4. RESULTS AND DISCUSSION
4.4 Cholesterol absorption
4.4.2 Comparison of protein expression in the BBM of wild type mice and ApoE
One of the most widely used mouse models to study dislipidemia is the apolipoprotein E–
deficient mice (ApoE-/- mice), in which targeted deletion of the apoE gene leads to severe hypercholesterolemia and spontaneous atherosclerosis. ApoE is synthesized in the liver and in macrophages and has a number of important anti-atherogenic functions. As a constituent of plasma lipoproteins, it serves as a ligand for the cell-surface lipoprotein receptors such as LDL-receptor (LDLr) and LDLr-related proteins (LRPs), thereby promoting the uptake of atherogenic particles from the circulation. Consequently, homozygous deletion of the apoE gene in mice results in a pronounced increase in the plasma levels of LDL and VLDL attributable to the failure of LDLr- and LRP mediated clearance of these lipoproteins (114).
The finding that ApoE strongly interacts with the BBM membrane (for details see § 4.4.1), added to the fact that many proteins involved in cholesterol absorption could also be identified using our protocol, encouraged us to investigate in more details the protein expression differences that could be observed in the BBM membrane of wild type mice versus ApoE knockout mice using the same proteomics approach as described above. Possible
differences in protein abundance could contribute to shed a better understanding in the mechanisms of high cholesterol absorption observed for the ApoE knockout mice.
The isolated BBM fractions of a pool of four male ApoE-deficient mice (B6.129P2-Apoetm1Unc/Crl) and four non-transgenic male mice of the same genetic background and age, were loaded in triplicate onto a 1D NuPAGE gel (see figure 4.16).
Figure 4.16: 1D SDS-PAGE gel representation of the BBM fractions of wild type and ApoE-KO mouse. Each fraction (30 μg total proteins per lane) was loaded in a 10 % 1D NuPAGE Bis-Tris gel run in MES buffer.
The gel bands of the fractions were cut and in-gel digested with trypsin. The extracted peptides of each band were then analyzed by LC-MS/MS and the proteins of each band were identified according to the criteria that have been described at the § 3.2.7.2-3. Identical bands of the compared BBM fractions were analyzed sequentially with washing intervals, to achieve the best possible technical reproducibility.
It was reinsuring that, in large, the identified BBM proteins in this experiment were identical to the BBM proteins that were identified in the previous study (see § 4.4.1). Most interestingly, several differences in protein abundance (based in peptide counts and number of different peptides) were clearly observed between wild type mice and ApoE KO mice. Since the study did not follow any formal quantification workflow, and only technical replicates were performed, we concentrated our interest only in proteins with major difference in abundance between the two types of mice, summarized in Table 4.7.
WT_1 WT_2 WT_3 ApoE_1 ApoE_2 ApoE_3
MW MW
97 64 51
39
28 191
-kDa WT_1 WT_2 WT_3 ApoE_1 ApoE_2 ApoE_3
MW MW
97 64 51
39
28 191 -kDa
Table 4.7: Examples of protein abundance differences between ApoE KO and wild type mice. Proteins labeled in yellow have been described to participate in cholesterol absorption while those showing a major change in abundance are highlighted in red.
According to the data presented in Table 4.7, the Ileal Sodium/bile acid cotransporter (IBAT) and apolipoprotein A-I were confidently identified in the wild type mice but were completely absent in the ApoE knockout mice. This finding was confirmed in all the technical triplicates.
These findings were further validated using Western Blot analysis, and the result for IBAT is shown in Fig. 4.17. Unfortunately, the Western Blot analysis for Apo-AI was inconclusive (complete absence of signal).
Figure 4.17: Western blot analysis of IBAT. Lane 1, Homogenized intestinal mucosa tissue from WT mice with the same genetic background as the ApoE KO. Lane 2, BBM isolated from the total mouse intestinal mucosa of control mice C57B/6J. Lane 3, ApoE knockout mice and (lane 4) WT mice with the same genetic background as the ApoE-KO mice. 1st antibody 1:500 goat polyclonal anti-IBAT (Cat. No: sc-27493), 2nd antibody 1:7500 Horseradis peroxidase donkey anti goat IgG. MW, molecular weight marker.
Due to its function, IBAT’s down-regulation was expected to have an impact on the bile acid metabolism in the ApoE-knockout mice. For this purpose, a separate in-house study investigated the bile acid pool size and composition in apoE-deficient mice compared to that in wild type mice. Results of this study showed that the liver and intestinal bile acid pool sizes were significantly increased in the apoE-deficient mice compare to the wild type mice (Evelyne Chaput, unpublished data; see Fig. 4.18). Thus, the increased bile acid production in the liver is consistent with disrupted bile acid re-uptake from the intestinal lumen, as expected from reduced IBAT protein expression, and increase the secretion of bile acids in the pancreatic fluids. The increased amount of bile acids in the intestinal lumen might explain why cholesterol absorption is higher in apoE KO mice, since the cholesterol molecule is better solubilized and more easily absorbed. This scenario is well supported by our data, but additional studies need to confirm these findings.
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Figure 4.18: Graphic representation of the total bile acids in liver (panel A) and small intestine (panel B) in ApoE knockout mice and wild type mice. The total bile acids are a sum of all the individual bile acids determined by GC-MS (Adapted from Evelyne Chaput, unpublished data).
It is worth mentioning that conflicting data have been reported for the ApoE KO mice in the literature. For example, in disagreement with our observations, Hakansson et al. (115) reported that the bile acid pool size of apoE KO does not differ from that of wild-type mice.
In addition, the authors report an increased IBAT activity based on the intestinal absorption of tauro-23-[75Se] selena-25-homocholic acid. In a contradictory report, and in agreement with our data, Woollett et al. (116) claim that plasma cholesterol and cholesterol absorption is higher in the ApoE KO mice than in the wild type. Interestingly, all these studies were based on gene data and on pharmacokinetic studies while our findings are based on protein-level datasets, which might be closer to the biological realm of the small intestine thann the above mentioned studies. Another interesting aspect is that most studies focus on the cholesterol absorption and the function of the intestine as a consequence of the cholesterol metabolism in the liver. Here, our data suggest that at least a part of the regulation of the cholesterol absorption happens in the small intestine independently of the liver metabolism. As a matter of fact, it could well be that the critical control elements of cholesterol metabolism are trigged at the small intestine level and that the liver is responding to this trigger rather than to lead it.