In order to investigate the role of P2Y₁₃ receptor in a dyslipidemic context, we studied the phenotype of P2Y₁₃ ^-/- mice fed high cholesterol diet (HCD) for 16 weeks. Body weight and liver weight were unchanged between P2Y₁₃ ^-/- and wild-type (WT, C57BL/6) mice maintained on HCD (data not shown) and plasma total cholesterol, HDL-C, LDL-C and triglycerides did not differ either (Table 1). However, on HCD feeding, hepatic total cholesterol content was significantly lower in P2Y₁₃ ^-/- than in WT mice, with decrease in both cholesterol ester and free cholesterol (Table 2). To further assess the effect of HCD on the metabolic phenotype of P2Y₁₃ ^-/- mice, we measured biliary flow and lipid secretion, which is considered an essential step in RCT 9 . As reported in Table 3, biliary flow was significantly decreased and biliary secretion of cholesterol, bile acid and phospholipid were also significantly reduced in P2Y₁₃ ^-/- as compared to WT mice after 16 weeks of HCD. We next measured the movement of ³H-cholesterol from macrophages to the feces, which is a surrogate well-established method to evaluate in vivo RCT 10 . We observed that macrophage-to-feces RCT was impaired in P2Y₁₃ ^-/- as compared to WT mice, as reflected by a ~60% reduction of total sterol recovered in feces (Figure 1C, -53±5 and -78±4 in % of neutral sterols and bile acids, respectively). This reduced RCT is most likely attributable to the described function of P2Y₁₃ receptor in hepatic HDL uptake 4 11 . Accordingly, P2Y₁₃ ^-/- mice fed HCD displayed a significant higher ³H- tracer present in plasma at 6 and 24 h (Figure 1A) and a trend to lower ³H-tracer recovered in the liver at 48 h, as compared to WT mice (Figure 1B). Macrophage-to-feces RCT experiments have frequently reported that ³H-tracer recovered in feces is more sensitive to evidence changes in RCT than ³H-tracer present in plasma and liver which is often unchanged 10 12 , probably because radioactivity recovered in feces represents the endpoint of RCT whereas radioactivity present in plasma and liver depends on cholesterol flux of a freely distributable tracer. This hence might explain the unchanged % of ³H-tracer present in plasma at 48 h and the lack of a significance in the decrease of ³H-tracer in the liver of P2Y₁₃ ^-/- compared to WT mice (Figure 1A, B).

Values are expressed as means ± SEM; n = 4 mice per group.

Values are expressed as means ± SEM; n = 10 mice per group.

*Indicates significant difference (p < 0.05) from control mice.

Values are expressed as means ± SEM; n = 10 mice per group.

*Indicates significant difference (p < 0.05) from control mice.

P2Y₁₃ deficiency results in a ~40% decrease of hepatic messenger RNA (mRNA) expression of the heterodimeric ATP-binding transporter Abcg5 and Abcg8 (Table 4), suggesting that the cholesterol derived from HDL that has been internalized through P2Y₁₃ receptor might use the Abcg5/g8 dependent pathway for apical biliary cholesterol secretion 13 . Hepatic gene expression of the other transporters controlling bile acid and phospholipid fluxes in the liver, such as biliary phospholipid transport protein (Abcb4/mdr3), bile salt export pump (Abcb11/bsep), basolateral transporter sodium taurocholate cotransporting polypeptide (Ntcp) and organic anion transport polypeptide (Oatp) were unchanged (Table 4). As reported in Tables 4 and 5, there was also no significant change in the expression of gene controlling bile acid production such as hepatic cholesterol 7 alpha-hydroxylase (Cyp7A1), hepatic sterol 27-hydroxylase (Cyp27A1), hepatic sterol 12-alpha-hydroxylase (Cyp8b1) and intestinal Fibroblast growth factor 15 (Fgf15). Furthermore, the reduction of bile acid secretion observed in P2Y₁₃ ^-/- mice compared to WT mice was mainly driven by lower bile flow (Table 3) since the concentration of bile acid in bile was not significantly reduced (data not shown) whereas both secretion and concentration of biliary cholesterol and phospholipid were decreased (Table 3 and data not shown). Altogether these data indicate that the effect of P2Y₁₃ deficiency on decreasing bile acid secretion into the bile cannot be attributed to a decrease of hepatic bile acids synthesis or apical transport but is more likely driven by the decrease of biliary flow. However, further investigation is required to determine the mechanism by which P2Y₁₃-mediated HDL endocytosis regulates biliary lipid secretion process. Indeed, although recent studies evidenced that HDL internalized by hepatocytes are transported to two intracellular pools – a rapid turnover retroendocytic pool involving endosomal recycling compartment (ERC) and a slow-turnover pool, involving multivesicular bodies, that is eventually further transported to lysosomes for degradation 14 , the process by which intracellular HDL trafficking governs biliary lipid secretion is still poorly characterized.

Real-time PCR was performed on individual livers of 3 h fasted mice (n = 10 mice per group). For all genes, the fold change was calculated by dividing the P2Y₁₃ ^-/- mice value by the wild-type mice value (e.g. an increase of 80% from wild-type is reported as 1.80). * and ** indicate significant difference (p < 0.05 and p < 0.01 respectively) from wild-type mice.

Real-time PCR was performed on individual intestine of 3 h fasted mice (n = 6 mice per group). For all genes, the fold change was calculated by dividing the P2Y₁₃.^-/- mice value by the wild-type mice value (e.g. an increase of 80% from wild-type is reported as 1.80).

Interestingly, hepatic mRNA expression of Abca1 and Abcg1, which contribute to cellular cholesterol efflux towards lipid-poor ApoA-I and HDL particles, respectively 13 , was significantly decreased in P2Y₁₃ ^-/- mice after 16 weeks of HCD (Table 4). These results might suggest that the decrease of hepatobiliary cholesterol secretion observed in P2Y₁₃ ^-/- mice fed a HCD is associated with a decrease of de novo HDL formation, thus explaining the unchanged plasma HDL-C levels per se, as previously proposed for P2Y₁₃ ^-/- mice fed a chow diet 4 . Alternatively, the P2Y₁₃-mediated HDL endocytosis pathway might efficiently drive cholesterol from HDL towards biliary lipid secretion but could be quantitatively negligible regarding the steady state concentration of plasma HDL-C. Accordingly, it is now well established that macrophage-derived cholesterol represents only a minor proportion of the total cholesterol transported by HDL particles but is essential to prevent foam cell formation 15 supporting the concept that the dynamics of HDL particles are essential for RCT but not necessary correlated to the static measure of plasma HDL-C concentration.

To assess HDL functionality in these mice, we tested whether P2Y₁₃ deletion would influence HDL functionalities regarding either their anti-inflammatory or anti-oxidative properties, or their capacity to elicit efflux from cholesterol-loaded macrophages. However, P2Y₁₃-deficient mice did not show any change in these respective HDL functionalities after 16 weeks of HCD (Figure 2), suggesting that P2Y₁₃ deletion neither interferes with HDL pleiotropic functions, nor HDL-mediated efflux from cholesterol-loaded macrophages. Furthermore, feeding the HCD for 16 weeks did not result in an increased lipid deposition in the aortic sinus of P2Y₁₃ ^-/- compared to WT mice (Figure 3). Not surprisingly, these results indicate that P2Y₁₃ deletion in C57BL/6 mice does not initiate atherosclerosis development. To formally assess the hypothesis whether P2Y₁₃ receptor plays a role in atherosclerosis development, mice lacking P2Y₁₃ would have to be crossed with a proatherogenic mouse model.

We previously demonstrated that chow-fed P2Y₁₃ deficient mice displayed a reduced uptake of HDL by the liver associated to decreased hepatic free cholesterol content and impaired biliary secretion of cholesterol and phospholipids 4 . These metabolic changes translated into a substantial ~45% decrease in the rate of macrophage-to-feces RCT 4 . In the present study, we observed that a chronic high cholesterol intake accentuates this metabolic phenotype, with a more dramatic impaired hepatobiliary RCT. Indeed, using HCD-fed P2Y₁₃ ^-/- compared to WT mice, we reported here that both free and esterified cholesterol content in the liver were decreased (Table 2), together with biliary flow and biliary lipid secretion (Table 3) and a ~60% reduction in the rate of macrophage-to-feces RCT (Figure 1).

P2Y₁₃ receptor is also expressed in the intestine 16 and could potentially also regulate intestinal cholesterol absorption or excretion. Our results indicates that the mRNA expression level of the principal genes involved in cholesterol absorption were unchanged between P2Y₁₃ ^-/- and WT mice maintained on HCD (Table 5), suggesting that cholesterol absorption is unchanged in P2Y₁₃ ^-/- mice.

Overall, our work emphasizes the importance of hepatic P2Y₁₃ activity when diet is rich in cholesterol, by regulating biliary lipid output and overall RCT without affecting HDL-C level per se or selected HDL functions. Thus, this study opens the way to reconsider pharmacological approaches to target HDL metabolism, particularly with regard to mechanistic aspects of RCT, by improving the flux of circulating HDL towards the liver (e.g., by stimulating P2Y₁₃) rather than increasing plasma HDL-C levels.

HDL: High density Lipoprotein; LDL: Low density lipoprotein; ApoA-I: apolipoprotein A-I; HCD: High cholesterol diet; RCT: Reverse cholesterol tansport; Ecto-F1ATPase: Ectopic F₁-ATPase; ATP: Adenosine triphosphate; ADP: Adenosine diphosphate; Abca1: ATP binding cassette subfamily A member 1; Abcb4: ATP binding cassette subfamily B member 4; Abcg1: ATP binding cassette subfamily G member 1; Abcg5: ATP binding Cassette subfamily G member 5; Abcg8: ATP binding Cassette subfamily G member 8; Oatp: Organic anion transport polypeptide; Ntcp: Sodium taurocholate cotransporting polypeptide; WT: Wild-type; EC: Esterified cholesterol; FC: Free cholesterol; TC: Total cholesterol.

The authors declare that they have no competing of interest.

LOM conceived the study and participated in its design and coordination. BR and JMB conceived P2Y₁₃ knockout mice and participated in the design of the study. LL, NS and GC carried out animal metabolic studies and participated in the design of the study. WA and UJFT carried out HDL functionality assays and analyzed the data. LOM, LL and NS have interpreted the overall data and drafted the manuscript. BP and ML revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.