Inhibition of hepatic bile salt uptake by Bulevirtide reduces atherosclerosis in Oatp1a1-/-Ldlr-/- mice

Abstract

Bile salts can strongly influence energy metabolism through systemic signaling, which can be enhanced by inhibiting the hepatic bile salt transporter Na⁺ taurocholate cotransporting polypeptide (NTCP), thereby delaying hepatic reuptake of bile salts to increase systemic bile salt levels. Bulevirtide is an NTCP inhibitor and was originally developed to prevent NTCP-mediated entry of Hepatitis B and D into hepatocytes. We previously demonstrated that NTCP inhibition lowers body weight, induces glucagon-like peptide-1 (GLP1) secretion, and lowers plasma cholesterol levels in murine obesity models. In humans, a genetic loss-of-function variant of NTCP has been associated with reduced plasma cholesterol levels. Here, we aimed to assess if Bulevirtide treatment attenuates atherosclerosis development by treating female Ldlr^-/- mice with Bulevirtide or vehicle for 11 weeks. Since this did not result in the expected increase in plasma bile salt levels, we generated Oatp1a1^-/-Ldlr^-/- mice, an atherosclerosis-prone model with human-like hepatic bile salt uptake characteristics. These mice showed delayed plasma clearance of bile salts and elevated bile salt levels upon Bulevirtide treatment. At the study endpoint, Bulevirtide-treated female Oatp1a1^-/-Ldlr^-/- mice had reduced atherosclerotic lesion area in the aortic root that coincided with lowered plasma LDL-c levels, independent of intestinal cholesterol absorption. In conclusion, Bulevirtide, which is considered safe and is EMA-approved for the treatment of Hepatitis D, reduces atherosclerotic lesion area by reducing plasma LDL-c levels. We anticipate that its application may extend to atherosclerotic cardiovascular diseases, which warrants clinical trials.

Keywords: LDL; bile acids and salts/metabolism; cholesterol; dyslipidemias; liver.

Fig. 1

Bulevirtide treatment delays clearance and increases levels of plasma bile salts in Oatp1a1^−/−Ldlr^−/− mice. Using CRISPR/CAS9 the mOatp1a1 locus was mutated in order to generate Oatp1a1^−/−Ldlr^−/− mice. A: sgRNA was targeted to exon 2 and 3 of the mOatp1a1 locus and together with Cas9 injected in the cytoplasm of a zygote derived from Ldlr^+/+ and Ldlr^−/− mice, after which the zygote was implanted into the oviducts of foster mice. Breeding was continued to obtain homozygous Oatp1a1^−/−Ldlr^−/− mice. B: Hepatic OATP1A1 abundance was measured in offspring to confirm knockout (KO) (third lane). The second lane served as a KO control. The gallbladder of male and female Oatp1a1^−/−Ldlr^−/− (blue circle) or Oatp1a1^+/+Ldlr^−/− mice (white circle) treated with a single dose of Bulevirtide was cannulated and [³H]sodium taurocholic acid (TC) was administered intravenously to measure [³H]-TC clearance from (C) plasma and (D) bile and to measure (E) bile flow, which was expressed as μL/minute/100 g body weight (n = 3–5/group). Data are presented as means ± SD. ∗Vehicle versus Bulevirtide. ∗ P < 0.05, according to two-way ANOVA and following Šídák’s multiple-comparisons test (C). Fig. 1A was created with BioRender.com.

Fig. 2

Bulevirtide treatment attenuates atherosclerosis development and lowers plasma cholesterol levels in Oatp1a1^−/−Ldlr^−/− mice. Female Oatp1a1^−/−Ldlr^−/− mice were injected subcutaneously with Na⁺ taurocholate co-transporting polypeptide (NTCP) inhibitor Bulevirtide (yellow circles or bars) or vehicle (black circles or bars) every day for 10 weeks. At study endpoint, (A) total plasma bile salt levels and (B) individual bile salt species were measured, including tauro-alpha-muricholic acid (TαMC), tauro-beta-muricholic acid (TβMC), tauroursodeoxycholic acid (TUDC), taurocholic acid (TC), taurochenodeoxycholic acid (TCDC), omega-muricholic acid (ΩMC), taurodeoxycholic acid (TDC), alpha-muricholic acid (αMC), beta-muricholic acid (βMC), cholic acid (CA), ursodeoxycholic acid (UDC), chenodeoxycholic acid (CDC), and deoxycholic acid (DC) (n = 14–15/group). C: Body weight was monitored throughout the study (n = 15–16/group). Cross-sections of the aortic root area were stained with Oil red O to visualize atherosclerotic lesions from which the area was determined. D: Representative images of each staining are displayed and (E) mean atherosclerotic lesion area was expressed relative to the total area of the aortic root (n = 9–14/group). Plasma (F) cholesterol (n = 13/group) and (G) triglyceride concentration (n = 8–9/group) was measured in very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). Data are presented as means ± SD. ∗Vehicle versus Bulevirtide. ∗ P < 0.05, according to Mann Whitney’s test (A), or student’s t test (B, E, F).

Fig. 3

Bulevirtide treatment does not alter hepatic expression of genes involved in bile salt or cholesterol metabolism in Oatp1a1^−/−Ldlr^−/− mice. Female Oatp1a1^−/−Ldlr^−/− mice were injected subcutaneously with Na⁺ taurocholate co-transporting polypeptide (NTCP) inhibitor Bulevirtide (yellow circles or bars) or vehicle (black circles or bars) every day for 10 weeks. Hepatic gene expression of (A) farnesoid X receptor (Nr1h4), (B) organic solute transporter beta (Slc51β), (C) fatty acid synthetase (Fasn), (D) small heterodimer partner (Nr0b2), (E) pregnane X receptor (Nr1i2), (F) cytochrome P450 family 7 subfamily A member 1 (Cyp7a1), (G) cytochrome P450 family 27 subfamily A member 1 (Cyp27a1), (H) cytochrome P450 family 8 subfamily B member 1 (Cyp8b1), (I) apolipoprotein A1 (Apoa1), (J) apolipoprotein B (Apob), (K) apolipoprotein C2 (Apoc2), (L) ATP binding cassette subfamily A member 1 (Abca1), (M) LDL receptor related protein 1 (Lrp1), (N) 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (Hmgcr), (O) ATP-binding cassette sub-family G member 5 (Abcg5), and (P) ATP-binding cassette sub-family G member 8 (Abcg8) was determined by quantitative polymerase chain reaction, normalized to glyceraldehyde-3-phosphate dehydrogenase (Gapdh) and hypoxanthine phosphoribosyltransferase (Hprt) and shown relative to the expression in the vehicle group (n = 13–15/group). Data are presented as means ± SD.

Fig. 4

Bulevirtide treatment does not alter food intake, fecal bile salt levels, or intestinal cholesterol absorption in Oatp1a1^−/−Ldlr^−/− mice. Female Oatp1a1^−/−Ldlr^−/− mice were injected subcutaneously with Na⁺ taurocholate co-transporting polypeptide (NTCP) inhibitor Bulevirtide (yellow circles) or vehicle (black circles) every day for 3 days after administration of an oral bolus of [¹⁴C]cholesterol and [³H]sitostanol in olive oil. A: Plasma bile salts were measured after 3 days and (B) food intake was monitored throughout the 3 days (C) Bile salt levels were measured in feces collected during the last 24 h of treatment. ³H and ¹⁴C activity were determined in feces samples after 24, 48, and 72 h after administration of the tracers from which (D) fecal [¹⁴C]cholesterol excretion and (E) intestinal uptake were calculated. F: In the liver individual bile salt species were measured, including tauro-alpha-muricholic acid (TαMC), tauro-beta-muricholic acid (TβMC), tauroursodeoxycholic acid (TUDC), taurohyodeoxycholic acid (THDC), taurocholic acid (TC), glycocholic acid (GC), taurochenodeoxycholic acid (TCDC), taurodeoxycholic acid (TDC), alpha-muricholic acid (αMC), beta-muricholic acid (βMC), glycodeoxycholic acid (GDC), cholic acid (CA), ursodeoxycholic acid (UDC), hyodeoxycholic acid (HDC), chenodeoxycholic acid (CDC), and deoxycholic acid (DC). G: Total bile salt content was calculated by adding individual species. H: Hepatic total cholesterol content was measured. Data are presented as means ± SD (n = 4–7/group). ∗Vehicle versus Bulevirtide. ∗ P < 0.05, according to student’s t test (A) or two-way ANOVA (D).