diabetes and development of arteriosclerosis Langer et al.

The mice were housed individually under conventional conditions. The protocols were approved by the Regierungsprsidium Tuebingen Germany and all procedures were conducted in accordance with the recommendation given by the animal care facility of the University Ulm. Atherosclerotic lesions were analysed in the aortic arch, diabetes and development of arteriosclerosis, and descending aorta as previously described [ 11 ].

Briefly, mice were perfused at physiological pressure with normal saline via the left ventricle, diabetes and development of arteriosclerosis, and the hearts and aortas were removed en bloc. The aortic arch was embedded in optical cutting temperature compound Tissue Freezing Medium, Jung, Germany. To evaluate intimal lesion size, frozen sections of aortic arch were incubated with Oil-red-O 0. Longitudinal cryostat sections of the aortic arch, and formalin fixed, pinned-out en face preparations of the descending aorta were prepared as described previously [ 1213 ].

Aortic lesions were stained with Oil-red-O according to the method of Paigen et al, diabetes and development of arteriosclerosis. We analysed mouse atherosclerotic lesions in longitudinal sections from a segment of the lesser curvature of the aortic arch defined using a perpendicular line dropped from the right side of the innominate artery and from the left side of the left subclavian diabetes and development of arteriosclerosis, as shown in Figure 2A.

Images were captured by a digital system, area of staining was measured using computer-assisted image quantification Image-Pro Plus software and the percentage of the total area with positive colour was calculated and recorded for each mouse. Measurements and evaluation of the atherosclerotic lesions were performed in a blinded fashion. A Representative photograph of a mouse aortic arch longitudinal section, diabetes and development of arteriosclerosis, used for the analysis of total wall area by computer-assisted image quantification.

B C-peptide deposition in the aortic arch. Representative longitudinal sections of the aortic arch show C-peptide deposition in atherosclerotic plaques in a control a and a C-peptide葉reated mouse b. Adjacent sections de and f stained with identical concentrations of type and class matched IgG show no immunoreactive C-peptide. Arrow indicates C-peptide positive areas. ApoE-deficient mice on chow or high fat diet alone served as controls. Each data point represents a value from a single mouse chow diet: After 12 weeks of C-peptide or water s.

Furthermore, glucose and insulin levels showed no differences between groups Table 2. No local side-effects of C-peptide injections were observed. Metabolic characteristics of mice on chow diet or high fat diet, as well as placebo or C-peptide treated mice on high fat diet. Markers for endothelial function: Basal C-peptide levels did not differ between groups 1.

After 12 weeks of treatment, s, diabetes and development of arteriosclerosis. Given that patients with early diabetes and insulin resistance exhibit elevated levels of C-peptide, diabetes and development of arteriosclerosis, these data suggest that our model may be suited to mimic the pathophysiological condition in these patients. C-peptide serum levels at baseline and after 12 weeks of C-peptide s.

C-peptide levels were measured with RIA in blood samples taken four hrs after the last application of dissolved peptide. To examine the deposition of C-peptide in early arteriosclerotic lesions, we used longitudinal sections of the aortic arch Fig.

Immunohistochemical analyses of the aortic arch showed prominent Diabetes and development of arteriosclerosis staining in C-peptide葉reated mice with only scarce deposition in the control group. Staining of sections diabetes and development of arteriosclerosis isomatched IgGs at similar concentrations showed no immunoreactivity, thus affirming the specificity of the detected signals Fig.

Computer-assisted image quantification revealed significantly higher C-peptide deposition in C-peptide葉reated mice compared to controls 2. As shown in Fig. Adjacent sections de and f stained with identical amounts of type and class matched IgG show no immunoreactivity. Arrow indicates Mac-3 positive area red.

A, B High power view of rectangle in A sections of C-peptide葉reated mice show immunoreactive C-peptide in the intima stained in red, indicated diabetes and development of arteriosclerosis arrows.

C, F Represent IgG-matching controls. Moreover, staining of lesions for Ki, a proliferation marker, revealed a trend towards a higher cell proliferation rate in C-peptide treated mice 0. C-peptide treatment increases smooth muscle cell content in the aortic arch of C-peptide treated mice. Finally, we investigated lipid deposition in lesions using Oil-red-O staining. In C-peptide葉reated mice, lipid deposition in the aortic arch was significantly higher compared with controls Moreover, there was a trend預lbeit not significant葉owards increased lipid deposition in en face preparations of the abdominal and thoracic aorta in C-peptide葉reated mice compared to control mice Fig.

A C-peptide treatment increases lipid content in the aortic arch. B C-peptide treatment increases plaque formation in mice. Representative microphotographs of thoracic and abdominal aortic specimen stained for lipid deposition with Oil-red-O in a control a and a C-peptide葉reated mouse b. Arrows indicate fat rich plaques. This study demonstrates for the first time that elevated serum levels of C-peptide increase macrophage and VSMC content in the vessel wall comparing levitra and viagara ApoE-deficient mice, thus leading to increased lesion formation in this model.

Previously, observational data from our group have demonstrated that C-peptide deposits in the subendothelial space in early lesions of patients with coumadin and nosebleeds, colocalizing there with inflammatory cells such as monocytes and lymphocytes [ 7diabetes and development of arteriosclerosis, 8 ]. Moreover, C-peptide擁nduced chemotaxis of these cells in vitroraising the hypothesis that C-peptide could contribute to early atherogenesis antibiotics and candida patients with diabetes and insulin resistance: To distinguish the effect of C-peptide on vessel wall inflammation and plaque formation from potentially confounding metabolic effects in diabetes and insulin resistance, we chose the model of non-diabetic ApoE-deficient mice and increased C-peptide concentration by twice daily s.

A similar approach has previously been used by Langer et al. In our model, C-peptide injections were well-tolerated without any side effects, leading to a four- to five-fold increase in serum C-peptide levels, thus mimicking elevated concentrations in patients with insulin resistance and early type 2 diabetes. Interestingly, in contrast to previous reports, C-peptide treatment of these mice did not affect glucose or insulin levels nor the lipid profile.

This difference to other studies may be due to the different animal models used [ 16 ]. In our experimental setting week-old ApoE-deficient mice were put on a Western type diet for 1 week to trigger lesion development. After this week, diabetes and development of arteriosclerosis, mice were treated with C-peptide for 12 weeks in parallel to a high fat diet, as shown before [ 1718 ].

Because we were interested to explore the effect of C-peptide on early lesion development, we choose a moderate diet and harvested animals already after 13 weeks of diet. In addition to that, we found a clear, albeit not significant trend to increased plaque formation in the en face preparation in the aorta.

Conflicting in vitro data exist on the role of C-peptide in SMC proliferation [ 101920 ] depending on the origin of SMCs as well as the experimental conditions chosen. Our previous in vitro data clearly demonstrated an effect of C-peptide on CD4-positive lymphocyte chemotaxis but due to methodological limitations with lack of an appropriate antibody for CD4-positive cells for immunohistochemistry in mice, we were not able to examine the effect of C-peptide on CD4-positive lesion lymphocyte content, but we see no differences for CD3-positive lesion lymphocyte content in our model.

This data are in contrast to several findings in which C-peptide reduce up-regulation of cell adhesion molecules under inflammatory conditions [ 2122 ]. The use of solvent water in control mice is a limitation of our study and treatment with scrambled C-peptide or peptide with endothelium affinity may have been a more appropriate control.

Still, previous in vitro data from our group demonstrated that scrambled C-peptide does not exhibit any effect in vascular cells. However, future experiments should use scrambled C-peptide or peptide with endothelium affinity as a negative control. Taken together, this study demonstrates that elevated C-peptide concentrations increase monocyte recruitment into the vessel wall in vivothus promoting lesion formation in a mouse model of arteriosclerosis.

Our findings illustrate for the first time a hitherto unapparent causal link between high levels of serum-C-peptide and the progression of atherosclerotic lesions in an animal model, thus giving further momentum to the hypothesis that C-peptide may contribute to lesion development in patients with insulin resistance and early type 2 diabetes. National Center for Biotechnology InformationU. J Cell Mol Med.

Published online Apr Received Mar 1; Accepted Jun This article has been cited by other articles in PMC.

 

Diabetes and development of arteriosclerosis

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