Diabetes and diabetes-associated lipid abnormalities have distinct effects on initiation and progression of atherosclerotic lesions

Abstract

Diabetes in humans accelerates cardiovascular disease caused by atherosclerosis. The relative contributions of hyperglycemia and dyslipidemia to atherosclerosis in patients with diabetes are not clear, largely because there is a lack of suitable animal models. We therefore have developed a transgenic mouse model that closely mimics atherosclerosis in humans with type 1 diabetes by breeding low-density lipoprotein receptor-deficient mice with transgenic mice in which type 1 diabetes can be induced at will. These mice express a viral protein under control of the insulin promoter and, when infected by the virus, develop an autoimmune attack on the insulin-producing beta cells and subsequently develop type 1 diabetes. When these mice are fed a cholesterol-free diet, diabetes, in the absence of associated lipid abnormalities, causes both accelerated lesion initiation and increased arterial macrophage accumulation. When diabetic mice are fed cholesterol-rich diets, on the other hand, they develop severe hypertriglyceridemia and advanced lesions, characterized by extensive intralesional hemorrhage. This progression to advanced lesions is largely dependent on diabetes-induced dyslipidemia, because hyperlipidemic diabetic and nondiabetic mice with similar plasma cholesterol levels show a similar extent of atherosclerosis. Thus, diabetes and diabetes-associated lipid abnormalities have distinct effects on initiation and progression of atherosclerotic lesions.

Figure 1

Diabetic mice demonstrated hyperglycemia throughout the study, and their metabolic control was improved by intense insulin therapy. Female LDLR^–/–;GP littermate mice were injected with saline (nondiabetic) or LCMV (diabetic) 1 week prior to changing the diet at week 0. The diets contained 0%, 0.12%, or 0.5% cholesterol (see key). One group of diabetic mice was treated with an intense insulin therapy (intense insulin) and fed the cholesterol-free diet. Blood glucose was measured at the indicated times. The number of animals per group is indicated within parentheses. NS, nonsignificant versus nondiabetic mice fed the cholesterol-free diet. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Diabetic mice exhibited severe insulitis and loss of immunoreactive insulin in β cells. Thirteen weeks after LCMV or saline injection, the pancreata of nondiabetic (A and B) and diabetic (C and D) mice were dissected, embedded, cross-sectioned, and stained with H&E (A and C). Immunoreactive insulin was detected by using a mouse monoclonal anti–insulin antibody in adjacent sections. This procedure results in a brown reaction product (B and D). The procedure was performed on 3–4 mice per group with similar results.

Figure 3

Diabetic mice fed cholesterol-rich diets developed diabetes-associated lipid abnormalities, while diabetic mice fed a cholesterol-free diet had no diabetes-induced lipid abnormalities. Plasma cholesterol profiles of nondiabetic and diabetic mice fed different diets were analyzed by FPLC (A, C, and D). A 100-μl aliquot of plasma obtained after 12 weeks on the diet was applied to a column filled with Superose 6HR. Each 250-μl fraction was subjected to cholesterol analysis. The results are presented as mean + SEM (A, C, and D) and mean ± SEM (B) of plasma samples obtained from 3 different animals. The major lipoprotein peaks (VLDL, LDL, and HDL) are indicated. Susceptibility of LDL to copper oxidation was measured in LDL fractions isolated from nondiabetic and diabetic mice fed the cholesterol-free diet (B). The appearance (lag phase and rate of formation) of conjugated dienes was measured by a spectrophotometer. Chol., cholesterol.

Figure 4

Diabetes caused a greater degree of atherosclerosis regardless of dietary cholesterol content (shown as percentage). After 12 weeks on diet, diabetic and nondiabetic LDLR^–/–;GP mice were killed by cardiac perfusion and their tissues fixed. The aorta was dissected, and the area covered by atherosclerotic lesions was identified by Sudan IV staining and image analysis. Results are expressed as mean + SEM (A and C) or as scatter plots (B). In C, subgroups of cholesterol-fed nondiabetic and diabetic mice with similar plasma cholesterol levels were selected for comparison of lesion area. The number of mice per group is indicated above each bar in A and C. Statistical analysis was performed by using unpaired Student’s t test or one-way ANOVA followed by the Newman-Keuls multiple-comparison test (*P < 0.05, **P < 0.01, and ***P < 0.001 for comparison of groups within brackets). Symbols in B are defined in Figure 1.

Figure 5

Diabetes caused lesion initiation in mice fed a cholesterol-free diet. Diabetic LDLR^–/–;GP mice fed the 0% cholesterol diet (A–F, H–I) and nondiabetic LDLR^–/–;GP littermates fed the 0% cholesterol diet (G and J) were perfusion fixed after 12 weeks on diet, as described in Figure 4. The BCA was dissected, paraffin embedded, and serial sectioned until maximal lesion size was identified. Sections were stained using a Movat’s pentachrome procedure (A–C, E, and G). Black represents nuclei and elastin, yellow represents collagen and reticular fibers, blue represents glycosaminoglycans, red represents muscle, and intense red represents fibrinoid and fibrin (hemorrhage). Some sections were used to detect macrophages (D and F), by using a rat monoclonal Mac-2 antibody, and others were used to detect AGEs (H and J) or used as negative controls (I). Representative sections are shown. Scale bars: 100 μm.

Figure 6

Diabetes caused advanced lesions in mice fed cholesterol-rich diets. Diabetic LDLR^–/–;GP mice fed the 0.12% cholesterol diet (A, B, and E), or 0.5% cholesterol diet (C), and nondiabetic LDLR^–/–;GP littermates fed the 0.12% cholesterol diet (F) or the 0.5% cholesterol diet (D) were perfusion fixed after 12 weeks on diet, as described in Figure 4. The BCA was dissected, paraffin embedded, and serial sectioned until maximal lesion size was identified. Sections were stained using a Movat’s pentachrome procedure (A–D). Black represents nuclei and elastin, yellow represents collagen and reticular fibers, blue represents glycosaminoglycans, red represents muscle, and intense red represents fibrinoid and fibrin (hemorrhage). Some sections were used to detect AGEs (E and F). Representative sections are shown. Note the intralesional hemorrhage (marked by arrows) in A and C. In B, erythrocytes in the lesion are indicated by open arrows. Scale bars: 100 μm (A, C–F); 20 μm (B). (G) A graphical representation of frequency of hemorrhage in lesions of similar size from diabetic and nondiabetic mice.

Figure 7

The combination of diabetes and cholesterol-rich diet caused occlusion of coronary microvessels and lipid embolism. Blocked coronary arteries and lipid embolism probably caused death in diabetic mice fed cholesterol-rich diets. (A) A Movat’s stained advanced lesion with cholesterol clefts and fibrous cap in the extension of the aortic sinus into the coronary arteries from a diabetic mouse. This mouse died after 11 weeks on the 0.12% cholesterol diet. (B) H&E staining of occluding intramyocardial lesions at the time of death from a diabetic mouse fed the 0.12% cholesterol diet for 11 weeks. (C) An aorta completely occluded by lipid at the time of death from a diabetic mouse fed the 0.5% cholesterol diet for 9 weeks. Analysis of cross sections stained with Oil red O confirmed that the aorta was completely occluded by lipid at a site without a lesion. (D) The same aorta as in C stained with Sudan IV. Scale bars: 100 μm (A); 20 μm (B); and 1 mm (C).