Induction of fatal inflammation in LDL receptor and ApoA-I double-knockout mice fed dietary fat and cholesterol

Abstract

Atherogenic response to dietary fat and cholesterol challenge was evaluated in mice lacking both the LDL receptor (LDLr(-/-)) and apoA-I (apoA-I(-/-)) gene, LDLr(-/-)/apoA-I(-/-) or double-knockout mice. Gender- and age-matched LDLr(-/-)/apoA-I(-/-) mice were fed a diet consisting of 0.1% cholesterol and 10% palm oil for 16 weeks and compared to LDLr(-/-) mice or single-knockout mice. The LDLr(-/-) mice showed a 6- to 7-fold increase in total plasma cholesterol (TPC) compared to their chow-fed mice counterparts, while LDLr(-/-)/apoA-I(-/-) mice showed only a 2- to 3-fold increase in TPC compared to their chow-fed controls. This differential response to the atherogenic diet was unanticipated, since chow-fed LDLr(-/-) and LDLr(-/-)/apoA-I(-/-) mice began the study with similar LDL levels and differed primarily in their HDL concentration. The 6-fold diet-induced increase in TPC observed in the LDLr(-/-) mice occurred mainly in VLDL/LDL and not in HDL. Mid-study plasma samples taken after 8 weeks of diet feeding showed that LDLr(-/-) mice had TPC concentrations approximately 60% of their 16-week level, while the LDLr(-/-)/apoA-I(-/-) mice had reached 100% of their 16-week TPC concentration after only 8 weeks of diet. Male LDLr(-/-) mice showed similar aortic cholesterol levels to male LDLr(-/-)/apoA-I(-/-) mice despite a 4-fold higher VLDL/LDL concentration in the LDLr(-/-) mice. A direct comparison of the severity of aortic atherosclerosis between female LDLr(-/-) and LDLr(-/-)/apoA-I(-/-) mice was compromised due to the loss of female LDLr(-/-)/apoA-I(-/-) mice between 10 and 14 weeks into the study. Diet-fed female and, with time, male LDLr(-/-)/apoA-I(-/-) mice suffered from severe ulcerated cutaneous xanthomatosis. This condition, combined with a complete depletion of adrenal cholesterol, manifested in fatal wasting of the affected mice. In conclusion, LDLr(-/-) and LDLr(-/-)/apoA-I(-/-) mice showed dramatic TPC differences in response to dietary fat and cholesterol challenge, while despite these differences both genotypes accumulated similar levels of aortic cholesterol.

Figure 1.

A: HDL cholesterol concentration in chow-fed LDLr^−/− mice (striped bar) and LDLr^−/−/apoA-I^−/− mice (solid bar). B: Sixteen-week diet-fed LDLr^−/− mice (striped bar) and LDLr^−/−/apoA-I^−/− mice (solid bar). HDL cholesterol was determined as described in Experimental Procedures. All values represent the mean ± SEM. Values with unlike superscripts indicate significant differences at P < 0.01.

Figure 2.

FPLC separation of plasma lipoprotein showing the distribution of total cholesterol (A and B) and triglyceride (C and D) in LDLr^−/− (open square) and LDLr^−/−/apoA-I^−/− mice (filled circle) fed chow (A and C) or a diet containing 0.1% cholesterol and 10% palm oil for 16 weeks (B and D). Approximately 200 μl of chow-fed mouse plasma or 100 μl of diet-fed mouse plasma was applied to two Superose-6 columns connected in tandem and run at 0.5 ml/min, as described in Experimental Procedures. Total cholesterol and triglyceride concentrations were measured in each individual fraction by enzymatic assay. Western blot analysis was performed on each FPLC fraction using 10 μl from chow-fed plasma fractions and 20 μl from diet-fed plasma fractions as described in Experimental Procedures.

Figure 3.

Coomassie-blue-stained 4% to 30% SDS-PAGE of the d < 1.25 g/ml fraction from mouse plasma. Lanes 1 and 2 show the d <1.25 g/ml tops from two different chow-fed LDLr^−/−/apoA-I^−/−mice; lanes 3 and 4 show the d < 1.25 g/ml tops from two different chow-fed LDLr^−/− mice; lanes 5 and 6 show the d < 1.25 g/ml tops from two different chow-fed LDLr^−/− mice; lanes 7 and 8 show the d < 1.25 g/ml tops from two different diet-fed LDLr^−/−/apoA-I^−/− mice. The d < 1.25 g/ml plasma fraction was isolated by density ultracentrifugation of 100 to 200 μl of mouse plasma as described in Experimental Procedures. Each lane is the equivalent of 10 μg of total d < 1.25 g/ml lipoprotein. The gel was stained/destained then photographed using an α Innotech Imager.

Figure 4.

Cutaneous xanthomatosis and severe pruritus in diet-fed LDLr^−/−/apoA-I^−/− and LDLr^−/− mice. A: External appearance of a 14-week diet-fed male LDLr^−/−/apoA-I^−/− mouse, compared to a 14-week diet-fed female LDLr^−/−/apoA-I^−/− mouse (B). Female mice began showing severe ulcerated lesions on the abdomen, neck, and front limbs after approximately 9 weeks of consuming the atherogenic diet (containing 0.1% cholesterol, 10% palm oil). C: The skin lesions affecting the double-knockout mice were distinct from the hair loss and scaly thickened skin occasionally seen in diet-fed LDLr^−/− mice (C). D: Frequency of ulcerated lesions in male and female LDLr^−/−/apoA-I^−/− mice after 14 weeks.

Figure 5.

Histopathology of dermal xanthomatosis in LDLr^−/−/apoA-I^−/− mice. A: H&E-stained section of skin from a 16-week diet-fed LDLr^−/− female mouse. B: Skin section from a 16-week diet-fed LDLr^−/−/apoA-I^−/− male mouse with a thickened dermal layer infiltrated with fat-laden macrophages and cholesterol clefts. C: Skin section from a diet-fed female LDLr^−/−/apoA-I^−/− mouse showing dramatic thickening of the dermis, associated with massive macrophage infiltration and cholesterol deposits apparently disrupting the subcutaneous adipose tissue layer.

Figure 6.

Adrenal sections showing zona fasiculata cells from chow- and diet-fed LDLr^−/− and LDLr^−/−/apoA-I^−/− mice. A and B: Sections from chow-fed male and female LDLr^−/− mice, respectively. C and D: Sixteen-week diet-fed male and female LDLr^−/− mice, respectively. E and F: Sections from chow-fed male and female LDLr^−/−/apoA-I^−/− mice, respectively. G and H: Sections from 16-week diet-fed male and female LDLr^−/−/apoA-I^−/− mice, respectively. Magnification, ×63.

Figure 7.

A: H&E-stained liver sections from chow-fed male LDLr^−/−/apoA-I^−/− mouse. B: A diet-fed male LDLr^−/− mouse. C: A diet-fed male LDLr^−/−/apoA-I^−/− mouse. D: A diet-fed female LDLr^−/−/apoA-I^−/− mouse. Magnification, ×40.