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

Abstract

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.

Keywords: Cholesterol absorption; Enterocyte; Ezetimibe; Liver; Statins.

Fig. 1

Effect of varying the dose and time of simvastatin treatment on cholesterol synthesis in the small intestine and liver of mice. In the first study 9-week old male BALB/c mice were fed for 7 days a basal rodent diet containing simvastatin at levels that provided a dose of either 20 or 200 mg/day/kg bw. The control group was fed the basal diet alone. Rates of cholesterol synthesis in the small intestine (A) and liver (B) were measured in vivo using [³H] water as described in Materials and Methods. In a separate study matching male BALB/c mice were fed simvastatin at a fixed dose (200 mg/day/kg bw) for 0.5, 1, or 7 days and used for the measurement of intestinal (C) and hepatic (D) sterol synthesis. All values are the mean ± SEM of data from 5 to 7 animals in each group. Different letters (a, b, c) denote statistically significant (p< 0.05) differences between valuesas determined by one-way analysis of variance (ANOVA) with dose of simvastatin or time of feeding as variables.

Fig. 2

Effect of varying the dose of Ro 48-8071 on small intestine and liver weights, intestinal and hepatic cholesterol concentrations, and rates of intestinal and hepatic sterol and fatty acid synthesis in mice. Female BALB/c mice at 7 to 8 weeks of age were fed a rodent chow diet containing varying levels of Ro 48-8071 to provide doses of about 5 to 20 mg/day/kg bw over a 7-day feeding period. For the small intestines in these mice, organ weight (A), total cholesterol concentration (B), rate of cholesterol synthesis (C), and rate of fatty acid synthesis (D) were measured as described in Materials and Methods. The same parameters for liver were also measured in these mice (E-H). All values are the mean ± SEM of data from 6 to 8 animals at each dose. Different letters (a, b, c) denote statistically significant (p< 0.05) differences between values as determined by one-way ANOVA with Ro 48-8071 dose as the variable.

Fig. 3

Effect of varying period of treatment with Ro 48-8071 on rates of intestinal and hepatic sterol synthesis in mice. Male BALB/c mice at 7 to 9 weeks of age were fed a rodent chow diet containing Ro 48-8071 at one dose (15 mg/day/kg bw) for periods varying from 12h to 7 days. The rate of sterol synthesis in the small intestine (A) and liver (B) was measured in vivo specifically at 12 h, and 1, 2, 3 and 7 days after the start of treatment. All values are the mean ± SEM of data from 6 to 8 animals at each time point. Different letters (a, b, c) denote statistically significant (p< 0.05) differences between values as determined by one-way ANOVA with time on Ro 48-8071 treatment as the variable.

Fig. 4

Histology and immunochemistry of the midsection of the small intestine, and relative levels of expression of mRNA for genes serving as markers for proliferation and apoptosis in the whole small intestine in mice given Ro 48-8071. The results of two separate experiments are shown here. In the first, two matching groups of male BALB/c mice (n=3 each) at 10 to 16 weeks of age were given the basal diet alone or containing Ro 48-8071 (20 mg/day/kg bw) for 16 days. A 1 cm portion of the midsection of the small intestine was taken for histology (H&E staining)(A and B) and for immunohistochemistry (C and D) as described. The second experiment involved the measurement in the mucosae of the whole small intestine of the relative mRNA expression levels of a constellation of genes as fully detailed in Fig. 9. The expression level of mRNA for four of these genes are shown here in panel E. For each gene the level of expression in the mice fed the basal diet alone was arbitrarily set at 1.0. The values here represent the mean ± SEM of data from 6 or 7 mice in each group. None of the values for the mice given Ro 48-8071 are significantly different (p< 0.05) from their corresponding control as determined by an unpaired Student’s t-test.

Fig. 5

Rate of sterol synthesis in the liver, various regions of the gastrointestinal tract, and other extrahepatic organs in mice and hamsters given Ro 48-8071. Two separate experiments with mice were carried out. In the first, female BALB/c mice at 7 weeks of age were fed the basal diet alone or containing Ro 48-8071 (20 mg/day/kg bw) for 7 days. Rates of sterol synthesis were measured in 6 organs as described in the preceding experiments. These rates are expressed as the nmol of [³H] water incorporated into sterols/h/g of tissue. In a follow-up study, other female BALB/c mice at 10 weeks of age were fed the same diets as in the first study except for 10 days. These were used specifically for the measurement of whole animal sterol synthesis (data shown in inset). These rates are expressed as the umol of [³H] water incorporated into sterols/h/100 g bw. For the experiment with hamsters, males at approx 12 weeks of age were used and treatment with Ro 48-8071 was for 5 days. All values from both experiments are the mean ± SEM of data from 5 animals per group. The asterisk indicates the value is significantly different (p< 0.05) (by unpaired Student’s t-test) than that for the matching group fed the basal diet alone.nm – not measured.

Fig. 6

Intestinal and hepatic cholesterol concentrations and rates of sterol synthesis in LDLR-deficient mice given Ro 48-8071. Female ldlr^−/− mice and matching ldlr^+/+ controls (129/Sv background) at 21 to 25 weeks of age were fed a rodent chow diet alone or containing Ro 48-8071 (20mg/day/kg bw) for 10 days. The total cholesterol concentration and rate of sterol synthesis in both the small intestine (A and B) and liver (C and D) were measured as described. All values are the mean ± SEM of data from 6 animals in each group. Different letters (a, b, c) denote statistically significant differences between (p< 0.05) values as determined by two-way ANOVA with genotype and Ro 48-8071 dose as variables.

Fig. 7

Hepatic and plasma cholesterol concentrations and relative level of expression in liver of mRNA for two genes involved in bile acid synthesis in mice fed a high cholesterol diet along with Ro 48-8071. Female BALB/c mice at 10 to 16 weeks of age were fed a rodent chow diet alone or containing added cholesterol, with or without Ro 48-8071 (20mg/day/kg bw) for 18 days. The basal chow diet (inherent cholesterol content 0.02% w/w) was supplemented with powdered cholesterol to a final level of 1.0 % w/w. Hepatic and plasma total cholesterol concentrations and mRNA levels were measured as described. The mRNA levels are expressed relative to those obtained for mice given the basal diet alone which, in each case, were arbitrarily set at 1.0. Values are the mean ± SEM of data from 4 animals per group (low cholesterol) or 5 animals per group (high cholesterol). Different letters (a, b) denote statistically significant (p<0.05) differences between values as determined by two-way ANOVA with dietary cholesterol level and Ro 48-8071 dose as variables.

Fig. 8

Parameters of intestinal cholesterol metabolism and rates of neutral sterol and bile acid excretion in mice given either ezetimibe, a cholesterol absorption inhibitor, or Ro 48-8071. The data presented here were derived from four separate groups of experiments, all carried out using female BALB/c mice at 7 to 10 weeks of age. In one study cholesterol absorption (A) and fecal neutral sterol excretion (B) were measured. Separate groups were used for measuring fecal bile acid excretion (C). The third lot of mice was used for determining relative intestine weight (D) and the unesterified cholesterol (UC) concentration in the whole small intestine (E). Another set of mice was used for measuring the rate of intestinal sterol synthesis in vivo (F). In all experiments the dose of both ezetimibe and Ro 48-8071 was 20 mg/day/kg bw. Values are the mean ± SEM of data from 7 to 10 animals per group (A, B, C, F) or 5 animals per group (D, E). Different letters (a, b, c) denote statistically significant (p< 0.05) differences between values using a one-way ANOVA with type of dietary agent as the variable.

Fig. 9

Relative levels of expression of mRNA for multiple genes in the small intestine of mice given ezetimibe or Ro 48-8071. The mucosa from the entire small intestine was used. Most, but not all, of the mice used for these analyses were the same as those used for the measurements described in Fig. 8A and Fig. 8B. The mRNA levels were measured as described in Materials and Methods and are expressed relative to those obtained for mice given the basal diet alone, which in each case were arbitrarily set at 1.0. Values are the mean ± SEM of data from 6 or 7 mice per group. Different letters (a, b, c) denote statistically significant (p< 0.05) differences between values using a one-way ANOVA with type of dietary agent as the variable.