François Briand

P2Y13 receptor is critical for reverse cholesterol transport.

A major atheroprotective functionality of high‐density lipoproteins (HDLs) is to promote “reverse cholesterol transport” (RCT). In this process, HDLs mediate the efflux and transport of cholesterol from peripheral cells and its subsequent transport to the liver for further metabolism and biliary excretion. We have previously demonstrated in cultured hepatocytes that P2Y13 (purinergic receptor P2Y, G protein–coupled, 13) activation is essential for HDL uptake but the potential of P2Y13 as a target to promote RCT has not been documented. Here, we show that P2Y13‐deficient mice exhibited a decrease in hepatic HDL cholesterol uptake, hepatic cholesterol content, and biliary cholesterol output, although their plasma HDL and other lipid levels were normal. These changes translated into a substantial decrease in the rate of macrophage‐to‐feces RCT. Therefore, hallmark features of RCT are impaired in P2Y13‐deficient mice. Furthermore, cangrelor, a partial agonist of P2Y13, stimulated hepatic HDL uptake and biliary lipid secretions in normal mice and in mice with a targeted deletion of scavenger receptor class B type I (SR‐BI) in liver (hypomSR‐BI–knockoutliver) but had no effect in P2Y13 knockout mice, which indicate that P2Y13‐mediated HDL uptake pathway is independent of SR‐BI–mediated HDL selective cholesteryl ester uptake. Conclusion: These results establish P2Y13 as an attractive novel target for modulating RCT and support the emerging view that steady‐state plasma HDL levels do not necessarily reflect the capacity of HDL to promote RCT. (HEPATOLOGY 2010)

Both the Peroxisome Proliferator‐Activated Receptor δ Agonist, GW0742, and Ezetimibe Promote Reverse Cholesterol Transport in Mice by Reducing Intestinal Reabsorption of HDL‐Derived Cholesterol

Peroxisome proliferator‐activated receptor δ (PPARδ) agonism increases HDL cholesterol and has therefore the potential to stimulate macrophage‐to‐feces reverse cholesterol transport (RCT). To test whether PPAR™ activation promotes RCT in mice, in vivo macrophage RCT was assessed using cholesterol‐loaded/3H‐cholesterol‐labeled macrophages injected intraperitoneally. PPAR™ agonist GW0742 (10 mg/kg per day) did not change 3H‐tracer plasma appearance, but increased fecal 3H‐free sterols excretion by 103% (p < 0.005) over 48 hours. Total free cholesterol efflux from macrophages to serum (collected from both control and GW0742 groups) was not different, although ABCA1‐mediated efflux was significantly higher with GW0742. The metabolic fate of HDL labeled with 3H‐cholesteryl ether or 3H‐cholesteryl oleate was also measured. While 3H‐cholesteryl ether tissue uptake was unchanged, the 3H‐tracer recovered in fecal free sterol fraction after 3H‐cholesteryl oleate injection increased by 88% with GW0742 (p < 0.0005). This was associated with a lower Niemann‐Pick C1 like 1 (NPC1L1) mRNA expression in the small intestine (p < 0.05). The same experiments in mice treated with ezetimibe, which blocks NPC1L1, showed a similar 2‐fold increase in fecal free sterol excretion after labeled macrophages or HDL injection. In conclusion, PPAR™ activation enhances excretion of macrophage or HDL‐derived cholesterol in feces through reduced NPC1L1 expression in mice, comparable to the effect of ezetimibe.

Biliary Sterol Secretion Is Required for Functional In Vivo Reverse Cholesterol Transport in Mice

BACKGROUND & AIMS High-density lipoproteins (HDLs) protect against atherosclerotic cardiovascular disease, mainly by promoting reverse cholesterol transport (RCT). Biliary sterol secretion supposedly represents the final step in RCT, but the relevance of this pathway has not been explored. We tested the dependency of RCT on functional biliary sterol secretion. METHODS Macrophage-to-feces RCT was studied in mice with abolished (bile duct ligation) or decreased biliary sterol secretion (adenosine triphosphate binding cassette transporter B4 (Abcb4)-/- mice, with and without administration of a liver X receptor [LXR] agonist) after intraperitoneal injection of (3)H-cholesterol-loaded primary macrophage foam cells from mice. Fecal tracer excretion and also fecal mass sterol excretion were measured. Metabolism and tissue uptake of HDL cholesteryl ester was assessed with HDL kinetic studies. RESULTS Bile-duct ligation completely abolished RCT from (3)H-cholesterol-loaded macrophages to feces (P < .001). In Abcb4-/- mice lacking biliary cholesterol secretion, RCT was decreased markedly; fecal (3)H-tracer excretion was almost absent within neutral sterols (P < .001) and reduced within bile acids (P < .05). LXR activation stimulated RCT in wild-type (5.5-fold; P < .001) but not Abcb4-/- mice, whereas mass fecal sterol excretion increased similarly in both models (P < .05). Kinetic studies revealed minimal uptake of HDL cholesteryl ester by the intestine, which decreased on LXR activation (P < .05). CONCLUSIONS Functional RCT depends on biliary sterol secretion; there is no compensatory increase in RCT via bile acids. The stimulating effect of LXR agonists on RCT requires biliary cholesterol secretion. These results have implications for therapies against atherosclerotic cardiovascular disease targeting the RCT pathway.

Liver X receptor activation promotes macrophage-to-feces reverse cholesterol transport in a dyslipidemic hamster model

Liver X receptor (LXR) activation promotes reverse cholesterol transport (RCT) in rodents but has major side effects (increased triglycerides and LDL-cholesterol levels) in species expressing cholesteryl ester transfer protein (CETP). In the face of dyslipidemia, it remains unclear whether LXR activation stimulates RCT in CETP species. We therefore used a hamster model made dyslipidemic with a 0.3% cholesterol diet and treated with vehicle or LXR agonist GW3965 (30 mg/kg bid) over 10 days. To investigate RCT, radiolabeled 3H-cholesterol macrophages or 3H-cholesteryl oleate-HDL were then injected to measure plasma and feces radioactivity over 72 or 48 h, respectively. The cholesterol-enriched diet increased VLDL-triglycerides and total cholesterol levels in all lipoprotein fractions and strongly increased liver lipids. Overall, GW3965 failed to improve both dyslipidemia and liver steatosis. However, after 3H-cholesterol labeled macrophage injection, GW3965 treatment significantly increased the 3H-tracer appearance by 30% in plasma over 72 h, while fecal 3H-cholesterol excretion increased by 156% (P < 0.001). After 3H-cholesteryl oleate-HDL injection, GW3965 increased HDL-derived cholesterol fecal excretion by 64% (P < 0.01 vs. vehicle), while plasma fractional catabolic rate remained unchanged. Despite no beneficial effect on dyslipidemia, LXR activation promotes macrophage-to-feces RCT in dyslipidemic hamsters. These results emphasize the use of species with a more human-like lipoprotein metabolism for drug profiling.

Modulation of HDL metabolism by the niacin receptor GPR109A in mouse hepatocytes

The niacin receptor GPR109A is a G(i)-protein-coupled receptor which mediates the effects of niacin on inhibiting intracellular triglyceride lipolysis in adipocytes. However, the role of GPR109A in mediating the effects of niacin on high density lipoprotein (HDL) metabolism is unclear. We found niacin has no effect on HDL-C in GPR109A knockout mice. Furthermore, niacin lowered intracellular cAMP in primary hepatocytes mediated by GPR109A. We used an adeno-associated viral (AAV) serotype 8 vector encoding GPR109A under the control of the hepatic-specific thyroxine-binding globulin promoter to specifically overexpress GPR109A in mouse liver. Plasma HDL-C, hepatic ABCA1 and the HDL cholesterol production rate were significantly reduced in mice overexpressing GPR109A. Overexpression of GPR109A reduced primary hepatocyte free cholesterol efflux to apoA-I; conversely, GPR109A deficient hepatocytes had increased ABCA1-mediated cholesterol efflux. These data support the concept that the HDL-C lowering effect of niacin in wild-type mice is mediated through stimulation of GPR109A in hepatocytes; such an effect then leads to reduced hepatocyte ABCA1 expression and activity, decreased cholesterol efflux to nascent apoA-I, and reduced HDL-C levels. These results indicate that niacin-mediated activation of GP109A in liver lowers ABCA1 expression leading to reduced hepatic cholesterol efflux to HDL.

Empagliflozin, via Switching Metabolism Toward Lipid Utilization, Moderately Increases LDL Cholesterol Levels Through Reduced LDL Catabolism.

In clinical trials, a small increase in LDL cholesterol has been reported with sodium–glucose cotransporter 2 (SGLT2) inhibitors. The mechanisms by which the SGLT2 inhibitor empagliflozin increases LDL cholesterol levels were investigated in hamsters with diet-induced dyslipidemia. Compared with vehicle, empagliflozin 30 mg/kg/day for 2 weeks significantly reduced fasting blood glucose by 18%, with significant increase in fasting plasma LDL cholesterol, free fatty acids, and total ketone bodies by 25, 49, and 116%, respectively. In fasting conditions, glycogen hepatic levels were further reduced by 84% with empagliflozin, while 3-hydroxy-3-methylglutaryl-CoA reductase activity and total cholesterol hepatic levels were 31 and 10% higher, respectively (both P < 0.05 vs. vehicle). A significant 20% reduction in hepatic LDL receptor protein expression was also observed with empagliflozin. Importantly, none of these parameters were changed by empagliflozin in fed conditions. Empagliflozin significantly reduced the catabolism of 3H-cholesteryl oleate–labeled LDL injected intravenously by 20%, indicating that empagliflozin raises LDL levels through reduced catabolism. Unexpectedly, empagliflozin also reduced intestinal cholesterol absorption in vivo, which led to a significant increase in LDL- and macrophage-derived cholesterol fecal excretion (both P < 0.05 vs. vehicle). These data suggest that empagliflozin, by switching energy metabolism from carbohydrate to lipid utilization, moderately increases ketone production and LDL cholesterol levels. Interestingly, empagliflozin also reduces intestinal cholesterol absorption, which in turn promotes LDL- and macrophage-derived cholesterol fecal excretion.

Upregulating Reverse Cholesterol Transport With Cholesteryl Ester Transfer Protein Inhibition Requires Combination With the LDL-Lowering Drug Berberine in Dyslipidemic Hamsters

Objective—This study aimed to investigate whether cholesteryl ester transfer protein inhibition promotes in vivo reverse cholesterol transport in dyslipidemic hamsters. Methods and Results—In vivo reverse cholesterol transport was measured after an intravenous injection of 3H-cholesteryl-oleate–labeled/oxidized low density lipoprotein particles (3H-oxLDL), which are rapidly cleared from plasma by liver-resident macrophages for further 3H-tracer egress in plasma, high density lipoprotein (HDL), liver, and feces. A first set of hamsters made dyslipidemic with a high-fat and high-fructose diet was treated with vehicle or torcetrapib 30 mg/kg (TOR) over 2 weeks. Compared with vehicle, TOR increased apolipoprotein E–rich HDL levels and significantly increased 3H-tracer appearance in HDL by 30% over 72 hours after 3H-oxLDL injection. However, TOR did not change 3H-tracer recovery in liver and feces, suggesting that uptake and excretion of cholesterol deriving from apolipoprotein E-rich HDL is not stimulated. As apoE is a potent ligand for the LDL receptor, we next evaluated the effects of TOR in combination with the LDL-lowering drug berberine, which upregulates LDL receptor expression in dyslipidemic hamsters. Compared with TOR alone, treatment with TOR+berberine 150 mg/kg resulted in lower apolipoprotein E–rich HDL levels. After 3H-oxLDL injection, TOR+berberine significantly increased 3H-tracer appearance in fecal cholesterol by 109%. Conclusion—Our data suggest that cholesteryl ester transfer protein inhibition alone does not stimulate reverse cholesterol transport in dyslipidemic hamsters and that additional effects mediated by the LDL-lowering drug berberine are required to upregulate this process.

High-Fat and Fructose Intake Induces Insulin Resistance, Dyslipidemia, and Liver Steatosis and Alters In Vivo Macrophage-to-Feces Reverse Cholesterol Transport in Hamsters

Reverse cholesterol transport (RCT) promotes the egress of cholesterol from peripheral tissues to the liver for biliary and fecal excretion. Although not demonstrated in vivo, RCT is thought to be impaired in patients with metabolic syndrome, in which liver steatosis prevalence is relatively high. Golden Syrian hamsters were fed a nonpurified (CON) diet and normal drinking water or a high-fat (HF) diet containing 27% fat, 0.5% cholesterol, and 0.25% deoxycholate as well as 10% fructose in drinking water for 4 wk. Compared to CON, the HF diet induced insulin resistance and dyslipidemia, with significantly higher plasma non-HDL-cholesterol concentrations and cholesteryl ester transfer protein activity. The HF diet induced severe liver steatosis, with significantly higher cholesterol and TG levels compared to CON. In vivo RCT was assessed by i.p. injecting ³H-cholesterol labeled macrophages. Compared to CON, HF hamsters had significantly greater ³H-tracer recoveries in plasma, but not HDL. After 72 h, ³H-tracer recovery in HF hamsters was 318% higher in liver and 75% lower in bile (P < 0.01), indicating that the HF diet impaired hepatic cholesterol fluxes. However, macrophage-derived cholesterol fecal excretion was 45% higher in HF hamsters than in CON hamsters. This effect was not related to intestinal cholesterol absorption, which was 89% higher in HF hamsters (P < 0.05), suggesting a possible upregulation of transintestinal cholesterol excretion. Our data indicate a significant increase in macrophage-derived cholesterol fecal excretion in a hamster model of metabolic syndrome, which may not compensate for the diet-induced dyslipidemia and liver steatosis.

CETP Inhibitor Torcetrapib Promotes Reverse Cholesterol Transport in Obese Insulin-Resistant CETP-ApoB100 Transgenic Mice

Insulin resistance and type 2 diabetes are associated with low HDL‐cholesterol (HDL‐c) levels, which would impair reverse cholesterol transport (RCT). A promising therapeutic strategy is to raise HDL with cholesteryl ester transfer protein (CETP) inhibitors, but their effects on RCT remains to be demonstrated in vivo. We therefore evaluated the effects of CETP inhibitor torcetrapib in CETP‐apolipoprotein (apo)B100 mice made obese and insulin resistant with a 60% high‐fat diet. High‐fat diet over 3 months increased body weight and homeostasis model of insulin resistance index by 30% and 846%, respectively (p < 0.01 for both vs. chow‐fed mice). Total cholesterol (TC) increased by 46% and HDL‐c/TC ratio decreased by 28% (both p < 0.05). Compared to vehicle, high‐fat‐fed mice treated with torcetrapib (30 mg/kg/day, 3 weeks) showed increased HDL‐c levels and HDL‐c/TC ratio by 41% and 37% (both p < 0.05). Torcetrapib increased in vitro macrophage cholesterol efflux by 22% and in vivo RCT through a 118% increase in 3H‐bile acids fecal excretion after 3H‐cholesterol labeled macrophage injection (p < 0.01 for both). Fecal total bile acids mass was also increased by 158% (p < 0.001). In conclusion, CETP inhibition by torcetrapib improves RCT in CETP‐apoB100 mice. These results emphasize the potential of CETP inhibition to prevent cardiovascular diseases. Clin Trans Sci 2011; Volume 4: 414–420

Diet-induced dyslipidemia impairs reverse cholesterol transport in hamsters

Eur J Clin Invest 2011; 41 (9): 921–928