Hyperglycemic Ins2AkitaLdlr⁻/⁻ mice show severely elevated lipid levels and increased atherosclerosis: a model of type 1 diabetic macrovascular disease

Abstract

Accelerated atherosclerosis is the leading cause of death in type 1 diabetes, but the mechanism of type 1 diabetes-accelerated atherosclerosis is not well understood, in part due to the lack of a good animal model for the long-term studies required. In an attempt to create a model for studying diabetic macrovascular disease, we have generated type 1 diabetic Akita mice lacking the low density lipoprotein receptor (Ins2(Akita)Ldlr⁻/⁻). Ins2(Akita)Ldlr⁻/⁻ mice were severely hyperglycemic with impaired glucose tolerance. Compared with Ldlr⁻/⁻ mice, 20-week-old Ins2(Akita)Ldlr⁻/⁻ mice fed a 0.02% cholesterol AIN76a diet showed increased plasma triglyceride and cholesterol levels, and increased aortic root cross-sectional atherosclerotic lesion area [224% (P < 0.001) in males and 30% (P < 0.05) in females]. Microarray and quantitative PCR analyses of livers from Ins2(Akita)Ldlr⁻/⁻ mice revealed altered expression of lipid homeostatic genes, including sterol-regulatory element binding protein (Srebp)1, liver X receptor (Lxr)α, Abca1, Cyp7b1, Cyp27a1, and Lpl, along with increased expression of pro-inflammatory cytokine genes, including interleukin (Il)1α, Il1β, Il2, tumor necrosis factor (Tnf)α, and Mcp1. Immunofluorescence staining showed that the expression levels of Mcp1, Tnfα, and Il1β were also increased in the atherosclerotic lesions and artery walls of Ins2(Akita)Ldlr⁻/⁻ mice. Thus, the Ins2(Akita)Ldlr⁻/⁻ mouse appears to be a promising model for mechanistic studies of type 1 diabetes-accelerated atherosclerosis.

Fig.1.

Ins2^AkitaLdlr^−/− mice demonstrate insulin-deficient diabetic phenotypes. Ins2^AkitaLdlr^−/− mice were bred to Ldlr^−/− mice to generate Ins2^AkitaLdlr^−/− and Ldlr^−/− littermates. Four-week-old Ins2^AkitaLdlr^−/− and Ldlr^−/− mice were fed a semisynthetic low-cholesterol AIN76a diet (0.02% cholesterol) for 16 weeks. (A) Plasma insulin and (B) glucose levels were measured by standard methods. (C, D) Intraperitoneal glucose tolerance tests (IPGTT) were performed after a 6 h fast on male (C) and female (D) Ins2^AkitaLdlr^−/− and Ldlr^−/− mice (n = 8-14 per group, **P < 0.01 and ***P < 0.001).

Fig.2.

Akita mutation induces hyperlipidemia in Ldlr^−/− mice. Four-week-old male and female Ldlr^−/− and Ins2^AkitaLdlr^−/− mice were fed a semisynthetic 0.02% cholesterol-AIN76a diet for 16 weeks. The plasma levels of (A) triglyceride and total cholesterol and (B) lipoprotein fraction cholesterols (VLDL, LDL, and HDL) were measured in male and female Ldlr^−/− and Ins2^AkitaLdlr^−/− mice (n = 8-14 per group, *P < 0.05, **P < 0.01, and ***P < 0.001).

Fig.3.

Atherosclerosis is accelerated in diabetic Ins2^AkitaLdlr^−/− mice. Four-week-old male and female Ldlr^−/− and Ins2^AkitaLdlr^−/− mice were fed a semisynthetic 0.02% cholesterol AIN76a diet for 16 weeks. (A, C) Quantitative analysis of the lesion size in the aortic root of male and female Ldlr^−/− and Ins2^AkitaLdlr^−/− mice (n = 7-13 per group, **P < 0.001 and *P < 0.05). (B, D) Representative oil red O-stained sections of atherosclerotic lesion area in the aortic root of male and female Ldlr^−/− and Ins2^AkitaLdlr^−/− mice.

Fig.4.

Hepatic genes involved in lipid homeostasis are significantly altered in Ins2^AkitaLdlr^−/− mice. Total RNA was isolated from the livers of 20-week-old male Ldlr^−/− and Ins2^AkitaLdlr^−/− mice. The expression levels of hepatic genes involved in lipid homeostasis and other atherosclerosis-related genes were measured by quantitative PCR (n = 5 per group, *P < 0.05, **P < 0.01, and ***P < 0.001).

Fig.5.

Akita mutation increases mRNA levels of pro-inflammatory cytokine genes in the livers of Ins2^AkitaLdlr^−/− mice. Total RNA was isolated from the livers of 20-week-old male Ldlr^−/− and Ins2^AkitaLdlr^−/− mice. The expression levels of pro-inflammatory cytokine genes were measured by quantitative PCR (n = 5 per group, *P < 0.05 and ***P < 0.001).

Fig.6.

Akita mutation increases the pro-inflammatory cytokine levels in the atherosclerotic lesions and artery walls of Ins2^AkitaLdlr^−/− mice. Sections of atherosclerotic lesion areas in the aortic root of male Ldlr^−/− and Ins2^AkitaLdlr^−/− mice were stained with rabbit antibodies against mouse Mcp1, Tnfα, or Il1β followed by fluorescein-labeled goat anti-rabbit secondary antibody (red). The nuclei were stained with DAPI (blue).