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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 3, 2002 as doi:10.1096/fj.02-0341fje. |
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Metabolism Unit, Center for Metabolism and Endocrinology, Department of Medicine, and Molecular Nutrition Unit, Center for Nutrition and Toxicology, NOVUM, Karolinska Institute at Huddinge University Hospital, Stockholm, Sweden; and
* Molecular Biology, Cell Biology and Biochemistry, Astra Zeneca R&D, Mölndal, Sweden
2Correspondence: CME, M63, Karolinska Institute at Huddinge University Hospital, S-141 86 Stockholm, Sweden. E-mail: mats.rudling{at}cnt.ki.se
SPECIFIC AIMS
Induced fecal loss of bile acids (BA) reduces plasma cholesterol, presumably through induction of hepatic low density lipoprotein receptors (LDLR). To evaluate the potential of non-LDLR-mediated lipid lowering by pharmacological stimulation of BA synthesis, we studied the effects of PR835, a drug inhibiting the intestinal BA transporter (Slc10a2), in mice devoid of both the LDLR and its ligand, apo E.
PRINCIPAL FINDINGS
Selective active ileal BA uptake maintains an efficient enterohepatic circulation of BA, and the apical sodium codependent transporter (Slc10a2; also known as ASBT or ileal BA transporter, IBAT) is of critical importance for this process. Treatment with BA binding resins such as cholestyramine or surgical procedures such as ileal resection or biliary fistulation have been used to derepress CYP7A1 activity, increase hepatic LDLR, and thereby stimulate LDL clearance and lower plasma LDL. However, direct stimulation of enzyme activity through Cyp7a1 overexpression or growth hormone treatment has been demonstrated to lower plasma cholesterol in mice lacking the LDLR. To evaluate the potential for non-LDLR-mediated lipid lowering by pharmacological stimulation of BA synthesis, we studied LDLR/apoE-deficient mice. To specifically inhibit SLC10A2, we used the drug PR835, a bensothiazepine derivative shown to inhibit taurocholate uptake in cells transfected with human SLC10A2.
1. PR835 reduces plasma lipid levels
There was a dose-dependent reduction of plasma cholesterol up to 40%, whereas triglycerides (TG) were unaltered (Fig. 1
a). Separation of plasma lipoproteins by FPLC (Fig. 1b, c
) showed that the reduction of cholesterol was within chylomicron/VLDL (
60%) and IDL/LDL (25%) fractions whereas HDL cholesterol was unaltered.
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2. PR835 increases Cyp7a1 and HMGR
Enzymatic activity and mRNA levels of Cyp7a1 were increased by PR835 treatment (Fig. 1d
). Analysis of the concentration of 7
-hydroxy-4-cholesten-3-one (C4), a plasma marker that reflects BA synthesis in the liver, confirmed this observation (Fig. 1e
). HMGR mRNA was increased (Fig. 1e
), whereas the concentration of free cholesterol in hepatic microsomes was not altered.
3. PR835 does not stimulate hepatic lipoprotein receptors
To screen for other hepatic structures that could explain this unexpected finding, we performed ligand blot analysis using rabbit 125I-ß-VLDL, which contains apoB-48, apoB-100, and apoE. No binding of these lipoproteins could be demonstrated in liver membranes from the treated animals, however. We also evaluated the expression of other members of the LDLR gene family such as the LDLR related protein (LRP), megalin, and the VLDL receptor. LRP and megalin were equally abundant in all animals whereas VLDL receptors were not detectable. Scavenger receptor B class I (SR-BI) was actually reduced by treatment (Fig. 1f
).
4. Combination therapy with atorvastatin potentiates the effect (of PR835)
Combined treatment resulted in a 64% reduction of plasma cholesterol (Fig. 2
a). FPLC analysis (Fig. 2b, c
) showed that atorvastatin reduced cholesterol only in LDL particles, whereas PR835 reduced both LDL and VLDL cholesterol. The combination of the two drugs resulted in a profound reduction of cholesterol, with LDL particles reduced by 70%.
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5. Combination therapy does not influence hepatic lipoprotein receptors
Combined treatment strongly increased Cyp7a1 transcript levels and enzyme activities by threefold (Fig. 2d
). The effects of treatment on bile acid production were somewhat less pronounced (Fig. 2e
). HMGR mRNA increased by 13-fold (Fig. 2e
); hepatic microsomal free cholesterol content was slightly reduced (15%) in animals treated with the combination.
Ligand blotting of hepatic membranes did not reveal any binding of ß-VLDL, and we were not able to detect any differences in abundance of RAP binding lipoprotein receptors. The expression of SR-BI was confirmed to be reduced after PR835 (Fig. 2f
).
6. Inhibition of Slc10a2 by PR835 does not alter its gene expression
PR835 alone did not alter the transcript levels of Slc10a2, whereas increased levels were seen after atorvastatin. The cytosolic ileal BA binding protein (I-BABP) was reduced after treatment with PR835 (Fig. 2g
).
CONCLUSIONS AND SIGNIFICANCE
The concept of lowering plasma lipoprotein cholesterol by interruption of the enterohepatic circulation of BA is well established. Accordingly, previous studies using Slc10a2 inhibitors have shown therapeutic effects in mice and rats with diet-induced hyperlipidemia already within 3 days of treatment. As for ileal resection and BA binding resins, this plasma lipid lowering effect has been linked to an induction of hepatic LDLR. Blocking the compensatory increase in cholesterol synthesis by statins results in a further increase in LDLR, which may explain the additional cholesterol lowering in this situation. The current finding of a drastic reduction of plasma VLDL and LDL cholesterol in response to inhibition of Slc10a2 in animals lacking both the LDLR and apoE was therefore quite unexpected (Fig. 3
). The effects could not be attributed to induction of other known hepatic lipoprotein receptors and indicate the presence of new points of targeting in lipid lowering therapy. The role of SLC10A2 inhibitors in the treatment of human hyperlipidemia will be important to explore further.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0341fje; to cite this article, use FASEB J. (December 3, 2002) 10.1096/fj.02-0341fje 
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P < 0.01; 
P < 0.001. Means and SE are shown.
