Hepatic entrapment of esterified cholesterol drives continual expansion of whole body sterol pool in lysosomal acid lipase-deficient mice

Abstract

Cholesteryl ester storage disease (CESD) results from loss-of-function mutations in LIPA, the gene that encodes lysosomal acid lipase (LAL). Hepatomegaly and deposition of esterified cholesterol (EC) in multiple organs ensue. The present studies quantitated rates of synthesis, absorption, and disposition of cholesterol, and whole body cholesterol pool size in a mouse model of CESD. In 50-day-old lal(-/-) and matching lal(+/+) mice fed a low-cholesterol diet, whole animal cholesterol content equalled 210 and 50 mg, respectively, indicating that since birth the lal(-/-) mice sequestered cholesterol at an average rate of 3.2 mg·day(-1)·animal(-1). The proportion of the body sterol pool contained in the liver of the lal(-/-) mice was 64 vs. 6.3% in their lal(+/+) controls. EC concentrations in the liver, spleen, small intestine, and lungs of the lal(-/-) mice were elevated 100-, 35-, 15-, and 6-fold, respectively. In the lal(-/-) mice, whole liver cholesterol synthesis increased 10.2-fold, resulting in a 3.2-fold greater rate of whole animal sterol synthesis compared with their lal(+/+) controls. The rate of cholesterol synthesis in the lal(-/-) mice exceeded that in the lal(+/+) controls by 3.7 mg·day(-1)·animal(-1). Fractional cholesterol absorption and fecal bile acid excretion were unchanged in the lal(-/-) mice, but their rate of neutral sterol excretion was 59% higher than in their lal(+/+) controls. Thus, in this model, the continual expansion of the body sterol pool is driven by the synthesis of excess cholesterol, primarily in the liver. Despite the severity of their disease, the median life span of the lal(-/-) mice was 355 days.

Keywords: cholesterol synthesis; esterified cholesterol; hepatomegaly; lysosomal storage disease; sterol balance.

Fig. 1.

Age-related changes in the body and liver weights of lal^+/+ and lal^−/− mice and illustration of massive liver enlargement and measurement of life span in lal^−/− mice. Body weight data (A and D) were obtained from large numbers of male and female mice over the age span of 21 to 168 days. Liver weight data (B, C, E, and F) were derived from mice at different ages that were used in various metabolic studies. Hepatomegaly characteristic of lal^−/− mice shown in male (G) and female (H) specimens. Values are means ± SE of data from a minimum of 5 mice at every time point. For the life span study (I) there was a total of 39 lal^−/− mice (18 females, 21 males).

Fig. 2.

Liver histology, lipid and triacylglycerol content, and mRNA expression levels for markers of macrophage presence, cytokines, and cell surface proteins in 49- to 52-day-old lal^+/+ and lal^−/− mice. These various parameters were measured as described. Additionally, plasma ALT activities were determined in mice of both genotypes at 50, 70, and 140 days of age. For the data in B and E, the measurements were in female mice. The liver histology (A), and triacylglycerol content (C) and mRNA expression analyses (D) were done in males. H&E, hematoxylin and eosin; ALT, alanine aminotransferase. Measurement bars in A equal 100 μm. The data in B, C, D, and E are means ± SE of data from 4–6 mice of each genotype. *Significantly different from value for matching lal^+/+ controls (P < 0.05).

Fig. 3.

Concentrations of esterified cholesterol (EC) and unesterified cholesterol in multiple organs of lal^+/+ and lal^−/− mice. The tissue cholesterol concentration data in various organs (A) are for 50-day-old male mice, whereas those specifically for the small intestine (B) are for female mice in the age range of 21–274 days. Values are means ± SE of data from 6 mice of each genotype (A), and from a minimum of 5 animals in each group (B), except at 52 days when there were 4 mice of each genotype, and also at 274 days when there were only 3 lal^−/− mice. *Significantly different from value for matching lal^+/+ controls (P < 0.05).

Fig. 4.

Relative weights (A), total cholesterol contents (B), and rates of cholesterol synthesis (C) for the liver and multiple other organs in 50-day-old female lal^+/+ and lal^−/− mice. Rates of cholesterol synthesis were measured in vivo as described. These rates (nmol·h⁻¹·g⁻¹) were multiplied by the respective whole organ weight to obtain synthesis in the entire organ. Similarly, whole organ cholesterol contents were calculated from the total cholesterol concentration (mg/g) multiplied by organ weight. Values are means ± SE of data for 6 mice of each genotype. *Significantly different from value for matching lal^+/+ controls (P < 0.05).

Fig. 5.

Rate of cholesterol synthesis in the liver vs. all extrahepatic organs combined and contribution of the liver to whole body cholesterol synthesis and content in lal^+/+ and lal^−/− mice at different ages. Cholesterol synthesis was measured in vivo as described in female mice at 21, 50, 98, and 165 days of age. The rate of synthesis in the liver and whole residual carcass was expressed 2 ways. In one, the rate was normalized per gram wet weight of tissue (nmol·h⁻¹·g⁻¹) (A and B), whereas in the other this value was multiplied by the respective whole organ weight to obtain synthesis in the entire liver vs. the remainder of the animal. The summation of these 2 values yielded synthesis in the whole mouse (nmol·h⁻¹·animal⁻¹) (C). Similarly, whole organ cholesterol contents were calculated from the total cholesterol concentration (mg/g) multiplied by organ weight (D). Values are means ± SE of data from 4–6 mice of each genotype. At 384 days there were 4 mice (2 males and 2 females) of each genotype. *Significantly different from value for matching lal^+/+ controls (P < 0.05). nm, Not measured.

Fig. 6.

Fecal lipid content, intestinal cholesterol absorption, and rates of fecal neutral sterol and bile acid excretion in 50-day-old lal^+/+ and lal^−/− mice. Two matching sets of mice were used for these measurements, one for fractional cholesterol absorption (D) and fecal neutral sterol excretion (E), the other for quantitation of fecal lipid content (C) and the rate of fecal bile acid excretion (F). Values are means ± SE of data for 6 mice of each genotype. *Significantly different from value for matching lal^+/+ controls (P < 0.05). M, male; F, female.

Fig. 7.

Relative mRNA levels for multiple genes in the livers of 50-day-old male lal^+/+ and lal^−/− mice. The livers used for these analyses were derived from the same mice used for the measurement of unesterified cholesterol (UC) and EC levels in multiple organs (Fig. 3A). The mRNA levels were normalized against the housekeeping gene cyclophilin and arithmetically adjusted to yield a unit of 1.0 for lal^+/+ controls. The names of the genes studied are as follows: LIPA, lysosomal acid lipase (LAL); NPC2, Niemann-Pick type C2; NPC1, Niemann-Pick type C1; SREBP2, sterol regulatory element-binding protein-2; HMG CoA SYN, hydroxymethylglutaryl-CoA synthase; HMG CoA RED, hydroxymethylglutaryl-CoA reductase; ABCA1, ATP-binding cassette A1; ABCG5, ATP-binding cassette G5; ABCG8, ATP-binding cassette G8; ABCG1, ATP-binding cassette G1; SOAT1, sterol O-acyltransferase 1 (ACAT1); SOAT2, sterol O-acyltransferase 2 (ACAT2); SREBP1c, sterol regulatory element-binding protein-1c; ACCa, acetyl-CoA carboxylase; SCD1, steroyl-CoA desaturase 1. Values are means ± SE of data for 6 mice of each genotype. *Significantly different from value for matching lal^+/+ controls (P < 0.05).