Amirfarbod Yazdanyar

Role of Phospholipid Transfer Protein in High-Density Lipoprotein-Mediated Reverse Cholesterol Transport

Reverse cholesterol transport (RCT) describes the process whereby cholesterol in peripheral tissues is transported to the liver where it is ultimately excreted in the form of bile. Given the atherogenic role of cholesterol accumulation within the vessel intima, removal of cholesterol through RCT is considered an anti-atherogenic process. The major constituents of RCT include cell membrane– bound lipid transporters, plasma lipid acceptors, plasma proteins and enzymes, and lipid receptors of liver cell membrane. One major cholesterol acceptor in RCT is high-density lipoprotein (HDL). Both the characteristics and level of HDL are critical determinants for RCT. It is known that phospholipid transfer protein (PLTP) impacts both HDL cholesterol level and biological quality of the HDL molecule. Recent data suggest that PLTP has a site-specific variation in its function. Moreover, the RCT pathway also has multiple steps both in the peripheral tissues and circulation. Therefore, PLTP may influence the RCT pathway at multiple levels. In this review, we focus on the potential role of PLTP in RCT through its impact on HDL homeostasis. The relationship between PLTP and RCT is expected to be an important area in finding novel therapies for atherosclerosis.

Liver phospholipid transfer protein (PLTP) expression with a PLTP-null background promotes very low-density lipoprotein production in mice†

It is known that plasma phospholipid transfer protein (PLTP) activity influences lipoprotein metabolism. The liver is one of the major sites of lipoprotein production and degradation, as well as of PLTP expression. To address the impact of liver‐expressed PLTP on lipoprotein metabolism, we created a mouse model that expresses PLTP in the liver acutely and specifically, with a PLTP‐null background. This approach in mouse model preparations can also be used universally for evaluating the function of many other genes in the liver. We found that liver PLTP expression dramatically increases plasma levels of non–high‐density lipoprotein (HDL) cholesterol (2.7‐fold, P < 0.0001), non‐HDL phospholipid (2.5‐fold, P < 0.001), and triglyceride (51%, P < 0.01), but has no significant influence on plasma HDL lipids compared with controls. Plasma apolipoprotein (apo)B levels were also significantly increased in PLTP‐expressing mice (2.2‐fold, P < 0.001), but those of apoA‐I were not. To explore the mechanism involved, we examined the lipidation and secretion of nascent very low‐density lipoprotein (VLDL), finding that liver PLTP expression significantly increases VLDL lipidation in hepatocyte microsomal lumina, and also VLDL secretion into the plasma. Conclusion: It is possible to prepare a mouse model that expresses the gene of interest only in the liver, but not in other tissues. Our results suggest, for the first time, that the major function of liver PLTP is to drive VLDL production and makes a small contribution to plasma PLTP activity. (HEPATOLOGY 2012)

Ezetimibe inhibits hepatic Niemann-Pick C1-Like 1 to facilitate macrophage reverse cholesterol transport in mice.

Objective—Controversies have arisen from recent mouse studies about the essential role of biliary sterol secretion in reverse cholesterol transport (RCT). The objective of this study was to examine the role of biliary cholesterol secretion in modulating macrophage RCT in Niemann-Pick C1-Like 1 (NPC1L1) liver only (L1LivOnly) mice, an animal model that is defective in both biliary sterol secretion and intestinal sterol absorption, and determine whether NPC1L1 inhibitor ezetimibe facilitates macrophage RCT by inhibiting hepatic NPC1L1. Approach and Results—L1LivOnly mice were generated by crossing NPC1L1 knockout (L1-KO) mice with transgenic mice overexpressing human NPC1L1 specifically in liver. Macrophage-to-feces RCT was assayed in L1-KO and L1LivOnly mice injected intraperitoneally with [3H]-cholesterol–labeled peritoneal macrophages isolated from C57BL/6 mice. Inhibition of biliary sterol secretion by hepatic overexpression of NPC1L1 substantially reduced transport of [3H]-cholesterol from primary peritoneal macrophages to the neutral sterol fraction in bile and feces in L1LivOnly mice without affecting tracer excretion in the bile acid fraction. Ezetimibe treatment for 2 weeks completely restored both biliary and fecal excretion of [3H]-tracer in the neutral sterol fraction in L1LivOnly mice. High-density lipoprotein kinetic studies showed that L1LivOnly mice compared with L1-KO mice had a significantly reduced fractional catabolic rate without altered hepatic and intestinal uptake of high-density lipoprotein–cholesterol ether. Conclusions—In mice lacking intestinal cholesterol absorption, macrophage-to-feces RCT depends on efficient biliary sterol secretion, and ezetimibe promotes macrophage RCT by inhibiting hepatic NPC1L1 function.

Liver-Specific Phospholipid Transfer Protein Deficiency Reduces High-Density Lipoprotein and Non–High-Density Lipoprotein Production in Mice

Objective—The liver is one of the critical organs for lipoprotein metabolism and a major source for phospholipid transfer protein (PLTP) expression. The effect of liver-specific PLTP deficiency on plasma lipoprotein production and metabolism in mice was investigated. Approach and Results—We created a liver-specific PLTP-deficient mouse model. We measured plasma high-density lipoprotein (HDL) and apolipoprotein B (apoB)–containing lipoprotein (or non-HDL) levels and their production rates. We found that hepatic ablation of PLTP leads to a significant decrease in plasma PLTP activity, HDL lipids, non-HDL lipids, apoAI, and apoB levels. In addition, nuclear magnetic resonance examination of lipoproteins showed that the deficiency decreases HDL and apoB-containing lipoprotein particle numbers, as well as very low-density lipoprotein particle size, which was confirmed by electron microscopy. Moreover, HDL particles from the deficient mice are lipid-poor ones. To unravel the mechanism, we evaluated the apoB and triglyceride production rates. We found that hepatic PLTP deficiency significantly decreases apoB and triglyceride secretion rates. To investigate the role of liver PLTP on HDL production, we set up primary hepatocyte culture studies and found that the PLTP-deficient hepatocytes produce less nascent HDL. Furthermore, we found that exogenous PLTP promotes nascent HDL production through an ATP-binding cassette A 1-mediated pathway. Conclusions—Liver-specific PLTP deficiency significantly reduces plasma HDL and apoB-containing lipoprotein levels. Reduction of production rates of both particles is one of the mechanisms.

All members in the sphingomyelin synthase gene family have ceramide phosphoethanolamine synthase activity

Sphingomyelin synthase-related protein (SMSr) synthesizes the sphingomyelin analog ceramide phosphoethanolamine (CPE) in cells. Previous cell studies indicated that SMSr is involved in ceramide homeostasis and is crucial for cell function. To further examine SMSr function in vivo, we generated Smsr KO mice that were fertile and had no obvious phenotypic alterations. Quantitative MS analyses of plasma, liver, and macrophages from the KO mice revealed only marginal changes in CPE and ceramide as well as other sphingolipid levels. Because SMS2 also has CPE synthase activity, we prepared Smsr/Sms2 double KO mice. We found that CPE levels were not significantly changed in macrophages, suggesting that CPE levels are not exclusively dependent on SMSr and SMS2 activities. We then measured CPE levels in Sms1 KO mice and found that Sms1 deficiency also reduced plasma CPE levels. Importantly, we found that expression of Sms1 or Sms2 in SF9 insect cells significantly increased not only SM but also CPE formation, indicating that SMS1 also has CPE synthase activity. Moreover, we measured CPE synthase Km and Vmax for SMS1, SMS2, and SMSr using different NBD ceramides. Our study reveals that all mouse SMS family members (SMSr, SMS1, and SMS2) have CPE synthase activity. However, neither CPE nor SMSr appears to be a critical regulator of ceramide levels in vivo.

Adipocyte Phospholipid Transfer Protein and Lipoprotein Metabolism

Objective—Phospholipid transfer protein (PLTP) is highly expressed in adipose tissues. Thus, the effect of adipose tissue PLTP on plasma lipoprotein metabolism was examined. Approach and Results—We crossed PLTP-Flox-&Dgr;Neo and adipocyte protein 2 (aP2)-Cre recombinase (Cre) transgenic mice to create PLTP-Flox-&Dgr;Neo/aP2-Cre mice that have a 90 and a 60% reduction in PLTP mRNA in adipose tissue and macrophages, respectively. PLTP ablation resulted in a significant reduction in plasma PLTP activity (22%), high-density lipoprotein-cholesterol (21%), high-density lipoprotein-phospholipid (20%), and apolipoprotein A-I (33%) levels, but had no effect on nonhigh-density lipoprotein levels in comparison with those of PLTP-Flox-&Dgr;Neo controls. To eliminate possible effects of PLTP ablation by macrophages, we lethally irradiated PLTP-Flox-&Dgr;Neo/aP2-Cre mice and PLTP-Flox-&Dgr;Neo mice, and then transplanted wild-type mouse bone marrow into them to create wild-type→PLTP-Flox-&Dgr;Neo/aP2-Cre and wild-type→PLTP-Flox-&Dgr;Neo mice. Thus, we constructed a mouse model (wild-type→PLTP-Flox-&Dgr;Neo/aP2-Cre) with PLTP deficiency in adipocytes but not in macrophages. These knockout mice also showed significant decreases in plasma PLTP activity (19%) and cholesterol (18%), phospholipid (17%), and apolipoprotein A-I (26%) levels. To further investigate the mechanisms behind the reduction in plasma apolipoprotein A-I and high-density lipoprotein lipids, we measured apolipoprotein A-I–mediated cholesterol efflux in adipose tissue explants and found that endogenous and exogenous PLTP significantly increased cholesterol efflux from the explants. Conclusions—Adipocyte PLTP plays a small but significant role in plasma PLTP activity and promotes cholesterol efflux from adipose tissues.

Sphingolipids and HDL Metabolism

The major sphingolipids in the human plasma are sphingomyelin, ceramide, sphingosine-1-phosphate, and glycosphingolipids. Sphingolipids are found to be associated with plasma lipoproteins and their presence can influence lipoprotein, including high-density lipoprotein (HDL), metabolism. Sphingolipids are also present on cell plasma membrane and they also affect lipoprotein, including HDL, production and catabolism. In this chapter, we will summarize what we know about sphingolipids and HDL metabolism.