Spontaneously diabetic Ins2(+/Akita):apoE-deficient mice exhibit exaggerated hypercholesterolemia and atherosclerosis

Abstract

Type 1 diabetes (T1D) increases the risk of adverse coronary events. Among risk factors, dyslipidemia due to altered hepatic lipoprotein metabolism plays a central role in diabetic atherosclerosis. Nevertheless, the likely alterations in plasma lipid/lipoprotein profile remain unclear, especially in the context of spontaneously developed T1D and atherosclerosis. To address this question, we generated Ins2(+/Akita):apoE(-/-) mouse by cross-breeding Ins2(+/Akita) mouse (which has Ins2 gene mutation, causing pancreatic β-cell apoptosis and insulin deficiency) with apoE(-/-) mouse. Ins2(+/Akita):apoE(-/-) mice developed T1D spontaneously at 4-5 wk of age. At 25 wk of age and while on a standard chow diet, diabetic Ins2(+/Akita):apoE(-/-) mice exhibited an approximately threefold increase in atherosclerotic plaque in association with an approximatelty twofold increase in plasma non-HDL cholesterol, predominantly in the LDL fraction, compared with nondiabetic controls. To determine factors contributing to the exaggerated hypercholesterolemia, we assessed hepatic VLDL secretion and triglyceride content, expression of hepatic lipoprotein receptors, and plasma apolipoprotein composition. Diabetic Ins2(+/Akita):apoE(-/-) mice exhibited diminished VLDL secretion by ~50%, which was accompanied by blunted Akt phosphorylation in response to insulin infusion and decreased triglyceride content in the liver. Although the expression of hepatic LDL receptor was not affected, there was a significant reduction in the expression of lipolysis-stimulated lipoprotein receptor (LSR) by ~28%. Moreover, there was a marked decrease in plasma apoB-100 with a significant increase in apoB-48 and apoC-III levels. In conclusion, exaggerated hypercholesterolemia and atherosclerosis in spontaneously diabetic Ins2(+/Akita):apoE(-/-) mice may be attributable to impaired lipoprotein clearance in the setting of diminished expression of LSR and altered apolipoprotein composition of lipoproteins.

Fig. 1.

En face analysis of atherosclerotic lesion area in the aortas from spontaneously diabetic Ins2^+/Akita:apolipoprotein (apo)E^−/− mice. Representative photomicrographs of the entire aorta (magnification ×10; A) and aortic arch (magnification ×40; B) showing enhanced atherosclerotic lesion in the diabetic mice compared with age-matched nondiabetic mice (25 wk of age, each group). C: atherosclerotic lesion area is expressed as the percentage of the total luminal surface area of the aortic arch in diabetic mice (n = 10) compared with nondiabetic mice (n = 7). The data shown in the bar graphs are means ± SE. *P < 0.01 compared with nondiabetic mice.

Fig. 2.

Changes in plasma lipid profile in spontaneously diabetic Ins2^+/Akita:apoE^−/− mice. A: plasma samples were obtained by heart puncture from diabetic Ins2^+/Akita:apoE^−/− mice and age-matched nondiabetic Ins2^+/+:apoE^−/− mice after a 5-h fast (25 wk of age; n = 4 mice/group). Total cholesterol (Total-C), HDL cholesterol (HDL-C), and triglyceride (TG) were measured, and non-HDL cholesterol (non-HDL-C) was calculated by subtracting HDL-C from Total-C. The data shown in the bar graphs are means ± SE. *P < 0.01 compared with nondiabetic mice. B: plasma samples were subjected to fast-performance liquid chromatography analysis, and cholesterol (top) and TG (bottom) were measured in each of the eluted fractions.

Fig. 3.

Hepatic VLDL secretion in diabetic Ins2^+/Akita:apoE^−/− mice under hyperinsulinemic euglycemic conditions. A: schematic of a hyperinsulinemic euglycemic clamp study; 20-wk-old diabetic Ins2^+/Akita:apoE ^−/− mice and nondiabetic littermate Ins2^+/+:apoE ^−/− mice (after a 5-h fast) were infused with insulin at a constant rate as indicated and with glucose at variable rates to maintain euglycemia. Triton WR-1339, an inhibitor of lipoprotein lipase, was injected intravenously at 90 min after infusions with insulin and glucose. Tail blood samples were collected at the indicated time points (●) to measure lipids before and after Triton WR-1339 injection. B: plasma insulin concentrations before (0 min) and during (210 min) the hyperinsulinemic euglycemic clamp study. C: the linear graphs show the changes in blood glucose concentrations during a 4.5-h hyperinsulinemic euglycemic clamp in diabetic mice (n = 7; ●) compared with nondiabetic control mice (n = 5; ○). D: the temporal changes in plasma levels of TG before and after Triton WR-1339 infusion in diabetic Ins2^+/Akita:apoE^−/− (n = 7; ●) compared with nondiabetic Ins2^+/+:apoE ^−/− mice (n = 5; ○) under hyperinsulinemic euglycemic conditions. The data shown in the linear graphs are means ± SE. NS, not significant. *P < 0.01 compared with nondiabetic mice.

Fig. 4.

Hepatic expression of insulin receptor (IR) signaling components and TG content in diabetic Ins2^+/Akita:apoE^−/− mice. A: immunoblot analysis of IR (β-subunit), phospho-Akt (p-Akt), and Akt from livers of 20-wk old nondiabetic (non-DM) Ins2^+/+:apoE ^−/− mice and diabetic (DM) Ins2^+/Akita:apoE^−/− mice at basal state and after a 4.5-h hyperinsulinemic euglycemic clamp, as described in Fig. 3. Mice were fasted for 5 h. The relative levels of p-Akt/Akt ratio are shown in the bar graph. The data are means ± SE; n = 3/group. *P < 0.05 compared with non-DM mice. B: hepatic TG content of 20-wk-old non-DM Ins2^+/+:apoE ^−/− mice and diabetic Ins2^+/Akita:apoE^−/− mice. Mice were fasted for 5 h. The data shown in the bar graph are means ± SE; n = 6–7/group. *P < 0.01 compared with non-DM mice.

Fig. 5.

The expression of hepatic lipoprotein receptors in diabetic Ins2^+/Akita:apoE^−/− mice. A: immunoblot analysis of the LDL receptor (LDLr) and lipolysis-stimulated lipoprotein receptor (LSR) expression in the liver extracts from 5-h-fasted 20-wk-old diabetic Ins2^+/Akita:apoE ^−/− mice (n = 6) and age-matched nondiabetic control Ins2^+/+:apoE ^−/− mice (n = 5). To normalize protein level, superoxide dismutase-1 (SOD-1) was used an internal control. B: the relative expression of hepatic lipoprotein receptors is shown in the bar graph. The data are means ± SE. *P < 0.01 compared with non-DM mice.

Fig. 6.

Diminished apoB-100 and increased apoB-48 and apoC-III in the plasma samples from diabetic Ins2^+/Akita:apoE^−/− mice. A: immunoblot analysis of the plasma apolipoproteins such as apoB-100, apoB-48, apoA-I, and apoC-III from 15-h-fasted 20-wk-old diabetic Ins2^+/Akita:apoE ^−/− mice and age-matched non-DM control Ins2^+/+:apoE ^−/− mice (n = 6/group). B: the relative levels of plasma apolipoproteins normalized to the mean of those in non-DM Ins2^+/+:apoE ^−/− control. The data shown in the bar graph are means ± SE. *P < 0.05 compared with non-DM mice.