Suppression of brain cholesterol synthesis in male Mecp2-deficient mice is age dependent and not accompanied by a concurrent change in the rate of fatty acid synthesis.

Mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2) are the principal cause of Rett syndrome, a progressive neurodevelopmental disorder afflicting 1 in 10,000 to 15,000 females. Studies using hemizygous Mecp2 mouse models have revealed disruptions to some aspects of their lipid metabolism including a partial suppression of cholesterol synthesis in the brains of mature Mecp2 mutants. The present studies investigated whether this suppression is evident from early neonatal life, or becomes manifest at a later stage of development. We measured the rate of cholesterol synthesis, in vivo, in the brains of male Mecp2-/y and their Mecp2+/y littermates at 7, 14, 21, 28, 42 and 56 days of age. Brain weight was consistently lower in the Mecp2-/y mice than in their Mecp2+/y controls except at 7 days of age. In the 7- and 14-day-old mice there was no genotypic difference in the rate of brain cholesterol synthesis but, from 21 days and later, it was always marginally lower in the Mecp2-/y mice than in age-matched Mecp2+/y littermates. At no age was a genotypic difference detected in either the rate of fatty acid synthesis or cholesterol concentration in the brain. Cholesterol synthesis rates in the liver and lungs of 56-day-old Mecp2-/y mice were normal. The onset of lower rates of brain cholesterol synthesis at about the time closure of the blood brain barrier purportedly occurs might signify a disruption to mechanism(s) that dictate intracellular levels of cholesterol metabolites including oxysterols known to exert a regulatory influence on the cholesterol biosynthetic pathway.

PRD125, a potent and selective inhibitor of sterol O-acyltransferase 2 markedly reduces hepatic cholesteryl ester accumulation and improves liver function in lysosomal acid lipase-deficient mice.

In most organs, the bulk of cholesterol is unesterified, although nearly all possess a varying capability of esterifying cholesterol through the action of either sterol O-acyltransferase (SOAT) 1 or, in the case of hepatocytes and enterocytes, SOAT2. Esterified cholesterol (EC) carried in plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to whether pharmacological inhibition of SOAT2 might reduce tissue EC accretion in CESD. When weaned at 21 days, Lal(-/-) mice, of either gender, had a whole liver cholesterol content that was 12- to 13-fold more than that of matching Lal(+/+) littermates (23 versus 1.8 mg, respectively). In Lal(-/-) males given the selective SOAT2 inhibitor PRD125 1,11-O-o-methylbenzylidene-7-O-p-cyanobenzoyl-1,7,11-trideacetylpyripyropene A in their diet (∼10 mg/day per kg body weight) from 21 to 53 days, whole liver cholesterol content was 48.6 versus 153.7 mg in untreated 53-day-old Lal(-/-) mice. This difference reflected a 59% reduction in hepatic EC concentration (mg/g), combined with a 28% fall in liver mass. The treated mice also showed a 63% reduction in plasma alanine aminotransferase activity, in parallel with decisive falls in hepatic mRNA expression levels for multiple proteins that reflect macrophage presence and inflammation. These data implicate SOAT2 as a potential target in CESD management.

Impact of physiological levels of chenodeoxycholic acid supplementation on intestinal and hepatic bile acid and cholesterol metabolism in Cyp7a1-deficient mice.

Mice deficient in cholesterol 7α-hydroxylase (Cyp7a1) have a diminished bile acid pool (BAP) and therefore represent a useful model for investigating the metabolic effects of restoring the pool with a specific BA. Previously we carried out such studies in Cyp7a1(-/-) mice fed physiological levels of cholic acid (CA) and achieved BAP restoration, along with an increased CA enrichment, at a dietary level of just 0.03% (w/w). Here we demonstrate that in Cyp7a1(-/-) mice fed chenodeoxycholic acid (CDCA) at a level of 0.06% (w/w), the BAP was restored to normal size and became substantially enriched with muricholic acid (MCA) (>70%), leaving the combined contribution of CA and CDCA to be <15%. This resulted in a partial to complete reversal of the main changes in cholesterol and BA metabolism associated with Cyp7a1 deficiency such as an elevated rate of intestinal sterol synthesis, an enhanced level of mRNA for Cyp8b1 in the liver, and depressed mRNA levels for Ibabp, Shp and Fgf15 in the distal small intestine. When Cyp7a1(-/-) and matching Cyp7a1(+/+) mice were fed a diet with added cholesterol (0.2%) (w/w), either alone, or also containing CDCA (0.06%) (w/w) or CA (0.03%) (w/w) for 18days, the hepatic total cholesterol concentrations (mg/g) in the Cyp7a1(-/-) mice were 26.9±3.7, 16.4±0.9 and 47.6±1.9, respectively, vs. 4.9±0.4, 5.0±0.7 and 6.4±1.9, respectively in the corresponding Cyp7a1(+/+) controls. These data affirm the importance of using moderate levels of dietary BA supplementation to elicit changes in hepatic cholesterol metabolism through shifts in BAP size and composition.

Deletion of sterol O-acyltransferase 2 (SOAT2) function in mice deficient in lysosomal acid lipase (LAL) dramatically reduces esterified cholesterol sequestration in the small intestine and liver.

Sterol O-acyltransferase 2 (SOAT2), also known as ACAT2, is the major cholesterol esterifying enzyme in the liver and small intestine (SI). Esterified cholesterol (EC) carried in certain classes of plasma lipoproteins is hydrolyzed by lysosomal acid lipase (LAL) when they are cleared from the circulation. Loss-of-function mutations in LIPA, the gene that encodes LAL, result in Wolman disease (WD) or cholesteryl ester storage disease (CESD). Hepatomegaly and a massive increase in tissue EC levels are hallmark features of both disorders. While these conditions can be corrected with enzyme replacement therapy, the question arose as to what effect the loss of SOAT2 function might have on tissue EC sequestration in LAL-deficient mice. When weaned at 21 days, Lal(-)(/)(-):Soat2(+)(/)(+) mice had a whole liver cholesterol content (mg/organ) of 24.7 mg vs 1.9mg in Lal(+/+):Soat2(+/+) littermates, with almost all the excess sterol being esterified. Over the next 31 days, liver cholesterol content in the Lal(-)(/)(-):Soat2(+)(/)(+) mice increased to 145 ± 2 mg but to only 29 ± 2 mg in their Lal(-)(/)(-):Soat2(-)(/)(-) littermates. The level of EC accumulation in the SI of the Lal(-)(/)(-):Soat2(-)(/)(-) mice was also much less than in their Lal(-)(/)(-):Soat2(+)(/)(+) littermates. In addition, there was a >70% reduction in plasma transaminase activities in the Lal(-)(/)(-):Soat2(-)(/)(-) mice. These studies illustrate how the severity of disease in a mouse model for CESD can be substantially ameliorated by elimination of SOAT2 function.

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

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.

Systemic administration of 2-hydroxypropyl-ß-cyclodextrin to symptomatic Npc1-deficient mice slows cholesterol sequestration in the major organs and improves liver function.

In Niemann-Pick type C (NPC) disease, loss-of-function mutations in either NPC1 or NPC2 result in progressive accumulation of unesterified cholesterol (UC) and glycosphingolipids in all organs, leading to neurodegeneration, pulmonary dysfunction and sometimes liver failure. There is no cure for this disorder. Studies using primarily NPC mouse models have shown that systemic administration of 2-hydroxypropyl-ß-cyclodextrin (2HPßCD), starting in early neonatal life, diminishes UC accumulation in most organs, slows disease progression and extends lifespan. The key question now is whether delaying the start of 2HPßCD treatment until early adulthood, when the amount of entrapped UC throughout the body is markedly elevated, has any of the benefits found when treatment begins at 7 days of age. In the present study, Npc1(-/-) and Npc1(+/+) mice were given saline or 2HPßCD subcutaneously at 49, 56, 63 and 70 days of age, with measurements of organ weights, liver function tests and tissue cholesterol levels performed at 77 days. In Npc1(-/-) mice, treatment with 2HPßCD from 49 days reduced whole-liver cholesterol content at 77 days from 33.0 ± 1.0 to 9.1 ± 0.5 mg/organ. Comparable improvements were seen in other organs, such as the spleen, and in the animal as a whole. There was a transient increase in biliary cholesterol concentration in Npc1(-/-) mice after 2HPßCD. Plasma alanine aminotransferase and aspartate aminotransferase activities in 77-day-old 2HPßCD-treated Npc1(-/-) mice were reduced compared with saline-treated controls. The lifespan of Npc1(-/-) mice given 2HPßCD marginally exceeded that of the saline-treated controls (99 ± 1.1 vs 94 ± 1.4 days, respectively; P < 0.05). Thus, 2HPßCD is effective in mobilizing entrapped cholesterol in late-stage NPC disease leading to improved liver function.

Impact of the loss of caveolin-1 on lung mass and cholesterol metabolism in mice with and without the lysosomal cholesterol transporter, Niemann-Pick type C1.

Caveolin-1 (Cav-1) is a major structural protein in caveolae in the plasma membranes of many cell types, particularly endothelial cells and adipocytes. Loss of Cav-1 function has been implicated in multiple diseases affecting the cardiopulmonary and central nervous systems, as well as in specific aspects of sterol and lipid metabolism in the liver and intestine. Lungs contain an exceptionally high level of Cav-1. Parameters of cholesterol metabolism in the lung were measured, initially in Cav-1-deficient mice (Cav-1(-/-)), and subsequently in Cav-1(-/-) mice that also lacked the lysosomal cholesterol transporter Niemann-Pick C1 (Npc1) (Cav-1(-/-):Npc1(-/-)). In 50-day-old Cav-1(-/-) mice fed a low- or high-cholesterol chow diet, the total cholesterol concentration (mg/g) in the lungs was marginally lower than in the Cav-1(+/+) controls, but due to an expansion in their lung mass exceeding 30%, whole-lung cholesterol content (mg/organ) was moderately elevated. Lung mass (g) in the Cav-1(-/-):Npc1(-/-) mice (0.356±0.022) markedly exceeded that in their Cav-1(+/+):Npc1(+/+) controls (0.137±0.009), as well as in their Cav-1(-/-):Npc1(+/+) (0.191±0.013) and Cav-1(+/+):Npc1(-/-) (0.213±0.022) littermates. The corresponding lung total cholesterol contents (mg/organ) in mice of these genotypes were 6.74±0.17, 0.71±0.05, 0.96±0.05 and 3.12±0.43, respectively, with the extra cholesterol in the Cav-1(-/-):Npc1(-/-) and Cav-1(+/+):Npc1(-/-) mice being nearly all unesterified (UC). The exacerbation of the Npc1 lung phenotype and increase in the UC level in the Cav-1(-/-):Npc1(-/-) mice imply a regulatory role of Cav-1 in pulmonary cholesterol metabolism when lysosomal sterol transport is disrupted.

Sustained and selective suppression of intestinal cholesterol synthesis by Ro 48-8071, an inhibitor of 2,3-oxidosqualene:lanosterol cyclase, in the BALB/c mouse.

The small intestine plays a fundamentally important role in regulating whole body cholesterol balance and plasma lipoprotein composition. This is articulated through the interplay of a constellation of genes that ultimately determines the net amount of chylomicron cholesterol delivered to the liver. Major advances in our insights into regulation of the cholesterol absorption pathway have been made using genetically manipulated mouse models and agents such as ezetimibe. One unresolved question is how a sustained pharmacological inhibition of intestinal cholesterol synthesis in vivo may affect cholesterol handling by the absorptive cells. Here we show that the lanosterol cyclase inhibitor, Ro 48-8071, when fed to BALB/c mice in a chow diet (20 mg/day/kg body weight), leads to a rapid and sustained inhibition (>50%) of cholesterol synthesis in the whole small intestine. Sterol synthesis was also reduced in the large intestine and stomach. In contrast, hepatic cholesterol synthesis, while markedly suppressed initially, rebounded to higher than baseline rates within 7 days. Whole body cholesterol synthesis, fractional cholesterol absorption, and fecal neutral and acidic sterol excretion were not consistently changed with Ro 48-8071 treatment. There were no discernible effects of this agent on intestinal histology as determined by H&E staining and the level of Ki67, an index of proliferation. The mRNA expression for multiple genes involved in intestinal cholesterol regulation including NPC1L1 was mostly unchanged although there was a marked rise in the mRNA level for the PXR target genes CYP3A11 and CES2A.

Ezetimibe markedly attenuates hepatic cholesterol accumulation and improves liver function in the lysosomal acid lipase-deficient mouse, a model for cholesteryl ester storage disease.

Lysosomal acid lipase (LAL) plays a critical role in the intracellular handling of lipids by hydrolyzing cholesteryl esters (CE) and triacylglycerols (TAG) contained in newly internalized lipoproteins. In humans, mutations in the LAL gene result in cholesteryl ester storage disease (CESD), or in Wolman disease (WD) when the mutations cause complete loss of LAL activity. A rat model for WD and a mouse model for CESD have been described. In these studies we used LAL-deficient mice to investigate how modulating the amount of intestinally-derived cholesterol reaching the liver might impact its mass, cholesterol content, and function in this model. The main experiment tested if ezetimibe, a potent cholesterol absorption inhibitor, had any effect on CE accumulation in mice lacking LAL. In male Lal(-/-) mice given ezetimibe in their diet (20 mg/day/kg bw) for 4 weeks starting at 21 days of age, both liver mass and hepatic cholesterol concentration (mg/g) were reduced to the extent that whole-liver cholesterol content (mg/organ) in the treated mice (74.3±3.4) was only 56% of that in those not given ezetimibe (133.5±6.7). There was also a marked improvement in plasma alanine aminotransferase (ALT) activity. Thus, minimizing cholesterol absorption has a favorable impact on the liver in CESD.

Ontogenic changes in lung cholesterol metabolism, lipid content, and histology in mice with Niemann-Pick type C disease.

Niemann-Pick Type C (NPC) disease is caused by a deficiency of either NPC1 or NPC2. Loss of function of either protein results in the progressive accumulation of unesterified cholesterol in every tissue leading to cell death and organ damage. Most literature on NPC disease focuses on neurological and liver manifestations. Pulmonary dysfunction is less well described. The present studies investigated how Npc1 deficiency impacts the absolute weight, lipid composition and histology of the lungs of Npc1(-/-) mice (Npc1(nih)) at different stages of the disease, and also quantitated changes in the rates of cholesterol and fatty acid synthesis in the lung over this same time span (8 to 70days of age). Similar measurements were made in Npc2(-/-) mice at 70days. All mice were of the BALB/c strain and were fed a basal rodent chow diet. Well before weaning, the lung weight, cholesterol and phospholipid (PL) content, and cholesterol synthesis rate were all elevated in the Npc1(-/-) mice and remained so at 70days of age. In contrast, lung triacylglycerol content was reduced while there was no change in lung fatty acid synthesis. Despite the elevated PL content, the composition of PL in the lungs of the Npc1(-/-) mice was unchanged. H&E staining revealed an age-related increase in the presence of lipid-laden macrophages in the alveoli of the lungs of the Npc1(-/-) mice starting as early as 28days. Similar metabolic and histologic changes were evident in the lungs of the Npc2(-/-) mice. Together these findings demonstrate an intrinsic lung pathology in NPC disease that is of early onset and worsens over time.