Diabetes and diabetes-associated lipid abnormalities have distinct effects on initiation and progression of atherosclerotic lesions (original) (raw)
Diabetic mice show elevated blood glucose and glycated hemoglobin levels. LCMV injection resulted in stable diabetes, which is defined as blood glucose levels greater than 13.9 mmol/l (250 mg/dl) in 90% of mice expressing the GP transgene but never in mice lacking GP. At the end of the study, the body weights of the diabetic mice were slightly lower than those of nondiabetic mice (5–13% lower), but all diabetic mice had gained weight during the course of the study (Table 1).
Average weights, blood glucose, glycated hemoglobin, plasma lipid levels, and plasma insulin levels in diabetic and nondiabetic mice
Blood glucose levels had increased by 1–2 weeks after LCMV injection and remained significantly elevated throughout the study in diabetic mice fed the different diets (Figure 1). Mean blood glucose levels and glycated hemoglobin levels were more than 2-fold higher in diabetic than in nondiabetic mice (Table 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.
One group of diabetic mice fed the cholesterol-free diet was treated intensely with insulin to achieve improved metabolic control (Figure 1; Table 1). Blood glucose levels in these mice were not significantly different from those of nondiabetic mice.
Diabetic mice demonstrate loss of endogenous insulin and insulitis. Cross sections of the pancreata from nondiabetic (Figure 2, A and B) and diabetic (Figure 2, C and D) mice were analyzed by immunohistochemistry using an anti–insulin antibody. Analysis of the pancreata from diabetic mice indicated a severe destruction of β cells and loss of immunoreactive insulin (Figure 2, C and D) compared with pancreata from nondiabetic mice (Figure 2, A and B). H&E staining also revealed insulitis, with characteristic infiltration of T cells (5) into remaining insulin-positive islets (Figure 2C). The number of insulin-positive islets per pancreatic cross section was 7-fold lower in diabetic mice than in nondiabetic mice (41.5 ± 3.6 islets per cross section in nondiabetic mice and 6.1 ± 2.0 islets per cross section in diabetic mice; P < 0.0001). There were no differences in the number of islets among diabetic mice fed the different diets (data not shown).
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.
Nonfasting insulin levels varied between 40 and 295 pmol/l in nondiabetic mice, between 18 and 332 pmol/l in insulin-treated diabetic mice, and between 79 and 296 pmol/l in intensely insulin-treated diabetic mice (Table 1). Diabetic mice that showed insulin levels in the high range (<100 pmol/l) generally had received new insulin pellets less than 2 weeks prior to the end of the study. Endogenous insulin levels in diabetic mice that had not received exogenous insulin during the last 2 months were as low as 23.4 ± 2.3 pmol/l (mean ± SEM, n = 4).
Diabetic mice fed a cholesterol-free diet show no lipid abnormalities. Nondiabetic and diabetic LDLR–/–;GP mice fed the cholesterol-free diet had similar triglyceride and cholesterol levels (Table 1). Accordingly, nondiabetic and diabetic mice had similar lipoprotein profiles at the end of the study. Thus, diabetes alone does not induce a lipid disorder when these LDLR–/– mice are fed a cholesterol-free diet (Figure 3A). Furthermore, the susceptibility of LDL to oxidation by CuSO4 in diabetic mice fed the cholesterol-free diet was not different from that of nondiabetic mice (Figure 3B). When mice were fed the cholesterol-free diet, the intensely insulin-treated group of diabetic mice did not have altered blood cholesterol levels in comparison with diabetic mice fed the same diet, but they did show a small but significant elevation of cholesterol levels in comparison with nondiabetic mice at the end of the study (Table 1).
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.
Diabetic mice fed cholesterol-rich diets show increased VLDL and triglyceride levels. Hyperlipidemia, more severe than that normally seen in LDLR–/– mice, developed when dietary fat and cholesterol contents were increased. Cholesterol levels did not change markedly between 6 and 12 weeks from the start of the study (Table 1), and stable hypercholesterolemia was reached prior to week 3 in most mice (data not shown). Diabetic mice fed the 0.12% or the 0.5% cholesterol diet showed markedly elevated cholesterol and triglyceride levels compared with nondiabetic mice (Table 1). These increases were due entirely to an increase in the levels of VLDL (Figure 3, C and D). Nonesterified fatty acids (NEFAs) also were significantly increased in diabetic as opposed to nondiabetic mice fed the 0.5% cholesterol diet (Table 1).
Diabetes causes more atherosclerotic lesions regardless of diet. Nondiabetic and diabetic LDLR–/–;GP mice developed aortic lesions, particularly in the arch and in the thoracic aorta distal to the arch. The diabetic mice that were fed the cholesterol-free diet showed significantly increased atherosclerotic lesion area, 2.8-fold over nondiabetic mice (Figure 4, A and B). Statistical univariate ANOVA showed that this increase could be explained by hyperglycemia (i.e., the increased lesion area was no longer statistically significant when corrected for hyperglycemia; P > 0.05) but not by total cholesterol (corrected P < 0.001). Intense insulin treatment of diabetic mice fed the cholesterol-free diet resulted in normalization of the lesion area (Figure 4, A and B).
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.
Diabetic mice that were fed the 0.12% and 0.5% cholesterol diets also showed significantly increased lesion area (2.2-fold and 2.5-fold over nondiabetic mice, respectively) (Figure 4, A and B). In these diabetic mice, the increase could be explained by the increased cholesterol levels (i.e., the increased lesion area was no longer statistically significant when corrected for plasma cholesterol levels; corrected P > 0.05). To further analyze the contribution of hyperglycemia as opposed to that of hyperlipidemia in these mice, subgroups of diabetic and nondiabetic mice with plasma cholesterol levels in the range of 30 mmol/l were selected for comparison of lesion areas. Diabetic mice were selected from the groups fed the 0.12% and the 0.5% cholesterol diets, whereas nondiabetic mice were selected from the groups fed the 0.5% and the 1.25% cholesterol diets. The results show that the lesion area in fat-fed nondiabetic and diabetic mice with similar extents of hyperlipidemia was similar (Figure 4C). These results suggest that hyperglycemia has no demonstrable effect on lesion area in the presence of severe hyperlipidemia and that the increased lesion area in diabetic mice fed the cholesterol-rich diets is due primarily to the increased cholesterol and triglyceride levels seen in these mice.
LCMV infection in mice lacking the GP transgene does not mimic the effects of diabetes. LCMV-injected control mice lacking the GP transgene showed no differences in lipid levels, glucose levels, or extent of atherosclerosis compared with mice injected with saline. The lack of effect of LCMV infection was apparent in mice fed the cholesterol-free diet, in mice fed the 1.25% cholesterol diet (Table 2), and in mice fed the 0.12% or 0.5% cholesterol diet (data not shown). Thus, the results obtained in diabetic mice are not due to LCMV infection per se.
LCMV infection does not affect levels of glucose, lipids, or atherosclerosis in mice lacking the GP transgene
Diabetic mice fed the cholesterol-free diet show increased lesion initiation, whereas mice fed cholesterol-rich diets show marked intralesional hemorrhage in the brachiocephalic artery. Diabetic mice fed the cholesterol-free diet demonstrated fatty-streak lesions characterized by macrophage infiltration in the brachiocephalic artery (BCA), as shown in Figure 5, A–F, whereas nondiabetic mice had significantly fewer fatty-streak lesions at this site (Figure 5G; Table 3). In addition, diabetic mice had a significantly higher frequency of glycosaminoglycan accumulation in the aortic media compared with nondiabetic mice, as demonstrated by Movat’s pentachrome stain (e.g., Figure 5, A and G; Table 3). To investigate possible mechanisms of the increased atherosclerosis in diabetic mice fed the cholesterol-free diet, AGEs were detected by immunohistochemistry. Most of the AGE immunoreactivity was observed in macrophage-rich areas, but some staining was also apparent in smooth muscle cells and in the extracellular tissue surrounding the vessel. No staining was present in the negative rabbit serum control (Figure 5I). Nondiabetic mice fed the cholesterol-free diet exhibited little AGE staining (Figure 5J), whereas diabetic mice showed positive AGE immunoreactivity localized primarily to macrophages (Figure 5H).
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.
Fatty streaks and glycosaminoglycan accumulation in diabetic mice fed a cholesterol-free diet, and intralesional hemorrhage in the BCA of cholesterol-fed diabetic mice
For mice fed the cholesterol-rich diets, lesion severity was markedly increased by the presence of diabetes. Diabetic mice on the 0.12% and 0.5% cholesterol-rich diets consistently exhibited intralesional hemorrhage (Figure 6, A–C), and erythrocytes were present in the lesions (Figure 6B). Conversely, intralesional hemorrhage was seldom found in nondiabetic mice fed cholesterol-rich diets at this 12-week time point (Figure 6D). AGE immunoreactivity was present in macrophages in lesions from both diabetic (Figure 6E) and nondiabetic mice (Figure 6F) that were fed cholesterol-rich diets.
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.
To address the question whether diabetic mice that were fed the cholesterol-rich diets exhibited more intralesional hemorrhage regardless of lesion size, the diabetic animals were matched with nondiabetic animals that were fed any of the three cholesterol-rich diets but with the same maximal cross-sectional area of lesions in the BCA (Figure 6G). Diabetic mice with a maximal lesion cross-sectional area of 119,231 ± 19,497 μm2 (mean ± SEM, n = 9) were matched with nondiabetic mice with a maximal lesion cross-sectional area of 114,783 ± 30,232 μm2 (n = 9; P = 0.91). In these diabetic mice, hemorrhage covered 60% ± 10% of the analyzed BCA lesion length but only 12% ± 4% (P < 0.001) of the lesion in nondiabetic mice, as shown graphically in Figure 6G. Therefore, the effect of diabetes on intralesional hemorrhage is not merely a reflection of the larger lesions in diabetic mice but is due to variables within the diabetic milieu.
Notably, diabetic mice fed the high-cholesterol diets showed increased mortality. Whereas 93% of diabetic mice fed the cholesterol-free diet survived, only 41% of diabetic mice fed the 0.12% diet survived, and 33% of diabetic mice fed the 0.5% cholesterol diet survived the 12-week study. In contrast, 100% of the nondiabetic mice survived, regardless of diet. The increased mortality in diabetic mice on these cholesterol-rich diets was not due to increased severity of the diabetic state (e.g., dehydration, hyperglycemia, and/or ketoacidosis). In some of the diabetic fat-fed mice that died or (when possible) were euthanized on an emergency basis during the course of the study, advanced lesions with cholesterol clefts were observed in the extension of the aortic sinus into the coronary arteries (Figure 7A), and coronary microvessels almost completely occluded by foam cells were present (Figure 7B). In addition, at least 30% of these animals showed complete occlusion of arteries (aorta, coronary microvessels, and/or pulmonary microvessels) caused by Sudan IV–positive neutral lipid deposits (Figure 7, C and D). The hearts of some of these diabetic mice also showed interstitial fibrosis, and the kidneys frequently contained microaneurysms (7); such changes are not likely to be fatal, however. No other abnormalities that may have caused death were found in these diabetic mice. The increased mortality in diabetic mice that were fed the cholesterol-rich diets is therefore probably due to occlusion of arteries by lipid embolism and occluding plaques.
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).