Clin Chem Lab Med 2014; 52(12): 1695–1727

Review

Indra Ramasamy* Recent advances in physiological lipoprotein metabolism

Abstract: Research into lipoprotein metabolism has devel- summarizes recent advances in knowledge of the molecu- oped because understanding lipoprotein metabolism has lar mechanisms regulating lipoprotein metabolism. important clinical indications. Lipoproteins are risk fac- tors for cardiovascular disease. Recent advances include Keywords: ABCA1; angiopoietin-like proteins; apoC; the identification of factors in the synthesis and secretion apoAI; apolipoprotein E (apoE); cholesterol ester transfer of triglyceride rich lipoproteins, chylomicrons (CM) and protein; chylomicron; LDL receptor; lecithin cholesterol very low density lipoproteins (VLDL). These included the acyl transferase; lipoprotein(a); lipoprotein lipase; micro- identification of microsomal transfer protein, the cotrans- somal transfer protein; propertin convertase subtilisin/ lational targeting of apoproteinB (apoB) for degradation kexin type 9; SR-BI; phospholipid transfer protein; very regulated by the availability of lipids, and the characteri- low density lipoproteins (VLDL). zation of transport vesicles transporting primordial apoB containing particles to the Golgi. The lipase maturation DOI 10.1515/cclm-2013-0358 factor 1, glycosylphosphatidylinositol-anchored high den- Received May 14, 2013; accepted July 8, 2013; previously published sity lipoprotein binding protein 1 and an angiopoietin-like online August 12, 2013 protein play a role in lipoprotein lipase (LPL)-mediated hydrolysis of secreted CMs and VLDL so that the right Abstract 1695 amount of fatty acid is delivered to the right tissue at the Background 1696 right time. Expression of the low density lipoprotein (LDL) Normal lipoprotein metabolism 1697 receptor is regulated at both transcriptional and post- Assembly and secretion of transcriptional level. Proprotein convertase subtilisin/ apoB containing lipoproteins 1697 kexin type 9 (PCSK9) has a pivotal role in the degradation Chylomicrons 1697 of LDL receptor. Plasma remnant lipoproteins bind to spe- Chylomicron assembly 1697 cific receptors in the liver, the LDL receptor, VLDL recep- Apolipoproteins and chylomicron synthesis 1698 tor and LDL receptor-like proteins prior to removal from Catabolism of chylomicrons 1701 the plasma. Reverse cholesterol transport occurs when Lipoprotein lipase and chylomicron metabolism 1701 lipid free apoAI recruits cholesterol and phospholipid to Cellular membrane receptors and chylomicron assemble high density lipoprotein (HDL) particles. The metabolism 1702 discovery of ABC transporters (ABCA1 and ABCG1) and Very low density lipoproteins (VLDL) 1702 scavenger receptor class B type I (SR-BI) provided further VLDL assembly 1702 information on the biogenesis of HDL. In humans HDL- Apolipoproteins and VLDL synthesis 1703 cholesterol can be returned to the liver either by direct Catabolism of VLDL 1704 uptake by SR-BI or through cholesteryl ester transfer pro- LDL receptor and apolipoproteins 1704 tein exchange of cholesteryl ester for triglycerides in apoB Lipoprotein(a) 1707 lipoproteins, followed by hepatic uptake of apoB contain- High density lipoprotein (HDL) 1708 ing particles. Cholesterol content in cells is regulated by Biosynthesis of HDL and reverse several transcription factors, including the liver X receptor cholesterol transport 1708 and sterol regulatory element binding protein. This review ABCA1 and HDL assembly 1708 Apolipoprotein AI and HDL assembly 1709

*Corresponding author: Indra Ramasamy, Department of Blood Cholesteryl ester transfer protein (CETP) Sciences, Worcester Royal Hospital, Charles Hasting Way, Worcester and phospholipid transfer protein 1712 WR5 1EP, UK, E-mail: [email protected] ABCG1 1713 1696 Ramasamy: Lipoprotein metabolism

Scavenger receptor BI (SR-BI) 1714 vascular events was reduced by about a fifth. Absolute Other factors in HDL remodeling 1715 reduction in cardiac mortality produced by lowering LDL Hormones and lipid metabolism 1715 cholesterol with statin therapy in a given population Clinical implications of lipoprotein metabolism 1716 depended chiefly on the absolute risk of death due to cor- Future directions 1717 onary exclusion. After adjustments for other risk factors, the risk ratio for upper versus lower tertiles of HDL-choles- terol was similar in participants allocated more intensive as well as those allocated less intensive statin therapy, Background suggesting that the risk reduction was similar in patients with higher HDL concentrations [6]. Cardiovascular disease (CVD) is a major cause of world- Hypertriglyceridemia is a heterogeneous disorder wide mortality. The main clinical entities of CVD are coro- with an unclear association with atherosclerosis and nary artery disease (CAD), ischemic stroke, and peripheral ischemic heart disease. It is difficult to distinguish the arterial disease (PAD). The risk factors for CVD are multi- effects of triglyceride on CVD risk from that of low HDL as factorial and include age, male gender, tobacco smoking, HDL is inversely correlated to triglycerides. Most studies lack of physical activity, dietary habits, blood pressure, that reported an association between fasting plasma tri- and dyslipidemias. glycerides and CVD risk found that after adjustment for The term dyslipidemias, elevated or decreased levels other risk factors (especially HDL) triglyceride was no of lipoproteins, covers a broad spectrum of lipid abnormal- longer an independent risk factor [7]. A meta-analysis of ities. Dyslipidemias may be secondary and related to other studies carried out between 1996 and 2007 reported a pos- diseases or primary and due to the interaction between a itive association between triglyceride levels and stroke [8]. genetic predisposition and environmental factors. Obser- The practice of measuring triglycerides in a fasting condi- vational studies indicated a continuous positive relation- tion to assess a relationship between cardiovascular risk ship between coronary disease risk and blood cholesterol and plasma triglyceride has been questioned as humans concentrations [1]. Further, research from experimental are in post-prandial state for a major part of the day. Mora animals, laboratory investigations, epidemiology, and et al. [9] suggest that HDL-cholesterol, triglycerides, total/ genetic forms of hypercholesterolemia indicate that ele- HDL-cholesterol ratio and apolipoprotein AI (apoAI) are vated total cholesterol and low density lipoprotein (LDL) better predictors of CVD when measured in a non-fast- cholesterol is a risk factor for ischemic heart disease [2]. ing state. In the Copenhagen City Heart Study stepwise The Prospective Study Collaboration reports that the increasing levels of non-fasting cholesterol and non-fast- relations between cholesterol and CVD holds true across ing triglycerides were similarly associated with stepwise age and blood pressure categories. Interestingly, total/ increasing risk of myocardial infarction, with non-fasting HDL-cholesterol ratio is more informative in this meta- triglycerides being the best predictor in women and non- analysis than high density lipoprotein (HDL), non-HDL or fasting cholesterol the best predictor in men. Surprisingly, total cholesterol. The analysis, however, reported no clear only increasing levels of triglyceride were associated with association between cholesterol and stroke. Stroke is a total mortality whereas increasing cholesterol levels were heterogeneous condition and various causes of ischemic not [10]. Using the same study cohort the authors found stroke may have different associations with cholesterol [3, that stepwise increasing levels of non-fasting triglycerides 4]. By contrast, meta-analysis of randomized trials of few were associated with risk of ischemic stroke in men and years of statin therapy have shown that reduction of LDL women [11, 12]. Increased levels of non-fasting triglycer- cholesterol by about 1.5 mmol/L reduces by about a third ide indicate the presence of increased levels of remnants the incidence not only of ischemic heart disease but also from chylomicrons (CM) and very low density lipoproteins of ischemic stroke, independently of age, blood pressure (VLDL). These cholesterol containing triglyceride rich or prerandomization lipid concentrations. The benefits of lipoproteins can penetrate the arterial endothelium and treatment were significant in the first year, but remained may get trapped within the subendothelial space leading in subsequent years. The reduction in ischemic stroke in to the development of atherosclerosis [13]. Several studies randomized trials of statins suggests a need for more anal- have raised the possibility that remnant particles may be ysis of blood lipids and stroke subtypes [5]. Further meta- associated with CVD risk [14, 15]. In meta-analyses of the analysis of randomized trials comparing more versus less effects of Gly188Glu and Asn291Ser substitutions in lipo- intensive statin therapy suggested that for each 1 mmol/L protein lipase Wittrup et al. [16] reported that in carriers reduction in LDL cholesterol the annual rate of major of both substitutions post-heparin lipoprotein lipase (LPL) Ramasamy: Lipoprotein metabolism 1697 activity was decreased with an increase in plasma triglyc- As there is a need for strict maintenance of tissue cho- erides. Accordingly, the risk of ischemic heart disease was lesterol and triglyceride levels the body relies on a complex increased in heterozygous carriers. In a further prospective homeostatic network to modulate the availability of cho- study, over 20 years, relatives in familial combined hyper- lesterol and triglyceride to tissues. In recent years there lipidemia families were at increased risk of CVD mortality. have been great advances in the diagnosis and treatment Among familial hypertriglyceridemia families the relative of abnormalities of lipid homeostasis. There is mounting risk was higher but did not reach statistical significance, interest in optimizing dyslipidemia management through probably because of smaller sample size. Baseline triglyc- individualization of additional treatment. Further, under- eride levels predicted subsequent CVD mortality in both standing hyperlipidemias becomes relevant with the world- families and the risk remained statistically significant wide epidemic of obesity, metabolic syndrome, and T2DM. after adjustment for total cholesterol [17]. Other studies A more complete understanding of the molecular mecha- suggest that LPL variants are associated with a differential nisms of lipoprotein metabolism should give an improved susceptibility to CVD [18, 19]. understanding of hyperlipidemias in humans and open new Fibrates are agonists of the peroxisome proliferator- avenues for individualization of treatment of dyslipidemias activator receptors (PPARs) and are selective for α recep- and other metabolic disorders leading to dyslipidemias. This tors. They have been shown to raise HDL-cholesterol, article will give an overview of recent studies and advance- lower total cholesterol and triglyceride concentrations ment in our knowledge of the metabolism of lipoproteins. and decrease LDL concentrations [20]. Several large scale trials of fibrate therapy have been completed, results, however, have been conflicting about the presence and magnitude of any cardiovascular protective effects. Sub- Normal lipoprotein metabolism group analysis of fibrate trials suggest that for patients with type II diabetes mellitus or the metabolic syndrome, Triglycerides and cholesteryl esters are transported in the fibrates may exert a greater cardio protective effect than form of lipoproteins. Triglyceride and cholesteryl esters, the general population [21]. The Action to Control Cardio- fat soluble vitamins comprise the core of the lipoproteins vascular Disease in Diabetes (ACCORD) trial investigated and are enveloped by a layer of phospholipids, free cho- the incremental effect of fenofibrate in patients with type lesterol and proteins. The proteins [apoproteins (apo) or 2 diabetes mellitus (T2DM) who were already being treated apolipoproteins] are critical regulators of lipid transport with simvastatin. The authors concluded that fenofibrate (Table 1) and play a role in lipoprotein assembly, lipid and simvastatin did not reduce the rate of fatal cardiovas- transport and lipid metabolism by mediating interactions cular events, non-fatal stroke as compared with simvasta- with receptors, enzymes and lipid transport proteins. An tin alone [22]. In the Fenofibrate Intervention and Event overview of lipid metabolism is given in Figure 1. Lowering in Diabetes (FIELD) trial treatment with fenofi- brate did not significantly reduce the risk of first myocar- dial infarction (MI) or coronary heart disease (CHD) death, Assembly and secretion of apoproteinB though it reduced non-fatal MI and revascularizations [23]. (apoB) containing lipoproteins A large meta-analysis including more than 45,000 indi- viduals suggested that therapy with fibrates can reduce Chylomicrons the risk of coronary events [24]. Analysis of the ACCORD study suggested that patients with a high triglyceride level Chylomicron assembly and low serum HDL-cholesterol level were a subset for Triacylglycerol is the predominant fat in the diet, contrib- which the greatest reduction in cardiovascular events was uting 90%–95% of the total energy derived from dietary achieved. This was confirmed in a further meta-analysis fat. Dietary fats also include phospholipids (predomi- which suggested that the greatest benefits of fibrates in nantly phophatidylcholine), cholesterol and fat soluble reducing the risk of vascular events was most pronounced vitamins. The activity of pancreatic lipase on triacylg- in patients with both high triglycerides and reduced HDL- lycerols generates 2-monoacylglycerols and free fatty cholesterol [25]. The Third Report of the National Cho- acids. Free fatty acids are absorbed from the intestinal lesterol Education Program (NCEP) Expert Panel (Adult lumen into the enterocyte for the biosynthesis of neutral Treatment Panel III) recommends that in persons with fats. A protein independent diffusion model and protein high serum triglycerides, elevated remnant lipoproteins dependent mechanisms have been proposed for the should be reduced in addition to lowering LDL [26]. transport of free fatty acids across the apical membrane 1698 Ramasamy: Lipoprotein metabolism

Table 1 Apolipoproteins.

Apolipoprotein Molecular weight Lipoproteins Metabolic functions Synthesis References

ApoAI 28,016 HDL, chylomicrons Structural component of HDL, LCAT Liver, intestine activator ApoAII 17,414 HDL, chylomicrons Liver [27] ApoAIV 46,465 HDL, chylomicrons Involved in chylomicron assembly and Intestine in [28, 29] secretion humans ApoAV HDL, VLDL, Effects on plasma triglyceride Predominantly [30, 31] chylomicrons concentrations are complex and variable. in the liver Activator of intravascular hydrolysis by LPL. Modulates hepatic triglyceride metabolism ApoB48 264,000 chylomicrons Necessary for assembly and secretion of Intestine chylomicrons from the small intestine ApoB100 540,000 VLDL, IDL, LDL Necessary for assembly and secretion of Liver VLDL from liver. Structural protein of VLDL, IDL and LDL. Ligand for LDL receptor ApoCI 6630 Chylomicrons, ApoCI inhibits lipoprotein binding to its Liver [32, 33] VLDL, IDL, HDL receptors. Potent inhibitor of cholesteryl ester transfer protein. ApoCII 8900 Chylomicrons, Activator of lipoprotein lipase VLDL, IDL, HDL ApoCIII 8800 Chylomicrons, Inhibits lipoprotein lipase; increases VLDL Synthesized in [32, 34–36] VLDL, IDL, HDL secretion. the liver and to ApoCIII can also stimulate several a lesser extent processes involved in atherogenesis and in the intestine vascular inflammation. Interferes with remnant lipoprotein clearance. ApoE 34,145 Chylomicrons, LDL receptor ligand for LDL and Predominantly [37, 38] VLDL, IDL, HDL chylomicron remnants. Ligand for LRP. Role in the liver in reverse cholesterol transport. Apo(a) 250,000–800,000 Lp(a) Liver

of the enterocyte. Bile salt micelles facilitate the transfer After ingestion of a meal, dietary fat are absorbed of cholesterol across the brush border membrane. Studies into the cells of the small intestine and incorporated with genetically modified animal models have identified into the core of nascent CMs. The human intestine is Niemann-Pick C1-like 1 (NPC1L1) protein as a cholesterol equipped to efficiently absorb dietary fat predominantly uptake transporter and ATP-binding cassette (ABC) pro- in the form of triacylglycerol and to form CMs and deliver teins ABCG5 and ABCG8 as cholesterol efflux transport- lipids in this form to peripheral tissues. The multistep ers. NPC1L1 and ABCG5/G8 are almost exclusively on the assembly of CMs within the enterocyte includes cellu- apical membranes of enterocytes in the intestine and lar lipid re-esterification, translocation of cellular lipid hepatocytes in the liver. This would allow for transport of pools, synthesis and post-translational modification of intracellular sterols back into the small intestine for fecal apoproteinB (apoB), and finally packaging lipid, and excretion [39]. Endoplasmic reticulum (ER) membrane apoB into CMs. Each of these lipoproteins contains 1 mol- localized enzymes, acyl-CoA:cholesterol acyltransferase ecule of apoB [42]. (ACAT) catalyze the esterfication of intracellular choles- terol; it has been suggested that esterification of choles- terol is important for entry of cholesterol into nascent Apolipoproteins and chylomicron synthesis CMs [40]. Fatty acids and monoacyl glycerol are utilized Apoprotein B48 (apoB48) is found on CMs. In humans, to resynthesize diacyl glycerol and triacylglycerol by apoB48 found in the small intestine is a truncated form acyl-CoA:monoacylglycerol acyltransferase and acyl- of apoB100 and is necessary for the intestinal secretion of CoA:diacylglycerol acyltransferase in the ER [41]. these lipoproteins. ApoB100 is found mainly in the liver. Ramasamy: Lipoprotein metabolism 1699

Intestine Adipose tissue/muscle

GPIHBP1 Free fatty acids B48 CII LPL CIII E Chylomicron remnants Chylomicron B48 E CIII

HSPG LRP LDLR

LPL LDL

CII IDL LPL E B100 VLDL LIVER

Figure 1 Pathways of triglyceride rich lipoprotein metabolism. Intestinal enterocytes absorb dietary lipids and package most of it into chylomicrons. Endothelial bound LPL hydrolyzes triglycerides in CM to generate chylomicron remnants which are rapidly cleared by the liver. Intrahepatic lipids are repackaged and secreted as VLDLs which are substrates for LPL. Remnants formed by hydrolysis of VLDL are either taken up by the liver or converted to LDL. The hepatic LDL receptor is responsible for clearing LDL from human plasma.

ApoB100 has 4536 amino acids. ApoB48 results from a ApoB like all secreted proteins is synthesized at the post-transcriptional modification of the apoB mRNA, surface of the ER. Once the ribosome is targeted and through the action of the APOB mRNA editing complex docked securely at the aqueous translocation channel the (APOBEC1) which forms a stop codon at approximately nascent chain is cotranslationally translocated across the 48% of the full length coding sequence [43]. The trun- ER membrane. The signal sequence which spans amino cated form of ApoB100, ApoB48 lacks the C-terminus acids 1–27 of apoB contacts the ER membrane, and translo- which contains the domain recognized by the LDL recep- cation of apoB occurs through the translocon, a proteina- tor. Unlike exchangeable apolipoproteins, apoB remains ceous channel in the ER membrane, formed mainly by the associated with lipoproteins from assembly/secretion to Sec61 protein. Entry of the N-terminus into the translocon lipoprotein remnant clearance. Given that the critical role causes reinitiation of the translation which is coordinated that apoB plays in lipoprotein transport, it is not surpris- with translocation until the full length protein enters the ing that regulation of the secretion of apoB containing ER lumen for further processing and transport [45]. MTP is lipoproteins is complex. Microsomal triglyceride transfer a heterodimeric protein complex possessing lipid transfer protein (MTP) activity is required for apoB containing lipo- activity, which functions in the small intestine and liver to protein assembly and secretion. Early studies suggested transfer lipids to assist in the formation of primordial apoB that a significant amount of newly synthesized apoB is lipoproteins. It has been shown to have three domains: 1) degraded. Unlike most secreted proteins, apoB levels are an apoB binding domain; 2) a lipid transfer domain; and primarily regulated through degradation. Studies of apoB 3) a membrane association domain [46]. The initial incor- degradation indicated that the turnover of the protein was poration of lipids into apoB by MTP may prevent apoB rapidly and negatively regulated by triglyceride synthesis. from degradation. The absence of either a lipid substrate It was found that when conditions were not favorable for or MTP may result in the improper folding of apoB and apoB assembly with lipids, apoB was degraded. Studies consequently its degradation [47]. However, it has been in several laboratories have demonstrated several points suggested that both enterocytes and hepatocytes may have along the intracellular processing of apoB where it can be different methods of apoB stabilization to achieve their targeted for lipoprotein assembly and secretion or degra- specific metabolic functions. It has been demonstrated in dation [44] (Figure 2). Caco-2 cells that mature apoB proteins accumulate in the 1700 Ramasamy: Lipoprotein metabolism

cis-Golgi network. The nascent CMs are larger (250 nm or more) in diameter while protein vesicles are smaller TG/PL/Chol (60–80 nm) [53], and it is unlikely that nascent CMs can MTP ER move across the ER because of physical constraints. This suggests that prechylomicron transport vesicles (PCTVs) apoB48 Prechylomicron particle may require specialized transport machinery for their export from the ER to the cis-Golgi. In addition, the inter- mittent nature of PCTV formation suggests that PCTV Breakdown budding may require proteins that differ from those used PCTV for protein vesicles. It has been suggested that an isoform of protein kinase C(ζ) (PKC) and vesicle-associated mem- brane protein 7 (VAMP7) play a role in the formation PCTVs and delivery to the cis-Golgi [54, 55]. Characteri- zation of PCTVs showed that various proteins including liver fatty acid binding proteins collocate with apoB48 [56]. CD36, described as a fatty acid translocase has been Golgi bodies demonstrated to be part of the PCTVs budding complex Secretion [57]. COPII coats are assembled from three components, i.e., Sar1b, Sec23/24 and Sec 13/31 complexes [58] and are needed to form lipid vesicles that can fuse with the cis-Golgi complex. The Sar1b protein rallies the COPII- complex to form a shell around the vesicles transporting CMs to the cis-Golgi complex [59]. It has been suggested that COPII proteins are required for fusion of PCTVs with Figure 2 Biosynthesis of chylomicrons. the cis-Golgi, but that, in the intestine, membrane bound In the intestinal cell newly synthesized apoB48 follows one of two lipid particles can bud from the ER in a COPII independent itineraries. In the presence of phospholipids, cholesterol and tria- manner [53]. N-ethylmaleimide-sensitive factor attach- cylglycerol, apoB48 is chaperoned by MTP, to form newly synthe- ment protein receptor (SNARE) proteins facilitate the tar- sized prechylomicron particles. In the absence of lipids it is rapidly degraded. The prechylomicron particle buds from the endoplasmic geting, docking and fusion of transport vesicles with their reticulum and is transported to the Golgi as the prechylomicron target membranes. SNARE proteins are type II integral transport vesicle. The budding process is facilitated by the fatty membrane proteins which are present on transport vesi- acid binding protein. Fusion with Golgi complex requires COPII and cles and their target membranes. The PCTV uses VAMP7 SNARE proteins. Within the Golgi complex the prechylomicron forms and syntaxin 5 with rBet1 and vti1a as the cis-Golgi SNARE the mature chylomicron particles. The mature chylomicron exits the Golgi complex in large transport vesicles to fuse with the basolat- proteins involved in the SNARE fusion complex. The lipid eral membrane and are secreted. The mature chylomicrons contain composition of CMs changes in the Golgi and it is likely apoAI, apoAIV and apoB48. that they acquire apoAI in this compartment [60]. MTP is found within the Golgi apparatus and takes part in triglyc- eride transfer within the Golgi apparatus [61]. Fully assem- bled lipoproteins are released by exocytosis of secretory apical regions as ‘ready to use’ stores and that a supply vesicles from the basolateral membranes of enterocytes. of lipids is required to mobilize these stores. This sug- In humans, apoAIV is a 46kd plasma glycoprotein gests that in the intestine, apoB stability may not be the synthesized predominantly by the intestine. ApoAIV only pathway for control of triglyceride secretion [48]. It enters the circulation on the surface of nascent CMs. It has has been suggested that apoB-MTP binding may lead to been suggested that apoAIV enhances particle expansion the unidirectional transfer of lipids to apoB [49]. MTP is and increases triglyceride secretion [62]. In the circula- modulated by several factors and typically regulated at tion, apoAIV is displaced from CMs by HDL-associated the transcriptional level [50, 51]. The newly synthesized apoC, and the exchange facilitates the activation of LPL chylomicron particle consists of apoB48, apoAIV, choles- by its cofactor apoCII [63]. teryl ester, triacylglycerol and phospholipid [52]. In humans, severe hyperchylomicronemia has Newly synthesized, correctly folded nascent lipopro- been associated with mutations in the apolipoprotein teins are transported from the ER unidirectionally to the AV (APOAV) gene [64]. ApoAV circulates at very low Ramasamy: Lipoprotein metabolism 1701 concentrations in the plasma in association with triglycer- in the blood. In humans, LPL and hepatic lipase (HL) have ide rich lipoproteins and HDL. Experimental data suggest been analyzed in plasma samples, collected after the that apoAV enhances the catabolism of triglyceride rich injection of heparin, a procedure used to release LPL and lipoproteins by stimulating LPL. One hypothesis is that HL from cellular binding. LPL is a member of the lipase ApoAV binds to CMs or VLDL and to endothelial proteo- family which includes HL and pancreatic lipase. All three glycans and LPL and stabilizes the endothelial lipolytic proteins are encoded by genes that share structural simi- system [30]. The question as to whether ApoAV is a key larities [71]. Due to its central role in lipid metabolism, determinant of triglycerides levels in humans remains understanding the mechanisms controlling LPL expres- conjectural. APOA5 is part of the APOA1/A4/C3/A5 gene sion becomes important. Changes in LPL expression are cluster located in chromosome 11. Studies in cell culture mainly through the action of hormones such as insulin, and animal models have identified several transcription glucocorticoids and adrenaline [72, 73]. Published data on factors including PPAR-α, liver X receptor-α (LXR-α), the points of control of LPL show its considerable com- hepatocyte nuclear factor 4-α (HNF4-α) and upstream plexity. LPL is regulated in a tissue/cell-specific manner, stimulatory factor (USF1) that contribute to the regulation and both transcriptional and post-transcriptional control of APOA5 expression [65, 66]. In a recent study, Lee et al. mechanisms have been identified in the regulation of LPL [67] identified cyclic AMP responsive element binding gene expression [74]. This is physiologically important protein H (CREB-H) as a regulator of several proteins because LPL directs fatty acid utilization according to the affecting lipolysis, including apoAV, apoCII and apoCIII. metabolic demands of each individual tissue. LPL gener- Three isoforms of PPARs (α, β, γ) have been described. ates fatty acids that are either used for fuel in muscle or PPARs are major regulators of lipid and glucose metabo- stored in the form of triglycerides in adipose tissue. For lism, allowing adaptation to changing nutritional envi- example, fasting decreases LPL activity in adipose tissue ronment. PPARα potentiates fatty acid metabolism in the but increases activity in cardiac tissue [73]. liver and PPARγ induces adipocyte differentiation [68]. LPL is glycosylated and exists as a dimer and is engaged in a number of different molecular interactions as part of its functions. Several effectors of LPL have Catabolism of chylomicrons been identified that modulate the function of LPL in vivo. In the lymph and blood, CMs acquire apoCI, apoCII, ApoCII is an essential cofactor of LPL activity and angi- apoCIII and apoE (Table 1). After gaining apoCII the acti- opoietin-like proteins (ANGPTL) 3 and 4 have been shown vator of LPL, chylomicron interacts with the enzyme and to inhibit LPL activity. The interaction between ANGPTL4 triglyceride hydrolysis is initiated. LPL mediated triglycer- and LPL results in the conversion of enzyme from cata- ide hydrolysis is accompanied by a reduction in the core lytically active dimers to inactive monomers The ANGPTL volume of the CMs and transfer of phospholipid, free cho- protein family currently has six members [75]. ANGPTL3 lesterol, apoCII and apoCIII back to HDL. As the chylomi- is expressed almost exclusively in the liver an organ that cron circulates, the core triglyceride undergoes hydrolysis expresses little or no LPL in adults, and is presumed to by endothelial bound LPL with entry of fatty acids into function as a circulating inhibitor of LPL. ANGPTL4 is muscle for energy production and adipocytes for storage. expressed in multiple tissues, with the highest level In the two-step model, the remaining relatively triglycer- in mice in the adipose tissue. ANGPTL4 expression in ide-depleted chylomicron ‘remnant’ particles which are adipose tissue is induced by fasting, suggesting it inhibits enriched in cholesteryl ester (from both dietary- and HDL- LPL in adipose tissue to reroute fat from adipose tissue to derived cholesteryl ester) and apoE enriched can interact other tissues including muscle during fasting [76]. with receptors on hepatocytes and be removed from the Recently two new proteins that are involved in circulation [69]. All CMs usually disappear from the circu- post-translationally regulating LPL activity have been lation within 12–14 h after a meal [70]. identified. The lipase maturation factor 1 (LMF1) plays an essential role in the formation of catalytically active LPL from newly synthesized polypeptides. The glyco- Lipoprotein lipase and chylomicron metabolism sylphosphatidylinositol-anchored high density lipopro- LPL is synthesized by a number of cells and tissues. The tein binding protein 1 (GPIHBP1) is critically involved major sites of LPL synthesis are the skeletal and cardiac in LPL-mediated hydrolysis of TG rich lipoproteins [77]. muscle and adipose tissue. LPL must transfer from its cell GPIHBP1 provides an important platform for LPL-medi- of origin to the luminal surface of capillary endothelial ated hydrolysis of chylomicron triglycerides. Davies et al. cells that are exposed to large triglyceride rich lipoproteins [78] showed that GPIHBP1 was effective in transporting 1702 Ramasamy: Lipoprotein metabolism

LPL from the basolateral to the apical surface of endothe- Other studies suggest that chylomicron remnant clear- lial cells. Recently, it has been suggested that LPL and ance is not impaired in FH subjects [88]. Using a lipid GPIHBP1 move bidirectionally across the cell surface, emulsion mimicking the composition of CMs, and meas- however, the understanding of the molecular processes uring metabolism using a stable isotope breath test, Watts of the transport remains incomplete [79]. In mice, et al. [89] suggest that the metabolism of CMs remnants is GPIHBP1 was expressed highly on the capillaries of lipo- not impaired in patients with FH. The role of LDL recep- lytic tissues such as adipose tissue, heart and skeletal tors in the metabolism of chylomicron remnants remains muscle. These findings suggest that GPIHBP1 is an impor- controversial. Experiments with mice and in vitro cell tant platform for the LPL-mediated processing of CMs in culture studies suggest that uptake with LDL receptors capillaries and that the differences in GPIHBP1 expres- occur rapidly (t1/2 ~10 mins) while the second LDL receptor sion could play a role in regulating the delivery of lipid independent pathway occurs less rapidly (t1/2 ~60 mins) nutrients to different tissues. The high affinity binding of [83, 90]. LPL to GPIHBP1 implies that GPIHBP1 can tear LPL from It has been suggested by other authors that in the endothelial heparin sulphate proteoglycans (HSPG) [80]. absence of LDL receptors other receptors such as the low Levels of expression of GPIHBP1 in liver are low, which density lipoprotein receptor-related proteins (LRP) may likely contributes to poor hepatic clearance of CMs until contribute to chylomicron remnant uptake, though LDL triglyceride hydrolysis transforms them into remnant receptors account for the majority of chylomicron remnant lipoproteins [81]. uptake under normal physiological circumstances [91]. The LRP is larger than and structurally similar to other members of the LDL receptor gene family. Whereas the Cellular membrane receptors and chylomicron LDL receptor appears to function solely in lipoprotein metabolism metabolism, the LRP appears to have several known The mechanism of hepatic clearance of chylomicron rem- ligands. It has been suggested that LRP may function as nants remains controversial. The particles first may be a multifunctional scavenger receptor, with a major func- sequestered in the liver perisinusoidal space (space of tion in the removal of proteinase and proteinase inhibitor Disse) where they may undergo further processing by HL complexes [92]. A role for LRP in lipoprotein metabolism and LPL, both of which are detectable in the space of Disse. has been further suggested by the fact that LRP binds not A group of hepatic lipoprotein receptors then endocytose only chylomicron remnants but also lipases [93]. Studies the lipoproteins leading to their lysosomal catabolism. in mice suggest that in an apoE deficient model, in which Multiple receptors clearing triglyceride rich remnants are LRP1 (the largest member of the low density lipoprotein thought to exist. Several authors have implicated HSPGs receptor family) mediated clearance of lipoprotein par- as receptors for lipoprotein remnants [82, 83]. Each HSPG ticles is impaired, further LRP1 dysfunction drives the consists of a polypeptide strand called the core protein, system to compensate by upregulation of LDR receptor onto which highly negative carbohydrate polymers called expression [94]. Binding studies suggest that apoAV inter- heparin sulphate are assembled. The carbohydrate side acts with two members of the LDL receptor family, LRP chains of hepatic HSPGs exhibit structures of high flexibil- and mosaic type-1 receptor, SorLA. Association of apoAV ity and negative charge that can increase ligand affinity with LRP or SorLA resulted in enhanced binding of human and direct lipoprotein remnants to the liver. The synde- CMs to receptor covered chips, leading to the hypothesis can-1 HSPG has been suggested as a major HSPG recep- that apoAV may influence lipid homeostasis by enhancing tor for remnant lipoproteins in the liver [84]. Several lipid receptor-mediated endocytosis of CMs [95, 96]. binding proteins such as LPL [85], HL and apoE [86] can bind to HSPGs and facilitate hepatic remnant clearance. The highly sulphated HSPGs in the liver result in nega- Very low density lipoproteins (VLDL) tive charges that can bind LPL and apoE. It is postulated that apoE interacts initially with HSPG prior to transfer to VLDL assembly receptors for internalization [86]. Early studies suggested that the metabolism of chy- Intestinal CMs and liver-derived VLDL represent the two lomicron remnants is severely impaired in patients with classes of triglyceride rich lipoproteins responsible for the familial hypercholesterolemia (FH) who lacked function- transfer of lipids to other cells of the body. CMs mediate ing LDL receptors. CMs require four LDL receptors for the transport of dietary lipids whereas VLDL delivers binding compared with one receptor per LDL particle [87]. endogeneous lipids to peripheral tissues. The production Ramasamy: Lipoprotein metabolism 1703 of VLDLs needs to be synchronized with their secretion the cytosolic surface of the ER can ingest ubiquitinylated to avoid adverse consequences such as hepatic steatosis apoB as it emerges in a retrograde manner from the trans- and higher concentrations of VLDLs in the blood. There locon. It is still unclear how full length glycosylated apoB, are three main sources that supply free fatty acids to the which is a large protein, can cross ER membrane into the liver: 1) free fatty acids from adipocytes; 2) chylomicron cytosol [102]. In addition, apoB interacts with an ensem- remnants; and 3) the intestine via the portal vein [97]. ble of molecular chaperones during and after transloca- Mobilized lipid storage pool in the liver, de novo synthe- tion. Molecular cytosolic chaperones such as Hsp70 and sis of fatty acids and phospholipids contribute to hepatic Hsp90 participate to varying degrees in the degradation VLDL synthesis. Phosphatidate phosphatase-1 converts of almost every ERAD substrate examined and it has been phosphatidate to diacylglycerol and plays a role in the bio- suggested that both are required for the interaction of synthesis of phospholipids and triacyl glycerol [98]. apoB with the proteasomal pathway [103]. In contrast, the Hsp110 chaperone protects apoB from degradation [104]. Further studies implicate the ER chaperone glucose-reg- Apolipoproteins and VLDL synthesis ulated protein/binding immunoglobulin protein (Grp78/ BiP) in ERAD of apoB100 [105]. It has been suggested that ApoB100 is a large hydrophobic protein which is the main cotranslational degradation of apoB may be initiated by structural component of VLDL and LDL. Models of the strong binding of BiP to the N-terminus followed by asso- secondary structure of apoB100 contain three α helical ciation of the AAA-ATPase, p97 with the C-terminus [106]. domains (1%–22%, 48%–56% and 89%–100% of full ERAD is not the only mechanism used to regulate length of apoB100) and two long β-sheet domains (22%– apoB. Two other processes have been suggested. Fisher

48% and 56%–89%) (NH2-βα1-β1-α2-β2-α3-COOH) [99]. et al. [107] demonstrated that the post-translational deg- Other studies have indicated that the first 17% of the N-ter- radation of apoB100 also occurs by a previously unknown minal end of apoB100 has the LPL binding domain, that mechanism, in a compartment separate from the ER, the domain between 66%–83% of the full length apoB100 known as post-ER presecretory proteolysis (PERPP). is the LDL receptor binding domain, and the sequences PERPP stimulation induces aggregation of apoB con- between 1% and 5.8% and between 9% and 16% are the taining lipoproteins which are then sorted to degrada- binding sites for MTP [100]. tion through the autophagy pathway. During autophagy If MTP and lipid availability is adequate, nascent damaged organelles, cytosolic proteins and protein apoB100, like apoB48 and other secretory proteins is aggregates are engulfed by double-membraned vesicles translocated efficiently across the ER membrane. There it (autophagosomes) which deliver their cargo for degra- can be assembled with lipids into VLDL in the ER lumen dation to the lysosomes [108]. It has been suggested that and secreted into the medium. If instead, there is inad- ERAD destroys apoB when triglycerides are limiting but equate lipid or MTP, cotranslational translocation of PERPP occurs when triglycerides are normal. Dietary pol- apoB100 is inefficient and newly synthesized apoB100 yunsaturated fatty acids (particularly the n-3 fatty acids can be degraded. It is believed that the complex structure enriched in fish oils) lower VLDL levels. The stimulation of apoB100 plays a role in its post-translational degrada- of PERPP by major polyunsaturated fatty acids appears tion, which is a predominant way in which the secretion of selective for apoB incorporated into VLDL particles [109]. apoB lipoproteins is controlled. In contrast to other secre- The second process has been demonstrated to occur in tory proteins, the stepwise translocation of apoB results vitro. Williams et al. [110] identified an unstirred water in the transient exposure of large domains to the cyto- layer around the plasma membrane from which apoB con- plasm as assayed by exogeneous proteases. It has been taining lipoproteins are taken up via the LDL receptor. In suggested that transient intermediates of nascent apoB addition, it has been suggested that the LDL receptor is are paused during their passage across the bilayer of ER. involved in hepatic VLDL assembly and that it promotes Both pause transfer sequences [101] and the presence of post-translational degradation thus interfering with β-sheet domains [100] have been indicated as influencing secretion of VLDL from the liver. This suggests a gatekeep- the translocation efficiency of apoB. Both the incomplete ing function for the receptor [111, 112]. nascent apoB polypeptides and full length glycosylated The current model of VLDL (and chylomicron) assem- apoB protein have been shown to be degraded by the ubiq- bly is in two steps. In the first step newly synthesized apoB uitin-proteasome system in the cytosol. The proteasome is lipidated during its translocation across the ER into is a multicatalytic machine that destroys ER-associated the lumen yielding the primordial apoB particle. In the degradation (ERAD) substrates. Proteosomes located at second step bulk transfer of corelipids from ER lumenal 1704 Ramasamy: Lipoprotein metabolism

lipid droplets to the primordial apoB particle is thought ER to take place post-translationally. Hepatic VLDL assembly and secretion is also influenced by de novo biosynthesis TG/PL/Chol of phospholipids and triacylglycerol [113]. Lipid droplets MTP were long considered simple storage lipid depots, but Pre- are now thought of as dynamic cellular organelles [114]. VLDL Various cytosolic lipid droplet-associated proteins, (per- Molecular chaperones lipin2, CideB and CGI-58) contribute to lipid metabolism, but the exact mechanism remains to be elucidated [115]. It is suggested that both CMs and VLDL utilize differ- ent vesicles and attendant mechanisms to be transported Proteosomal degradation from ER to the cis-Golgi. Unlike CMs, VLDLs depart from the ER in a COPII dependent fashion [116]. Furthermore, it is suggested that VLDL exits the ER as a poorly lipidated VLDL transport vesicle LDL-sized particle, and thus smaller vesicles are able to accommodate immature VLDL [117]. Although it is estab- Golgi bodies lished that the first step of VLDL assembly occurs in the ER, the location of additional steps in VLDL maturation Autophagy by PERPP is less clear. Several studies have suggested that ER is Secretion the final site of VLDL maturation to VLDL2 (Sf 20–100) or

VLDL1 (Sf > 100) [118], whereas others have implicated the Golgi complex as a second site of maturation [119]. The VLDL transport vesicle fuses with liver cis-Golgi to deliver VLDL to the Golgi lumen. The components of the SNARE complex that play a role in docking and fusion of the VLDL transport vesicles have been identified as Sec22b (vesicle SNARE), syntaxin 5, rBet1 and Gos28 (target mem- brane SNARE) [120]. The ER to Golgi movement of VLDL is prerequisite for their ultimate secretion from hepatocytes Figure 3 Biosynthesis of VLDL. (Figure 3). Biosynthesis of VLDL starts when apoB100 is translocated to the Although other functions have been characterized for lumen of the ER and interacts with MTP. In the absence of lipids and the VLDL-associated apolipoproteins apoE, apoCIII and MTP, apoB100 is targeted for proteosomal degradation. MTP driven lipidation gives rise to pre-VLDL particles. Interaction with molecu- apoV, studies have implicated these apolipoproteins in lar chaperones enhance/modify apoB100 degradation. Pre-VLDL is the assembly and secretion of the fully lipidated VLDL, transported to the Golgi apparatus in a COPII dependent fashion. independent of their role in lipoprotein clearance [35, Pre-VLDL is converted to VLDL in the Golgi prior to secretion. Pre- 63, 121]. Experimental evidence suggests that apoE may VLDL which is not converted to mature lipoprotein are sorted out facilitate the early stage of VLDL assembly in the ER and for autophagy through PERPP. Chol, cholesterol; PL, phospholipid; TG, triglycerides. apoCIII plays a role in the post-ER state of the assembly process. The mechanism by which apoAV can attenuate the production or secretion of VLDL is still unclear [122]. Studies with transgenic mice suggest that by binding to the LDL receptor, apoE and apoB100 containing lipids are Catabolism of VLDL cleared from the plasma [123].

Once in the plasma VLDL is hydrolyzed by LPL to generate smaller denser particles and subsequently intermediate LDL receptor and apolipoproteins density lipoproteins (IDL). During lipolysis these remnants become enriched with HDL-derived apoE, a high affinity Plasma apoE originates primarily from the liver and, to a ligand for LDL receptor. IDL particles can undergo further small but functionally significant extent, macrophages. catabolism by LPL to become LDL with loss of apoE. The ApoE contains 299-amino acid residues. The three iso- sole remaining protein apoB100 binds to LDL receptors. forms, apoE2, apoE3 and apoE4 differ only at positions 112 Ramasamy: Lipoprotein metabolism 1705 and/or 158. ApoE3 contains a cysteine at position 112 and generated within the lysosome suppresses 3-hydroxy- an arginine at 158, while apoE2 contains cysteine at both 3-methylglutaryl coenzyme A reductase (HMG-CoA) activ- sites and apoE4 contains arginine at these sites. ApoE3 ity, the enzyme which catalyzes the rate limiting step in is considered the wild type isoform in humans because cholesterol production. Expression of LDL receptor is of its high allelic frequency and lack of human disease regulated at both transcriptional and post-transcriptional phenotype [124]. It was proposed that apoE contains two levels which influence synthesis, LDL receptor stability, domains an N-terminal domain (a four-helix bundle) and endocytosis and trafficking. LXR are nuclear receptors C-terminal domain linked by a flexible hinge region. The that are activated in response to cellular cholesterol levels. N-terminal is responsible for LDL receptor binding but They inhibit cholesterol uptake by inducing the expres- in isolation binds to lipids. The C-terminal possesses the sion of the inducible degrader of the LDL receptor (IDOL) major lipoprotein binding sites. X-ray crystallographic an E3 ubiquitin ligase that mediates ubiquitination and structure revealed a salt bridge between Arg158 and degradation of the LDL receptor [129]. The IDOL pathway Asp154 in apoE3 that is absent in apoE2. Further in apoE2 appears to be active in macrophages, adrenals, intes- an alternative salt bridge forms between Arg150 and tine and liver [130]. IDOL contains two distinct domains: Asp154 effectively eliminating the availability of Arg150 a C-terminal RING E3 ligase domain and an N-terminal for interaction with the LDL receptor. Lipid association FERM (Band 4.1, ezrin, -radixin-moeisin) domain which is required for biologically active apoE to bind to the LDL is responsible for target recognition [131]. Receptor bound receptor. It is generally thought that the protein undergoes LDL is internalized by endocytosis. Rudenko et al. [132] a lipid binding-induced conformational change, though crystallized the three dimensional structure of the extra- the ultimate conformation adopted by apoE remains cellular domain at an acidic pH. At neutral pH the ligand unsolved [37]. Studies with point mutants suggested that binding repeats are predicted to be extended away from the receptor binding region lies in the vicinity of residues the EGF domains and accessible to lipoproteins. When the 136–150. However, residues outside the receptor binding pH falls as occurs in the endosome, the ligand binding region can also affect receptor binding activity. Mutagen- domain forms a physical association with the EGF pre- esis studies suggested that Arg172 contributed to receptor cursor homology domain. The acid dependent conforma- binding activity [125]. The major role for apoE is to func- tional change in the LDL receptor releases the lipoprotein tion as a ligand for cell surface receptors. As a player in from the ligand binding domain and signals the recep- lipoprotein metabolism, apoE binds to LDL receptor, LRP1 tor to return to the cell surface. When cell cholesterol and the VLDL receptor [126]. increases the production of LDL receptors is reduced. In The two physiological ligands of LDL receptor apoE mammals the cholesterol content of the cell membranes is and apoB100 bind to the ligand binding domain of the controlled by a family of membrane bound transcription LDL receptor which consists of seven repeats of approxi- factors designated as sterol-regulated transcription pro- mately 40 amino acid cysteine rich tandem repeats. The teins (SREBPs). In cholesterol-depleted cells the SREBPs ligand binding domain is located at the N-terminus of the are escorted by SREBP cleavage activating proteins (SCAP) protein and is followed by the epidermal growth factor in budding vesicles to the Golgi complex where they are (EGF) precursor domain. The EGF precursor domain processed by two proteases (site-1 protease and site-2 pro- contains two cysteine rich EGF domains of 40 amino tease) to release a fragment that enters the nucleus and acids (EGF-A and EGF-B) separated from a third EGF- activates transcription of multiple target genes includ- repeat (EGF-C) by a 280-amino acid β-propeller domain. ing those encoding HMG-CoA reductase and all other Immediately downstream of the EGF precursor homol- enzymes of cholesterol biosynthesis as well as the LDL ogy is a threonine and serine rich region to which mul- receptor. SREBPs are weak activators of gene expression tiple O-linked sugars are attached which is followed by by themselves and function in a synergistic manner with the transmembrane domain and a relatively short cyto- more generic transcriptional co-regulatory factors. When plasmic tail that contains all the sequences required for LDL-derived cholesterol enters the cells, SCAP senses the receptor clustering in clathrin-coated pits and for the excess cholesterol through its membranous sterol sensing internalization of the receptor [127, 128]. domain, changing its conformation so that SCAP/SREBP LDL receptors are gathered in clathrin-coated pits. complex is no longer incorporated into ER transport vesi- Receptor bound LDL remained on the surface of cells cles [133, 134]. SREBP-1c and SREBP-2 are the predominant for < 10 min and once in the cell the protein component of isoforms of SREBP in liver and most other intact tissues. LDL was digested to amino acids within 60 min. Lysosomal SREBP-1c favors the fatty acid biosynthetic pathway and digestion of LDL releases amino acids and the cholesterol SREBP-2 favors cholesterolgenesis [135]. Using IDOL-null 1706 Ramasamy: Lipoprotein metabolism cells Scotti et al. [136] suggest that the LXR-IDOL pathway EGF-A domain suggests binding site for EGF-A domain lies was activated more rapidly in response to sterol con- in the catalytic domain of PCSK9 [144]. Binding of PCSK9 centrations which suggested a role for IDOL in an acute to EGF-A was calcium dependent and increased dramati- response to changing cholesterol levels. Micro-RNAs cally with reduction in pH from 7 to 5.2. The data suggest (miRNAs) are short 21–24 nucleotide long, non-protein a model in which the binding of PCKS9 to EGF-A interferes coding RNAs that are important post-transcriptional reg- with an acid dependent conformational change (i.e., at ulators of gene expression. By binding to the 3′ untrans- the acidic pH of endosomes) required for receptor cycling. lated region of the protein-coding mRNA transcripts As a consequence the LDL receptor is rerouted from the they can reduce translation from these transcripts and in endosome to the lysosome where it is degraded [145]. A some cases lead to their degradation. The genetic locus second binding site between the positively charged C-ter- of SREBP2 and SREBP1 contain highly conserved miRNAs minal domain of PCSK9 and the negatively charged ligand that regulate cholesterol export and fatty acid oxidation. binding site of the LDL receptor with a much greater Two miRNAs, miR-33a and miR-33b are encoded within binding at pH 5.4 has been identified. This constitutes a introns of SREBP2 and SREBP1, respectively. miR-33 second step driving the LDL receptor towards degrada- reduces cholesterol export by directly targeting the tran- tion [146, 147]. Association of PCSK9 with LDL particles in scripts of the ABCA1 protein and reduces β-oxidation plasma lowers the ability of PCSK9 to bind to cell surface by directly targeting transcripts for the β-subunit of the LDL receptors, blunting PCSK9-mediated LDL receptor mitochondrial trifunctional protein (hydroxyacyl-coen- degradation. An N-terminal region of the PCSK9 prodo- zyme A dehydrogenase/3-ketoacyl-coenzyme A thiolase/ main (amino acid residues 31–52) was required for binding enoyl-coenzyme A hydratase β-subunit), the liver-specific to LDL in vitro [148]. An endogenous inhibitor annexin isoform carnitine palmitoyltransferase 1A and the carni- A2 interacts with the C-terminal of PCSK9 preventing tine O-octanoyltransferase [137]. PCSK9 from interacting with LDL receptor [149]. It is likely Propertin convertase subtilisin/kexin type 9 (PCSK9) that there are other key tissue-specific protein partners promotes degradation of LDR. PCSK9 is mainly expressed which would allow for different levels of LDL receptor in in liver, with lower levels of expression in the kidney, intes- tissues. The mechanism by which an LDL receptor with tine and brain [138] and is present in human plasma [139]. bound PCSK9 is excluded from the recycling tubules of PCSK9 is a 692-amino acid glycoprotein that contains a the sorting endosome and is rerouted to the lysosomes 22-residue signal sequence followed by a prodomain (resi- is not determined. LDL receptors that bind PCSK9 at the dues 31–152) and a catalytic domain (residues 153–454) cell membrane are internalized by the clathrin dependent that shares structural homology with the proteinase K endocytic pathway, but fail to enter the recycling pathway. family of subtilisin-like serine proteases. The cysteine The PCSK9-LDL receptor pathway is routed to the lys- and histidine rich C-terminal domain consists of residues osomes via a pathway that does not require ubiquitina- 455–692 which are three tightly packed modules named tion of the cytoplasmic tail of the receptor and does not M1, M2 and M3. PCSK9 undergoes autocatalytic cleav- involve the proteosomal or autophagy systems [150]. Two age in the ER that severs the covalent attachment of the propertin convertases, furin and PC5/6A, cleave PCSK9 prodomain with the catalytic domain. Nevertheless, the at the Arg218-Gln219 peptide bond, though it is controver- prodomain remains tightly associated with the catalytic sial whether cleavage affects PCSK9 function [139, 151]. domain as PCSK9 moves through the secretory pathway In addition a further protein, the LDL receptor adaptor [140]. PCSK9 gene transcription is under the control of protein may affect LDL receptor stability or recycling [152]. SREBPs and a highly conserved hepatocyte nuclear factor Yamamoto et al. [153] were the first to clone and 1 binding site has been identified as a sequence motif for characterize VLDL receptor cDNAs from rabbit heart and PCSK9 transcription. When cholesterol is depleted SREBP human macrophage cells [154]. VLDL receptor mRNA are is activated increasing the expression of LDL receptor and highly abundant in heart, muscle, adipose tissue and PCKS9 genes at the same time [141]. In cultured cells the brain and are barely detectable in the liver where LDL addition of recombinant PCSK9 to the medium results in receptors are expressed abundantly, suggesting that this LDL receptor degradation, providing evidence that PCSK9 receptor may be responsible for the entry of triglyceride can promote the degradation of LDL receptor by acting on rich lipoproteins into muscle and fat. In contrast to the the cell surface [142]. The proteolytic activity of PCSK9 is LDL receptor the VLDL receptor binds apoE2 as well as not required for PCSK9 action on the LDL receptor [143]. apoE3 particles and its expression is not down regulated PCSK9 binds specifically to the EGF-A domain. Study of by intracellular lipopoproteins. In vitro and in vivo studies the crystal structure of the complex between PCKS9 and have shown that the VLDL receptor binds VLDL but not Ramasamy: Lipoprotein metabolism 1707

LDL and functions as a peripheral lipoprotein receptor for stabilized by three intramolecular disulfide bonds and are VLDL. The VLDL receptor functions in concert with LPL found in plasminogen, prothrombin, tissue plasminogen and apoE [155]. Niemeier et al. [156] showed that the VLDL activator and a variety of proteases involved in blood coagu- receptor mediated the uptake of chylomicron remnants lation and fibrinolysis. The kringle domains of apo(a) con- and that the uptake was increased by the addition of LPL tains 11 kringle types, the last of which shares 85% amino and apoE. In the adipocyte, PPAR-γ regulates an array of acid homology with plasminogen kringle 5, the other 10 genes involved in lipid retention in adipose tissue. In vitro kringle types are similar but not identical to each other studies suggest that VLDL receptor expression is upregu- and share 78%–88% homology with plasminogen kringle lated by PPAR-γ [157]. 4, called kringle 4 types 1–10. Kringle 4 type 1 and kringle Following hydrolysis, the intracellular fate of VLDL 4 types 3–10 are present as a single copy in apo(a) parti- and CMs is more complex than the degradation of LDL. In cles, whereas kringle 4 type 2 is present in multiple copies, contrast to the lysosomal degradation of LDL-derived apoB, the number varying from 3 to 40. The varying number of Heeren et al. [158] showed that triglyceride rich lipopro- kringle 4 type 2 repeats is a major determinant of apo(a) tein-derived apoE followed a recycling pathway. Analysis molecular weight heterogeneity. Apo(a) size is inversely of secreted proteins identified 80% of recycled triglyc- correlated with Lp(a) levels, with subjects carrying small eride rich lipoprotein proteins in the HDL fraction [159]. apo(a) isoforms having increased apo(a) secretion [163]. Other studies suggest that at least a portion of the apoE Metabolic studies have shown that inverse association is present in the Golgi apparatus and is rerouted through between Lp(a) levels and apo(a) isoform size is due to dif- the secretory pathway, and that apoE deficient VLDL may ferences in hepatic secretion of apo(a) with subjects car- serve as an acceptor [160]. Recycling of apoE may be medi- rying small apo(a) isoforms having an increased apo(a) ated by Golgi-derived secretory vesicles or apoE may be secretion [164]. There is profound interindividual varia- resecreted from peripheral endosomal compartments. tion in Lp(a) levels for a given apo(a) size suggesting that In human hepatoma cells, triglyceride rich lipoprotein factors other than gene size predict size levels. A pentanu- derived apoE colocalizes with apoAI in endosomes. The cleotide repeat sequence (TTTTAn) ≈1 kb upstream of the authors suggest that apoAI may be targeted to endosomes LPA gene was found to affect Lp(a) levels [165]. In order to containing apoE/cholesterol complexes to promote recy- correlate the role of apo(a) gene polymorphisms to apo(a) cling and efflux. Further, these findings may indicate that gene regulation, it has been proposed that liver-specific apoE recycling provides an efficient mechanism to re-sup- transcriptional activators and repressors might contribute ply plasma with apoE containing HDL particles [161]. Dis- to the differential expression of apo(a) gene, in an individ- similar recycling of apoE isoforms could contribute to the ual-specific manner [166]. Both transcriptional and post- development of apoE4-associated diseases [162]. Studies transcriptional mechanisms may play roles in regulation suggest that apoE directs the intracellular routing of inter- of Lp(a) levels. Boerwinkle et al. [167] suggest that 69% nalized remnant lipoproteins, with the smaller VLDL par- of the variability in plasma Lp(a) levels is accounted for ticles directed to the perinuclear regions of the mouse by the number of apo(a) kringle 4 type 2 repeats and an macrophage whereas the larger remnants remain closer to additional 22% by other sequences within the gene coding the plasma membrane [160]. for apo(a). In addition to apo(a) and apoB100, Lp(a) par- ticles also contain apoAI, apoE and traces of apoAII [168]. Single nucleotide polymorphisms in the LPA gene locus and repeat polymorphisms in the promoter region of the Lipoprotein(a) LPA gene which increase Lp(a) levels have been shown to be associated with increased CHD risk [169, 170]. Further, Lipoprotein(a) [Lp(a)] consists of an LDL-like lipoprotein the risk for CHD increased continuously with a decreas- bound to a plasminogen-like glycoprotein, apo(a) which ing number of kringle 4 repeats [171]. However, although is disulfide linked to apoB. Levels of Lp(a) may vary thou- Lp(a) plasma levels are inversely correlated to the size sand-fold among individuals and levels are partly deter- of the apo(a) protein, the relative contribution of size mined by polymorphisms in the LPA gene coding for the (number of kringle repeats) and Lp(a) levels to CHD risk apo(a) moiety of Lp(a). Apo(a) is composed of multiple is not known. In contrast to other lipoprotein levels, Lp(a) kringle containing domains and a serine protease domain. levels are largely mediated by the LPA gene with small LPA gene which encodes apo(a) is thought to have been to negligible effects of diet [172]. Numerous studies have generated by duplication of the neighboring plasminogen found an association between high Lp(a) concentration gene. Kringles are triple-loop structural motifs that are and CHD risk. Further confirmed by Lippi et al. [173] who 1708 Ramasamy: Lipoprotein metabolism reviewed epidemiologic evidence linking Lp(a) with CVD body is transported to the liver and converted to bile acids and venous thromboembolism. The increased risk of CHD that are extensively used in the enterohepatic circulation. associated with high Lp(a) was independent of other HDL particles mediate the transport of cholesterol from established risk factors for CHD [174–177]. Risk estimation peripheral tissues to the liver in a process termed reverse however, may be higher in subjects with elevated Lp(a) cholesterol transport [188]. Excess accumulated choles- and other cardiovascular risk factors [178]. terol is toxic to cells. As in humans, cells other than those Pathways regulating the synthesis and degradation of in steroidogenic tissues and the liver cannot metabolize Lp(a) are not well understood. Early studies suggest that cholesterol, reverse cholesterol transport may reduce human hepatocytes secrete the apo(a)-apoB100 complex intracellularly accumulated cholesterol and contrib- [179]. Other studies suggest that apo(a) is synthesized ute to cholesterol homeostasis at the cellular and whole by hepatocytes and associates with LDL after secretion body level. About 70% of the total HDL protein is apoAI, to form Lp(a) in the sinusoids of the liver. Lp(a) assem- although it contains a variety of other proteins. The liver bly involves an initial non-covalent interaction between and intestine synthesize and secrete apoAI into the circu- amino acids 680–780 of apoB100 and kringle 4 types 5–8 lation as a lipid free or poorly lipidated protein [189]. Thus followed by formation of a disulfide bond [180]. Cys4326 of lipid free apoAI is released from the cells and interacts APOB has been shown to be required for the covalent link with cellular ABCA1 for assembly of HDL particles. HDLs between apo(a) and apoB100 [181]. It has been suggested are heterogeneous in size and composition [27]. Mass that the liver is the major organ of clearance of Lp(a) and spectrometric analysis of HDL confirmed the presence of that apo(a) moiety plays a role in the plasma clearance of several apolipoproteins [AI, AII, AIV, B, (a), CI, CII, CIV, Lp(a). ApoE and the receptors that mediate the binding D, E, F, H, J, LI and M] as well as proteins associated with and uptake of apoB100 containing lipoproteins have been inflammation, clotting, complement regulation as well as proposed as having a role in Lp(a) catabolism. However, proteolytic enzymes. As the plasma abundance of most of the nature of the major receptor(s) that mediate the plasma these proteins is insufficient to allow one copy per HDL clearance and tissue uptake of Lp(a) remains to be deter- molecule, it is likely that specific proteins are bound to mined [182]. Clearance of Lp(a) is not well understood, distinct HDL particles. This heterogeneity may translate but the LDL receptor or apo(a) size does not appear to be into distinct functionality of HDL subclasses [190, 191]. the main determinant of clearance [183]. A putative physi- ApoAII is the other major apolipoprotein in HDL. Early ological role of Lp(a) remains to be identified. Apo(a) has studies suggested that the proportion of apoAI and apoAII potent lysine binding domains, similar to those on plas- apolipoproteins varied in different density subfractions minogen and binds to damaged endothelial cells. In vitro of HDL: particles containing apoAI and apoAII in nearly studies have identified domains in apo(a) that mediate constant ratio and particles containing no apoAII [192]. inhibitory effects on fibrin clot lysis [184]. The mechanisms In mice approximately 30% of steady state plasma HDL is by which Lp(a) increases CHD risk is not well defined but contributed by the intestine [193]. may include a prothrombotic effect due to the similarity of apo(a) to the fibrinolytic enzyme plasminogen, and the atherogenic effect mediated by the preferential binding ABCA1 and HDL assembly of oxidized phospholipids by Lp(a) as well Lp(a) deposi- tion in the arterial wall [185, 186]. Animal models suggest Different steps that control the delivery and disposal of that the proteolytic breakdown products of apo(a) retain cholesterol are regulated by membrane transporters of the anti-angiogenic and anti-tumoral properties, which would ABC superfamily. The human genome contains 48 distinct suggest a beneficial effect of this protein [187]. ABC transporters that are grouped into seven subclasses labeled ABCA through ABCG. Structurally the ABCs fall into two groups: whole transporters having two similar structural units joined covalently and half-transporters High density lipoprotein (HDL) of single structural units that form active heterodimers or homodimers. Four members of this family have been Biosynthesis of HDL and reverse cholesterol shown to have a major impact on lipoprotein metabolism: transport ABCA1 is a 2261-amino acid integral membrane whole transporter protein that mediates the export of choles- Cholesterol is an important constituent of cell membranes terol and phospholipids to lipid poor lipoprotein; ABCG1 and is a precursor of steroid hormones. Cholesterol in the a homodimeric half transporter that mediates cellular Ramasamy: Lipoprotein metabolism 1709 transport cholesterol transport to lipidated lipoprotein poor apolipoprotein in the extracellular fluid is more rel- particles and ABCG5 and ABCG8 form heterodimers that evant for ABCA1 efflux (Figure 4). restrict intestinal absorption and promote biliary excre- tion of sterols [194]. In intestinal cells, ABCA1 has been shown to regu- Apolipoprotein AI and HDL assembly late the efflux of cholesterol from the basolateral but not the apical membrane [195]. ABCA1 is expressed only on Human apoAI is a 243-amino acid protein, the first 43 the basolateral surface of the hepatocytes [196]. Studies residues are encoded by exon3 and the 44–243 region by suggest that dietary/biliary and newly synthesized cho- exon4. Residues 44–243, are composed of eight 22 amino lesterol contribute to the origins of intestinal HDL-choles- acid and two-11 amino acid repeats that are predicted to terol [197]. form amphipathic α-helices. It has been suggested that The most critical step of ‘reverse cholesterol transport’ α-helix formation especially in the C-terminal region is the release of cholesterol from cells, and the reduction provides the energy source necessary for the high affin- of cellular cholesterol. The ACAT reaction and cholesterol ity binding of apoAI to lipids [203]. Each of the 10 helical release are active systems to protect the cells from mem- repeats has one face enriched in hydrophobic amino acids brane toxic excess accumulation of free cholesterol. The and the opposing face enriched in negatively charged same source of cholesterol is the preferred substrate for amino acids. The area between the hydrophilic and hydro- ACAT- and ABCA1-mediated lipid secretion. Cholesterol phobic faces of the helix is enriched in positively charged enrichment in HDL can be regulated by the modulation of amino acids, usually lysine and arginine. This structure is cellular factors. Both ACAT and PKC activities are involved thought to be suited to lipid binding. The N-terminus resi- in regulating the rate of mobilization of ABCA1-mediated dues (1–187) form a globular domain, while the C-termi- cholesterol release by apoAI. PKC inhibitors and activa- nus (188–243) consists largely of a series of amphipathic tors modulate both cholesterol content in the HDL gener- α-helices [204]. Several studies including an analysis of ated by the apolipoprotein-cell interaction and the change the crystal structure of apoAI suggest that apoAI consists in ACAT accessible cholesterol pool in certain cells under of two helical domains: a four-helix antiparallel bundle certain conditions [198]. formed by the N-terminal three quarters of apoAI and a The generation of new HDL particles is mediated by two-helix bundle adopted by the C-terminal end [205]. The the interaction of helical apolipoproteins with cellular central region corresponding to residues 121–186 is impor- ABCA1. The efficiency of reverse cholesterol transport tant for the activation of the enzyme LCAT [206]. In con- depends on the specific ability of apoAI to promote cho- trast to the crystal structure which suggests 80% helical lesterol efflux, bind lipids, activate lecithin cholesterol content, hydrogen exchange and mass spectrometry indi- acyl transferase (LCAT) and form mature HDL that inter- cate a helical content of approximately 50% [207]. It is acts with specific receptors and lipid transfer proteins. As likely that apoAI structure is dynamic (molten globular) HDL particle shape can affect the activities of HDL remod- and its relative instability may explain the ability of apoAI eling and activities of LCAT and cholesteryl ester transfer to unfold and remodel during HDL formation and matura- protein (CETP) understanding apoAI spatial arrangement tion. However, in true solution the first 98 residues of the on disc and sphere HDL is important. Early studies sug- N-terminus are probably not folded into the helical struc- gested that free apolipoproteins, apoAI, apoAII and apoE ture apparent in the crystal form [208]. Saito et al. [38] pro- can interact directly with macrophages preloaded with posed a two-step mechanism for lipid binding of apoAI: cholesteryl ester in culture to form HDL-like particles in an initial lipid binding step occurs through the flexible the medium [199]. Other exchangeable apolipoproteins carboxyl-terminal domain followed by a major conforma- including apoCII, apoCIII and apoE have been shown to tional reorganization of the amino-terminal helix bundle, stimulate ABCA1-mediated lipid efflux [200]. On the basis converting the hydrophobic helix-helix interactions to of studies using synthetic peptide mimics of apolipopro- helix-lipid interactions. In humans the monomeric form teins it appears that the key structural motif necessary of apoAI has been hypothesized as the predominant form for an apolipoprotein to function as a lipid acceptor is of apoAI in plasma and that ABCA1-mediated lipid release the presence amphipathic helices [201]. Studies in mice is reduced when apoAI is in a self-associated state [209]. suggest that in addition to its role in clearing TG rich Although it is known that the interaction of apoAI lipoproteins apoE participates in the biogenesis of apoE with functional ABCA1 results in the lipidation of apoAI containing HDL particles with the participation of ABCA1 and the formation of HDL the mechanism of this inter- [202]. It is likely, however, that the concentration of lipid action is not resolved. Some studies have pointed to the 1710 Ramasamy: Lipoprotein metabolism

Peripheral tissue

ABCA1 ABCG1 SRBI

ABCG5/8 Bidirectional ABCA1 ApoAI Cholesterol flux apoAI Cholesterol efflux ABCA1 ApoAI Discoidal HDL

ABCG5/8 LCAT Cholesterol ABCG1 ACAT Cholesteryl ester

SRBI Intestine

Spherical HDL Liver

ApoAI CETP/PLTP

LPL/HL LDL

VLDL

Figure 4 Biosynthesis of HDL. Biosynthesis of HDL begins with the interaction of lipid poor apoAI with ABCA1. Lipidated apoAI is the major acceptor for SR-BI and ABCG1 pathways. CETP promotes the transfer of cholesteryl esters from HDL to VLDL and LDL. PLTP transfers phospholipid between VLDL and HDL and remodels HDL into larger and smaller particles with the dissociation of lipid poor or lipid free apoAI. Solid arrows indicate cholesterol/ cholesteryl ester transfer.

importance of apoAI/ABCA1 interactions whereas others It is now accepted that ABCA1 is expressed in mul- have postulated apoAI/lipid interactions for the overall tiple cells and tissues, in the liver, macrophages, brain binding process. Other studies suggest two binding sites and various other tissues [213, 214]. The ABCA1 protein for apoAI are created at the surface of cells containing contains two transmembrane domains of six α-helices functional ABCA1. Cross linking studies suggest that apart and two intracellular nucleotide binding domains. Its from ABCA1, apoAI does not bind to any other cellular ABC consists of Walker A and Walker B motifs [215]. To protein, and that the other binding site for apoAI is a lipid maintain lipid homeostasis ABCA1 is tightly regulated site [210]. It has been suggested that the presence of a func- both transcriptionally and post-translationally. Expres- tional ABCA1 site leads to the formation of a major lipid sion of ABCA1 is regulated primarily by the LXR/retin- containing site for the binding and lipidation of apoAI. oid X receptor (RXR) system. The LXRs act as cholesterol The structural requirements for apoAI to associate with sensors and induce genes that protect the cell from cho- plasma membrane are unknown, though studies with lesterol overload [216]. The cloning and sequencing of the deletion mutants suggest that the C-terminal is required human ABCA1 gene [217] identified a cholesterol response for a productive complex with ABCA1 [211]. Apolipopro- element upstream of the transcriptional site of the human tein M (apoM) a 26 kDa protein has been hypothesized to ABCA1 gene [218]. Chinetti et al. [219] have reported that function catalytically to transfer lipid onto nascent HDL PPAR-α and PPAR-γ agonists induce ABCA1 mRNA expres- during or after their formation by ABCA1 [212]. sion and apoAI-mediated cholesterol efflux in normal Ramasamy: Lipoprotein metabolism 1711 macrophages. Cyclic AMP induces the ABCA1 gene tran- bundle domain and the hydrophobicity of the C-terminal scription in certain types of cells especially in macrophage domain are important determinants of both nascent HDL cell lines [220]. Calpain-mediated proteolysis is a further particle size and their rate of formation [231]. These dis- regulatory factor for ABCA1. ApoAI interacts with ABCA1 coidal particles are the major progenitors of the spherical before endocytosis inhibiting calpain-mediated proteoly- HDL particles that form the major fraction of circulating sis. Consequently, ABCA1 is recycled to the cell surface HDL in human plasma. In addition to the HDL particles without degradation. ApoAI therefore increases cell that each contain several apoAI molecules, some of the surface ABCA1 [221]. In one study, prevention of phos- apoAI in human plasma is present in a monomeric form. phorylation of ABCA1 threonine and serine residues by This species is called preβ1-HDL or lipid poor apoAI. PKC2-enhanced apoAI binding was observed [222]. Several These preβ1-HDL particles have been shown to react with intracellular proteins have been examined to determine ABCA1 leading to their conversion into larger discoidal whether they modulate ABCA1 activity. Of particular inter- particles [232]. est is caveolin-1, the main structural protein of caveolae, In the double belt model of discoidal HDL particles the cholesterol rich invaginated microdomains at the the apoAI molecules are packed around the edge of the surface of most peripheral cells. However, the effects of disc in an antiparallel orientation with their amphipathic caveolin-1 on cholesterol efflux are still controversial. The α-helical segments interacting with the phospholipid acyl overall effect is likely to be cell specific. Overexpression chains with an overall horseshoe shape [233]. This struc- of caveolin-1 in certain cells results in the enhancement of ture is consistent with crystal structures derived from N- cholesterol efflux [223, 224]. or C-terminal truncated apoAI molecules [234, 235]. Most Previous work suggested that a significant basal level of the effort to define lipidated apoAI structure has been of phopholipidation of apoAI occurs in the absence of directed at reconstituted/recombinant HDL which can be ABCA1 with both intracellular and peri-cellular lipidation reproducibly prepared in homogeneous size and composi- of apoAI [225]. ABCA1 was localized to both the plasma tion. Different models have been suggested for two apoAI membrane and endocytic compartments which cycle bound to recombinant HDL. There is considerable agree- between plasma membrane and other endocytic compart- ment between the different models, as all models suggest ments [226]. Models suggest that ABCA1 and apolipopro- that the central region of the bound apoAI assumes an tein containing vesicles endocytose to intracellular lipid antiparallel double belt with the amphipathic helix 5 of deposits where ABCA1 pumps lipids into vesicle lumen each strand adjacent to the same region of the second for release by endocytosis termed retro endocytosis [194]. molecule of apoAI. Differences between the models The relative contribution of cell surface versus intracellu- appear to involve interaction of the N- and C-terminal lar events in cholesterol efflux warrant further investiga- regions of the two monomers: 1) the solar flares model tion [227]. Recent work suggests that HDL biogenesis takes allows for regions of the apoAI to be more solvent acces- place on the cell surface rather than on endosomes [228]. sible; and 2) belt buckle model in which the N- and C-ter- A generally accepted model for the mechanism by which minal are closely associated but fold back to the central apoAI/ABCA1 interaction creates nascent HDL particles region of the apoAI belt [236]. Wu et al. [237] using small does not exist, though several models have been proposed. angle neutron scattering suggest an anti-parallel double One model suggests that membrane phospholipid translo- superhelix wrapped around an ellipsoidal lipid phase. It cation via ABCA1 induces bending of the membrane bilayer has been suggested that α-helices in apoAI in the lipid to create high curvature sites to which apoAI can bind and free and lipid-associated state, probably unfold and solubilize membrane phospholipid and cholesterol to refold. This dynamic behavior and structural flexibility of create nascent HDL particles [229]. The identification of apoAI facilitates the remodeling of HDL particles, which substrate specificity of ABCA1 and whether phospholipid is essential for their maturation and metabolism [238]. and cholesterol efflux are coupled will require additional Recently, a battery of molecular tools have been used to studies, but it is probable that the overall transport activ- refine the conformation of apoAI on HDL and a variety of ity of ABCA1 may be modulated by the availability of the physical chemical methods have been used to study recon- substrate in the plasma membrane [230]. stituted HDL and the results have been consistent with Nascent HDL particles are heterogeneous with the double belt model. Molecular dynamic simulations respect to size and lipid content. The major nascent HDL are consistent with salt bridge pairing and the ‘sticky’ particles comprise of discoidal particles containing two, N-terminal hypothesis. Both the crystal and molecular three or four apoAI molecules per particle. It has been dynamic simulation models suggest that pairwise helix suggested that the stability of the apoAI N-terminal helix repeats are uniquely designed to create a gap that exposes 1712 Ramasamy: Lipoprotein metabolism both acyl chains and unesterified cholesterol, resulting phospholipid in the form of minimal surface patches of in a LCAT presentation tunnel [239]. Sorci-Thomas et al. lipid bilayer [248]. During lipid metabolism apoAI moves [240] characterized apoAI containing nascent HDL par- between HDL and triglyceride rich lipoproteins [249]. ticles from ABCA1 expressing human embryonic kidney cells. They suggest that the smaller nascent HDL particles carried two molecules of apoAI and that larger choles- Cholesteryl ester transfer protein (CETP) and phospholipid terol rich nascent HDL particles contained three apoAI transfer protein molecules. The discoidal nascent HDL particles are the progenitors of spherical HDL2 and HDL3 particles that CETP is a 476-amino acid protein that mediates the trans- form the two major subpopulations of circulating HDL fer of neutral lipids including cholesteryl ester from HDL in human plasma. HDL2 is large, light, cholesteryl ester to VLDL and LDL in exchange for triglycerides, with sub- rich and HDL3 is small, dense cholesteryl ester poor and sequent uptake of LDL by hepatic LDL receptors. This protein rich [241]. In plasma nascent HDL is modified by pathway is commonly called the indirect reverse cho- LCAT to generate cholesteryl ester rich particles that are lesterol transport pathway. CETP is secreted primarily larger and more spherical. LCAT reacts with unesterified by the liver and adipose tissue and circulates primarily cholesterol in HDL transferring the 2-acyl group of lecithin associated with HDL [250]. Triglyceride enrichment of or phosphatidylethanolamine to the free hydroxyl residue HDL enhances the clearance of apoAI from plasma. HL- of cholesterol to generate cholesteryl esters [242]. Chemi- mediated hydrolysis of HDL results in decreased HDL par- cally and physically defined reconstituted spherical HDL ticle size. Accelerated clearance of HDL and apoAI ensues particles can be formed by incubating nascent HDL with with decreased plasma levels of HDL and apoAI [251, 252]. LCAT. Spherical HDL particles are the more mature form CETP may consequently exert both pro-atherogenic and of HDL and contain a large amount of neutral lipids, pre- anti-atherogenic actions. Lipid transfer inhibitor protein, dominantly cholesteryl ester. During maturation, choles- also known as apolipoprotein F (apoF), modulates CETP teryl ester molecules formed by LCAT at the HDL surface function by preferentially blocking CETP activity on LDL. partition into the particle core. Concurrently, the particle By selectively blocking lipid transfer to and from LDL, changes from the more elliptical nascent HDL to a spheri- apoF enhances the net flux of cholesteryl ester from HDL cal form. The spheroidal HDL is the main form of HDL to VLDL. As VLDL has a relatively short plasma lifetime, responsible for cholesterol transport to the liver [243]. this flux is proposed to facilitate the clearance of HDL- Despite spherical HDL being the most abundant cir- derived cholesteryl ester, thus promoting reverse choles- culating form of HDL, relatively few studies have experi- terol transport. ApoF does not bind to CETP, but rather mentally addressed its structure in detail because of its inhibits its activity by preventing CETP binding to the lipo- size and compositional heterogeneity. Using cross linking protein surface [253]. ApoF exists in two forms, one active chemistry and mass spectrometry Silva et al. [244] deter- and the other inactive. Active apoF is bound to LDL and mined that the general structural organization of apoAI it has been suggested that the conversion of apoF to its was similar overall between discs and spheres. Other active form depends on LDL composition [254]. Electron studies suggest a conformational difference between microscopy studies suggest that the N-terminal domain apoAI on spheres versus discs. Using small angle neutron of CETP penetrates into HDL and that the C-terminal end scattering Wu et al. [245] suggest that the protein compo- penetrates into LDL and VLDL. Thus apoAI and apoB or nent of spherical HDL is a hollow structure that cradles other proteins are not essential. Electron microscopy the non-spherical compact lipid core, and that the lipid show virtually no CETP complexes bridging two HDLs, component is only partially surrounded by the protein LDLs or VLDLs, implying CETP domain specificity for wrapper. They suggest that a model in which three apoAI HDL (N-terminal) and LDL and VLDL (C-terminal) The chains are arranged as a combination of a helical dimer co-existence of ternary complexes of HDL-CETP-LDL and and folded back hairpin was more consistent with cross HDL-CETP-VLDL and lipid transfer are consistent with the linking and mass spectrometry analysis. Other investi- mechanistic model of cholesterol ester transfer through a gators have looked at computational models as a means tunnel within CETP, though at this point the continuous of studying the structure and dynamics of spherical hydrophobic tunnel between the distal portions of the N- HDL structure [246, 247]. Molecular dynamic structure and C-terminal domains of CETP is hypothetical. Changes modeling to investigate the dynamics of HDL synthesis in hydrophobicity along the central cavity are suggested suggests that incremental twisting of the apoAI double as one of the dynamic forces which favor a cholesteryl helix structure incorporates increasing concentrations of ester N- to C-terminus transfer [255]. Both CETP structure Ramasamy: Lipoprotein metabolism 1713 and circulating molar concentrations of lipoproteins are of cells with lipid free apoAI through the action of ABCA1 thought to contribute to CETPs physiological preference alone. This implies a potential synergistic relationship for HDL [256]. Prior data using size exclusion chromatog- between ABCA1 and ABCG1 in peripheral cholesterol raphy show that incubation of HDL with LDL or VLDL and export, where ABCA1 lipidates lipid poor apoAI to gen- a CETP source form smaller pre-β HDL particles with the erate nascent HDL which in turn serve as substrates for dissociation of apoAI [257]. ABCG1-mediated cholesterol export. Although no human PLTP transfers phospholipid between HDL and VLDL phenotype has been associated with ABCG1 ablation, tar- as well as different particle-sized HDL. PLTP is a 476-amino geted disruption of ABCG1 in mice on a high fat diet led acid glycoprotein with an apparent molecular weight of to extensive lipid accumulation in tissue macrophages 80 kDa. PLTP exists in the plasma as a functionally active whereas overexpression of ABCG1 protected against diet form that can transfer phospholipid as well as an inactive induced lipid deposition. No change in plasma HDL was form that lacks this capability. PLTP is expressed ubiqui- found in these mice, suggesting that the most important tously in human tissues [258]. PLTP also remodels HDL action of ABCG1 may be in tissue macrophages [267]. Wang into large and small particles in a process that is accom- et al. [268] found that LXR agonist treatment of mouse panied by the dissociation of lipid poor or lipid free apoAI. macrophages increased the mass of cholesterol released

Remodeling is enhanced markedly in HDLs that contain from cells to HDL2, this increased cholesterol mass efflux triglyceride in their core [259]. Evidence for the impor- was markedly reduced in ABCG1 deficient macrophages. tance of PLTP in HDL metabolism comes from studies on In addition LXR activation induces the redistribution of PLTP knockout mice, which show that PLTP is essential ABCG1 from intracellular sites to the plasma membrane for maintaining normal HDL levels in plasma [260]. The in human macrophages. Several N-terminal variants of mechanism of remodeling of HDL by PLTP is not fully ABCG1 have been proposed based on different transla- understood. Studies with reconstituted HDL suggest that tional sites. Inhibition of protein kinase A resulted in an remodeling involves the formation of a large unstable increase in cholesterol export from cells with selective sta- fusion product, which either rearranges into small parti- bilization of an ABCG1 isoform [269]. ABCG1 is expressed cles that is not accompanied by the dissociation of apoAI in several tissues including brain, spleen, lung, placenta, or loses molecules of apoAI to form a more stable, large macrophages and the liver [270]. It has been proposed conversion product [261]. It can be postulated that the that ABCG1 may not serve as a direct transporter of mem- small HDL and apoAI particles generated serve as accep- brane cholesterol to acceptors but rather play a role by tors for cell-derived cholesterol. Other studies suggest enriching the cell membrane with cholesterol that can that ABCA1 is required for PLTP lipid efflux activity and be incorporated into a variety of acceptor particles, i.e., that this may involve direct interactions between PLTP ABCG1 effluxes cellular cholesterol by a process that is not and ABCA1 [262]. If there is redundancy of phospholipid dependent upon interaction with an extracellular protein transfer activity CETP would be expected to ameliorate [271]. However, macrophages may have special mecha- the low HDL state of PLTP deficiency. Studies with PLTP nisms to handle the massive amounts of cholesterol that knockout mice, bred with a CETP transgene suggest that they can take up through scavenger receptors or phago- in vivo there is no redundancy of function between PLTP cytosis. An alternative hypothesis is that the primary and CETP [263]. function of ABCG1 may be to control intracellular sterol homeostasis. ABCG1 present in endosomes and recycling endosomes suggests a mechanism by which ABCG1 trans- ABCG1 fers sterols to the inner leaflet of these vesicles before their fusion to the plasma membrane. This would result ABCA1 is a key regulator of cholesterol and phospho- in redistribution of these sterols to the outer leaflet of the lipid transport to lipid free apolipoproteins [264] forming plasma membrane such that they can desorb in a non- nascent HDL but is unlikely to be involved in cholesterol specific manner to multiple lipid acceptors [272]. transport to mature HDL. ABCG1 has been identified as the Concentrations of HDL with apoE are low in humans more likely mediator of cholesterol transport to HDL but [273]. Matsuura et al. [274] showed that HDL-apoE from not to lipid poor apoAI [265]. Gelissen et al. [266] showed CETP deficient humans can effectively accept unesterified that a range of phospholipids containing acceptors other cholesterol from macrophages. HDL from CETP deficient than HDL subclasses are efficient in mediating the export patients had markedly increased content of apoE and of cholesterol via ABCG1. Acceptors for ABCG1-mediated LCAT. HDL-apoE in the presence of LCAT promoted HDL cholesterol transport can be generated from incubation particle expansion, and this cholesterol efflux was ABCG1 1714 Ramasamy: Lipoprotein metabolism dependent. In addition, removal of the apoE resulted in levels, mostly due to increased cholesterol in HDL parti- a loss of ability of HDL to promote cholesteryl ester accu- cles. These HDL particles are larger and more heterogene- mulation suggesting that apoE may play a key role. Other ous in size than those in wild type animals, and they are studies suggest that apoE has an important role as an enriched in apoE [286]. Increased SR-BI expression also LCAT activator in mouse apoB lipoproteins [275]. In an enhanced cholesterol efflux to exogenously added apoE x-ray crystal model of apoE bound to phospholipids, it has [287]. In other studies lipid free apoE only partially com- been suggested that apoE molecule was folded into half petes with the binding of HDL to SR-BI, whereas the lipi- in a belt-like manner around a spheroidal phospholipid dated apoE competes fully [288]. SR-BI-mediated transfer molecule, forming a circular horseshoe two helices thick of unesterified cholesterol between cells and HDL is bidi- and one helix wide but not a complete belt [276]. rectional and sensitive to HDL phospholipid content and composition. SR-BI-mediated uptake should be greatest for free cholesterol and cholesteryl ester but significant Scavenger receptor BI (SR-BI) catabolism of HDL phospholipid and triglyceride can occur through SR-BI-mediated uptake [289, 290]. Based ABCA1, ABCG1 and scavenger receptor BI (SR-BI) make up on studies with mutant forms of SR-BI and apoAI, it has the known proteins that play a role in cholesterol efflux. As been suggested that SR-BI-mediated transport of lipids far as the mechanism of efflux and the preferred acceptors between cells and lipoproteins involves two sequential ABCA1 remains unique. ABCA1 uses lipid free and lipid steps: 1) lipoprotein binding; and 2) binding-dependent poor exchangeable apolipoproteins as its preferred accep- yet distinct SR-BI-mediated lipid transfer [291, 292]. Other tors. In contrast, ABCG1 and SR-BI use HDL and not lipid studies suggest that transfection mediated SR-BI overex- free apolipoproteins as cholesterol acceptors. SR-BI is an pression by COS cells can alter the lipid organization of 82 kDa membrane glycoprotein containing a large extra- cell membranes and that the proposed binding independ- cellular domain and two transmembrane domains with ent SR-BI-mediated efflux might be a consequence of lipid short cytoplasmic amino and carboxy terminal domains organization [293, 294]. Pagler et al. [295] suggest that ret- [277]. The tissue distribution of SR-BI which is predomi- roendocytosis of HDL is mediated by SR-BI and that res- nantly expressed in liver, adrenal gland and ovary is com- ecretion of HDL particle is accompanied by an efflux of patible with it playing a role in HDL-derived cholesteryl cellular cholesterol. ester transport to the liver and steroidogenic tissues [278]. The potential structural and/or functional interac- Although the liver and steroidogenic tissues are the sites tion between ABCA1 and SR-BI in regulating cholesterol of greatest SR-BI expression, SR-BI is expressed in other efflux has been explored. Using a murine macrophage cell sites throughout the body. SR-BI is expressed in the intes- line Chen et al. [296] showed that the expression of SR-BI tine, lung, endothelial cells and macrophages. SR-BI is expression reduced ABCA1-mediated efflux of cholesterol a multiligand receptor, however, it does not bind a wide but not phospholipid. The authors suggest that SR-BI array of polyanionic molecules that are ligands of other expression produced a reuptake pathway in which choles- scavenger receptors. There are now many members of terol released from the cells to lipoproteins by ABCA1 is a superfamily of scavenger receptors that have diverse incorporated back into the cell by SR-BI-mediated influx. structures, expression patterns and functions [279]. The Another possibility is that SR-BI reorganizes membrane promoter region of the SR-BI gene contains consensus lipids, sequestering cholesterol and making it unavailable DNA sequences that bind several transcription factors for ABCA1 efflux. In a further study co-expression of SR-BI including steroidgenic factor (SF-1) [280], SREBP-1 [281], with ABCG1 inhibited the ABCG1-mediated net cholesterol LXR [282] and liver receptor homolog 1 [283]. Stimulation efflux to HDL [297]. Other studies suggest that ABCG1 of PPAR-α increases SR-BI mRNA levels and protein in and SR-BI in hepatocytes do not contribute to cholesterol macrophages [284]. Taken together the data suggest that efflux to apoAI or the accompanying nascent HDL forma- activation of multiple signaling pathways may participate tion. In contrast, ABCA1 in hepatocytes is essential for in modulating SR-BI expression. The ability of SR-BI to nascent HDL formation [298]. In human macrophage foam mediate cholesterol efflux was established by studies in cells cholesterol efflux stimulated by LXR agonist requires SR-BI-transfected cultured cell lines [285]. Direct evidence SR-BI and ABCA1 but not ABCG1 [299]. It is likely that the for the role of SR-BI in HDL metabolism in vivo has come contributions of ABCA1, ABCG1 and SR-BI to cholesterol from studies in which the levels of SR-BI in mice have been efflux differ in a tissue- and species-specific manner [300]. manipulated. SR-BI knockout mice have, relative to wild As SR-BI-mediated transport does not appear to be type control animals, a 2.5-fold increase in total cholesterol dependent on ATP, it is likely that net flux of lipids is Ramasamy: Lipoprotein metabolism 1715 driven by a concentration gradient between cells and role of hormones on lipid metabolism is beyond the scope extracellular donor/acceptor particles. It is likely that of this review and a brief overview of the subject will be SR-BI mediates efflux to lipid poor HDL from macrophages given. loaded with non-metabolizable cholesterol and from the Lipid metabolism is endogeneously controlled by plasma membrane of hepatocytes and steroidogenic cells pancreatic, pituitary and adrenal hormones. Insulin is the down a concentration gradient determined by intracellu- primary regulator of blood glucose concentration. Insulin lar cholesterol metabolism. promotes the storage of substrates in fat, liver and muscle In vitro studies have shown that in addition to HDL, by stimulating lipogenesis, glycogen and protein synthe- SR-BI binds apoB containing lipoproteins [301]. Studies sis and inhibiting lipolysis, glycogenolysis and protein with mice suggest that, at least in rodents, SR-BI may have breakdown [307]. Insulin appears to be one of the key other roles in post-prandial lipid metabolism. It has been players ‘directing’ the flow of fatty acids to or from tissues suggested that SR-BI may have a role in facilitating chy- for utilization and storage. Glucagon decreases hepatic lomicron remnant metabolism or function as an initial triglyceride synthesis and secretion [308]. Glucagon has recognition site for chylomicron remnants [302] and may been shown to decrease hepatic lipoprotein secretion and facilitate the metabolism of VLDLs [303]. Recently, it has inhibit particle clearance but does not regulate intesti- been shown that HDL-SR-BI complex serves to direct nal lipoprotein metabolism in humans [309]. The role of signal initiation by SR-BI that leads to activation of diverse glucagon and the glucagon receptor in regulating lipopro- kinase cascades that may contribute to the cardiovascular tein metabolism is, however, still not clear. benefits of the lipoprotein [304]. In the liver, insulin has been shown to acutely inhibit the synthesis of VLDL particles in healthy individuals by a mechanism that involves an increase in apoB degrada- Other factors in HDL remodeling tion and decreased expression of MTP, thus leading to a suppression of VLDL release. Studies suggest that insulin Remodeling of HDL particles by plasma factors is an impor- signaling through FoxO1 plays an important role in regu- tant part of HDL metabolism. Endothelial lipase is synthe- lating hepatic MTP expression and VLDL production [310]. sized primarily by the vascular endothelial cells and to a Insulin favors the degradation of apoB in a pre-secretory lesser extent by macrophages and smooth muscle cells. In compartment. Insulin-mediated suppression involves humans’ plasma endothelial cell mass was inversely cor- the activation of phosphatidylinositide-3-kinase by the related with HDL-cholesterol levels [305]. HL can hydro- insulin receptor transduced through insulin receptor sub- lyze triglycerides in all lipoproteins but is predominant strates; a process that correlates with the translocation in the conversion IDL to LDL, and can also convert post- of activated phosphatidylinositide-3-kinase to intracel- prandial triglyceride rich HDL into post-absorptive triglyc- lular membranes [311]. Insulin-induced suppression of eride poor HDL [306]. intestinal lipoprotein secretion is both direct and indirect through suppression of circulating free fatty acids [312]. Thyroid hormones can influence lipid metabolism by: 1) inducing HMG CoA reductase; 2) controlling SREBP-2 Hormones and lipid metabolism which regulates LDL receptor expression; 3) increasing CETP activity; and 4) upregulating the synthesis of apoAV In the forward transport system triglyceride rich lipopro- [313–316]. ANGPTL3 gene is negatively regulated by trio- teins from the liver (VLDL) and intestine (CM) deliver fatty dothyronine leading to the speculation that triodothyro- acids to peripheral tissues. LPL plays a role in VLDL fatty nine can lower plasma triglycerides by suppressing the acid release and in its subsequent conversion to LDL. Con- ANGPTL3 gene thus activating LPL which plays a central versely in the reverse transport system HDL transports role in lipid metabolism [317]. Thyroid hormone interaction excess cholesterol from extrahepatic cells, e.g., mac- with the nuclear thyroid hormone receptor accounts for rophages to the liver where it can be recycled or catabo- several of the lipid reduction effects by thyroid hormones, lized to bile acids. Lipid (and carbohydrate) homeostasis though recent work suggests that non-classic mechanism in higher organisms is under the control of an integrated involving cytosolic second messengers including calcium system that has the capacity to rapidly respond to meta- and phosphoinositide-3-kinase, could contribute to the bolic changes. Regulation of lipoprotein production is an effect of thyroid hormones on lipid homeostasis [318]. interplay between systemic free fatty acid delivery, hormo- Hydrolysis of the triacylglycerol reserves in adipo- nal and nutritional factors. A detailed discussion of the cytes provides fatty acids during times of energy demand 1716 Ramasamy: Lipoprotein metabolism such as fasting and exercise. Abnormalities of adipose Early work on the LDL receptor leads to new ways of tissue lipolysis are associated with the development of thinking on cholesterol metabolism. FH inherited as an metabolic diseases. Catecholamines stimulate lipolysis autosomal dominant disorder, was found to be caused by in humans. Catecholamines stimulate adenyl cyclase a genetic defect in the LDL receptor [133]. Several muta- leading to increased cyclic AMP concentrations and cyclic tions in the LDL receptor binding domain of apoB100 AMP dependent phosphorylation of hormone sensitive have been described that are associated with hypercho- lipase, one of the key enzymes controlling lipolysis [319]. lesterolemia with an autosomal codominant inheritance Glucocorticoids may also participate in the stimulation of pattern [327]. In contrast, many non-sense, frameshift lipolysis in adipose tissue by stimulating the lipase des- and splicing mutations in the APOB gene causing the for- nutrin [320]. In the fed state insulin inhibits lipolysis by mation of truncated apoB can cause familial hypobetali- activation of phosphodiesterase that reduces cyclic AMP poproteinemia [328]. In 2003 mutations in the PCSK9 levels [321]. gene was identified in two French families with an Several hormones have been implicated in the tran- autosomal dominant form of FH [138]. Statins currently sition from fasted to fed state. Intestinal lipid absorption form the standard of care in hypercholesterolemia. Com- virtually ceases in the post-absorptive state, whereas bination therapy with statins is well established and secretion of hepatic VLDL persists. In the fasted state the ezetimibe is often used as an additional LDL choles- majority of triglycerides in VLDL were from circulating terol lowering agent. Two cases of homozygous ‘loss of free fatty acid from adipose tissue lipolysis [322]. Post- function’ mutations in PCSK9 have been described. The prandially the majority of the chylomicron triglycerides carriers, who lacked PCSK9 were healthy and fertile sug- are sequestered by extrahepatic tissues (mainly adipose) gesting that pharmacologic inhibition of PCSK9 may be following hydrolysis by the insulin-stimulated LPL [323]. safe [329]. In some cases FH is inherited by mutations in Intestinally derived hormones glucagon-like peptide-1 and the LDL receptor adapter protein1, a protein that inter- gastric inhibitory polypeptide enhance glucose stimu- acts with the cytoplasmic tail of the LDL receptor [330]. lated insulin secretion by pancreatic β cells [324]. During Newer more potent methods of LDL cholesterol reduc- fasting states growth hormone increases lipolysis and tion are currently being tested. Mimetic peptides and free fatty acid levels [325]. Studies suggest that monosac- monoclonal antibodies that specifically target PCSK9 charides can acutely enhance intestinal lipoprotein par- are in development as are inhibitors of CETP and MTP. ticle production [326]. The transition from fed to fasted Antisense oligonucleotides to specifically silence or state involves nutrient and hormonal signals to intestine, reduce the expression of apoB as a means of lowering liver and adipose tissue. During the post-prandial state VLDL are under investigation [331]. Statins activate the this ensures efficient absorption, transport and storage SREBP pathway and lower the formation of LXR ligands of lipids and in the fasted state this ensures subsequent with subsequent decreased ABCA1 expression [332]. As redistribution and utilization of stored lipids. there is cooperativity and cross talk between the dif- ferent lipoprotein metabolism pathways it is likely that future research will focus on combination therapy with the newer agents, as more effective methods of lowering Clinical implications of lipoprotein LDL cholesterol. metabolism As a therapeutic approach to increasing HDL research- ers have focused on injections of reconstituted HDL, apoAI Recent progress in the understanding of lipoprotein mimetics or full length apoAI. ABCA1 has been identified metabolism has fostered understanding of the genetic as defective in Tangier disease patients with HDL defi- basis of dyslipidemias. Severe dyslipidemias can be ciency [333]. monogenic disorders with a Mendelian inheritance and A few individuals with hypertriglyceridemia have rare present in childhood. More commonly they develop in monogenic disorders with loss of function mutation in LPL, later life and are an interplay between genetic variants APOCII, APOAV, LMF1 or GPIHBP1 genes. Most patients and environmental variants such as poor quality of life with hypertriglyceridemia, however, have a complex and imbalanced caloric intake or certain medications. The genetic etiology. Genome wide association studies have current review focuses on the molecular basis of normal identified several common genetic variants associated lipoprotein metabolism but will be incomplete without a with hypertriglyceridemia at genome wide significance brief account of the more recent advances in the inherited levels including APOAV, LPL and APOB genes [334]. As the form of dyslipidemias. genetic basis for hypertriglyceridemia becomes available Ramasamy: Lipoprotein metabolism 1717 this might be used to predict the individual’s response to 3. Amarenco P, Steg G. The paradox of cholesterol and stroke. diet and drug therapy. Lancet 2007;370:1803–4. 4. Amarenco P, Lavallee P, Touboul P. Stroke prevention, blood cholesterol and statins. Lancet 2004;3:271–8. 5. Cholesterol Treatment Trialists Collaboration. Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of Future directions data from 90056 participants in 14 randomised trials of statins. Lancet 2005;336:1267–78. Multiple genes and complex pathways are involved in 6. Cholesterol Treatment Trialists Collaboration. Efficacy and safety lipoprotein metabolism and cholesterol and lipid homeo- of more intensive lowering of LDL cholesterol: a meta-analysis stasis. These include pathways of intestinal chylomicron of data from 170000 participants in 26 randomised trials. Lancet secretion and liver VLDL secretion and cholesterol home- 2010;376:1670–81. 7. Emerging Risk Factors Collaboration. Major lipids, apolipo- ostasis with HDL providing a driving force for reverse proteins and risk of vascular disease. J Am Med Assoc cholesterol transport. Hormonal and nutritional factors 2009;302:1993–2000. further regulate lipoprotein metabolism. 8. Labreuche J, Taboul JP, Amarenco P. Plasma triglyceride levels There is growing evidence using genome wide asso- and risk of stroke and carotid atherosclerosis: a systematic ciation studies that several common genetic variants of review of the epidemiological studies. Atherosclerosis genes are associated with lipid traits in humans. Under- 2009;203:331–45. 9. Mora S, Rifai N, Buring JE, Ridker PM. Fasting compared with standing the mechanisms of lipoprotein metabolism is the nonfasting lipids and apolipoproteins for predicting incident basis for exploring the pathophysiology of dyslipidemias cardiovascular events. Circulation 2008;118:993–1001. and has important clinical applications. The central 10. Langsted A, Freiberg JJ, Tybjaerg-Hansen A, Schnohr P, question is how this explosion of knowledge may benefit Jensen GB, Nordestgaard BG. Nonfasting cholesterol and human health. It is likely that newer drugs can be used triglycerides and association with risk of myocardial infarction and total mortality: the Copenhagen City Heart Study with in combination with statins to reduce LDL cholesterol and 31 years of follow up. J Intern Med 2011;270:65–75. further decrease CVD risk. Novel compounds under inves- 11. Varbo A, Nordestgaard BG, Tybjaerg-Hansen A, Schnohr P, tigation are those that inhibit VLDL production, those that Jensen GB, Benn M. Nonfasting triglycerides, cholesterol increase LDL receptor expression, or those that modulate and ischaemic stroke in the general population. Ann Neurol LDL cholesterol content. Despite our increased molecular 2011;69:628–34. understanding of lipoprotein metabolism much, however, 12. Nordestgaard BG, Benn M, Schnohr P, Tybjaerg-Hansen A. Nonfasting triglycerides and risk of myocardial infarction, remains to be learned. As knowledge of lipoprotein metab- ischemic heart disease and death in men and women. J Am Med olism increases, this will in future, open possibilities for Assoc 2007;298:299–308. targeted individualized treatment for dyslipidemias. 13. Freiberg JJ, Tybjaerg-Hansen A, Jensen JS, Nordetgaard BG. Nonfasting triglycerides and risk of ischemic stroke in the Conflict of interest statement general population. J Am Med Assoc 2008;300:2142–52. 14. Phillips NR, Waters MD, Ravel RJ. Plasma lipoproteins Authors’ conflict of interest disclosure: The authors stated and progression of coronary artery disease evaluated by that there are no conflicts of interest regarding the publica- angiography and clinical events. Circulation 1993;88:2762–70. 15. McNamara JR, Shah PK, Nakajima K, Cupples LA, Wilson PW, tion of this article. Ordovas JM, et al. Remnant-like particle (RLP) cholesterol is Research funding: None declared. an independent cardiovascular risk factor in women: Employment or leadership: None declared. results from the Framingham Heart Study. Atherosclerosis Honorarium: None declared. 2001;154:229–36. 16. Wittrup HH, Tybjaerg-Hansen A, Nordestgaard BG. Lipoprotein lipase mutations, plasma lipids and lipoproteins, and risk of ischemic heart disease. A meta-analysis. Circulation 1999;99:2901–7. References 17. Austin MA, McKnight B, Edwards KL, Bradley CM, McNeely MJ, Psay BM, et al. Cardiovascular disease mortality in familial 1. Cholesterol Treatment Trialists Collaboration. Protocol for a forms of hypertriglyceridemia: a 20 year prospective study. prospective collaborative overview of all current and planned Circulation 2000;101:2777–82. randomised trials of cholesterol treatment regimens. Am J 18. Hokanson JE. Functional variants in the lipoprotein lipase gene and Cardiology 1995;75:1130–4. risk of cardiovascular disease. Curr Opin Lipidol 1999;10:393–9. 2. Prospective Studies Collaboration. Blood cholesterol 19. Benlian P, De Gennes JL, Foubert L, Zhang H, Gagne SE, and vascular mortality by age, sex, and blood pressure: a Hayden M. Premature atherosclerosis in patients with familial meta-analysis of individual data from 61 prospective studies with chylomicronemia caused by mutations in the lipoprotein lipase 55000 vascular deaths. Lancet 2007;370:1829–39. gene. New Engl J Med 1996;335:848–54. 1718 Ramasamy: Lipoprotein metabolism

20. Birjmohun RS, Hutten BA, Kastelein JP, Stroes ES. Efficacy 36. Sacks FM, Zheng C, Cohn JS. Complexities of plasma apolipo- and safety of high density lipoprotein cholesterol-increasing protein C-III metabolism. J Lipid Res 2011;52:1067–70. compounds. J Am Coll Cardiol 2005;45:185–97. 37. Chen J, Li Q, Wang J. Topology of human lipoprotein E3 uniquely 21. Rubins HB, Rubins SJ, Collins D, Nelson DB, Elam MB, regulates its diverse biological functions. Proc Natl Acad Sci Schaefer EJ, et al. Diabetes, plasma insulin and cardiovascular 2011;108:14813–8. disease: subgroup analysis from the Department of Veteran’s 38. Saito H, Dhanasekaran P, Nguyen D, Holvoet P, Lund-Katz S, affairs high density lipoprotein intervention trial (VA-HIT). Arch Phillips MC. Domain structure and lipid interaction in human Int Med 2002;162:2597–604. apolipoproteins A-I and E, a general model. J Biol Chem 22. Ginsberg HN, Elam MB, Lovato C, Crouse JR 3rd, Leiter LA, 2003;278:23227–32. Linz P, et al. for the ACCORD Study Group. Effects of combination 39. Brown MJ, Liquing Y. Protein mediators of sterol transport lipid therapy in type 2 diabetes mellitus. N Engl J Med across intestinal brush border membranes. Subcell Biochem 2010;362:1563–74. 2010;51:337–80. 23. The FIELD investigators. Effects of long-term fenofibrate therapy 40. Iqbal J, Hussain MM. Intestinal lipid absorption. Am J Physiol on cardiovascular events in 9795 people with type 2 diabetes Endo Metab 2009;296:E1183–94. mellitus (the FIELD study): randomised controlled trial. Lancet 41. Black DD. Development and physiological regulation of 2005;366:1849–61. intestinal lipid absorption. I. Development of intestinal 24. Jun M, Foote C, Lu J, Neal B, Patel A, Nicolls SJ, et al. Effects of lipid absorption: cellular events in chylomicron assembly fibrates on cardiovascular outcomes: a systemic review and and secretion. Am J Physiol Gastrointestin Liver Physiol meta-analysis. Lancet 2010;375:1875–84. 2007;293:G519–24. 25. Lee M, Saver J, Towfighi A, Chow J, Ovbiagele B. Efficacy of 42. Adiels M, Olofsson S, Taskinen MR, Boren J. Overproduction of fibrates for cardiovascular risk reduction in persons with very low-density lipoproteins is the hallmark of the dyslipidemia atherogenic dyslipidemia: a meta-analysis. Atherosclerosis in the metabolic syndrome. Arterioscler Thromb Vasc Biol 2011;217:492–8. 2008;28:1225–36. 26. Third report of the National Cholesterol Education Program 43. Lo CM, Nordskog BK, Nauli AM, Zheng S, Vonlehmden SB, Yang Q, (NCEP) expert panel on detection, evaluation, and treatment of et al. Why does the gut choose apolipoprotein B48 but not B100 high blood cholesterol in adults (Adult Treatment Panel III) final for chylomicron formation? Am J Physiol 2008;294:G344–52. report. Circulation 2002;106:3143–21. 44. Fisher EA, Ginsberg HN. Complexity in the secretory pathway: 27. Rosenson RS, Brewer HB, Chapman MJ, Fazio S, Hussain MM, the assembly and secretion of apoliprotein B-containing Kontush A, et al. HDL measures, particle heterogeneity, lipoproteins. J Biol Chem 2002;277:17377–80. proposed nomenclature, and relation to atherosclerotic cardio- 45. Ginsberg HN, Fisher EA. The ever expanding role of degradation vascular events. Clin Chem 2011;57:392–410. in the regulation of apolipoprotein B metabolism. J Lipid Res 28. Kalogeris TJ, Fukagawa K, Tso P. Synthesis and lymphatic 2009;50:S162–6. transport of intestinal apolipoprotein A-IV in response to graded 46. Liang J, Ginsberg HN. Microsomal triglyceride transfer protein doses of triglyceride. J Lipid Res 1994;35:1141–51. binding and lipid transfer activities are independent of each 29. Lu S, Yao Y, Meng S, Cheng X, Black DD. Overexpression of other, but both are required for secretion of apolipoprotein B apolipoprotein A-IV enhances lipid transport in newborn swine lipoproteins from liver cells. J Biol Chem 2001;276:28606–12. intestinal epithelial cells. J Biol Chem 2002;277:31929–37. 47. Jiang ZG, Liu Y, Hussain MM, Atkinson D, McKnight CJ. 30. Merkel M, Heeren J. Give me A5 for lipoprotein hydrolysis. J Clin Reconstituting initial events during the assembly of apolipo- Invest 2005;115:2694–6. proteins B-containing lipoproteins in a cell free system. J Mol 31. Blade AM, Fabritus MA, Li H, Weinberg RB, Shelness GS. Biol 2008;383:1181–94. Biogenesis of apolipoproteins A-V and its impact on VLDL 48. Morel E, Demignot S, Chateau D, Chambaz J, Rousset M, triglyceride secretion. J Lipid Res 2011;52:237–44. Delers F. Lipid-dependent bidirectional traffic of apolipoprotein 32. Shachter NS. Apolipoproteins C-I and C-III as important B in Polarized enterocytes. Mol Biol Cell 2004;15:132–41. modulators of lipoprotein metabolism. Curr Opin Lipidol 49. Hussain MM, Shi J, Dreizen P. Microsomal triglyceride transfer 2001;2:297–304. protein and its role in apoB-lipoprotein assembly. J Lipid Res 33. de Barros JP, Boualam A, Gautier T, Dumont L, Veges B, 2003;44:22–32. Masson D, et al. Apolipoprotein CI is a physiological regulator of 50. Iqbal J, Dai K, Seimon T, Jungreis R, Oyadomari M, Kuriakose G, cholesteryl ester transfer protein activity in human plasma but et al. IRE1 inhibits chylomicron production by selectively not in rabbit plasma. J Lipid Res 2009;50:1842–51. degrading MTP mRNA. Cell Metabol 2008;7:445–55. 34. Ginsberg HN, Le NA, Goldberg IJ, Gibson JC, Rubinstein A, 51. Dai K, Hussain M. NR2F1 disrupts synergistic activation of the Wang-Iverson P, et al. Apolipoprotein B metabolism in subjects MTTP gene transcription by HNF-4a and HNF-1a. J Lipid Res with deficiency of apolipoproteins CIII and AI. Evidence 2012;53:901–8. that apolipoprotein CIII inhibits catabolism of triglyceride- 52. Mansbach CM, Gorelick F. Development and physiological rich lipoproteins by lipoprotein lipase in vivo. J Clin Invest regulation of intestinal lipid absorption II. Dietary lipid 1986;78:1287–95. absorption, complex lipid synthesis and the intracellular 35. Sundaram M, Zhnong S, Khalil MB, Zhou H, Jiang ZG, Zhao Y, packaging and secretion of chylomicrons. Am J Physiol et al. Functional analysis of the missense APOC3 mutation Gastrointest Liver Physiol 2007;293:G645–50. Ala23Thr associated with human hypotriglyceridemia. J Lipid 53. Siddiqi SA, Gorelick FS, Mahan JT, Mansbach CM. COPII proteins Res 2010;51:1524–34. are required for Golgi fusion but not for endoplasmic reticulum Ramasamy: Lipoprotein metabolism 1719

budding of pre-chylomicron transport vesicle. J Cell Sci 71. Hide WA, Chan L, Wen-Hsuing L. Structure and evolution of the 2002;116:415–27. lipase superfamily. J Lipid Res 1992;33:167–78. 54. Siddiqi SA, Manbach CM. PKC?-mediated phosphorylation 72. Enerback S, Gimble JM. Lipoprotein lipase gene expression: controls budding of pre-chylomicron transport vesicles. J Cell physiological regulators at the transcriptional and post Sci 2008;121:2327–38. transcriptional level. Biochim Biophys Acta 1993;1169:107–25. 55. Siddiqi SA, Mahan J, Siddiqi S, Gorelick FS, Manbach CM. 73. Braun JE, Severson DL. Regulation of the synthesis, Vesicle-associated membrane protein 7 is expressed in processing and translocation of lipoprotein lipase. Biochem J intestinal ER. J Cell Sci 2005;119:943–50. 1992;287:337–47. 56. Neeli I, Siddiqi SA, Siddiq S, Mahani J, Lagakos WS, Binas B, 74. Mead JR, Irvine SA, Ramji DP. Lipoprotein lipase: structure, et al. Liver fatty acid binding protein initiates budding of function, regulation, and role in disease. J Mol Med pre-chylomicron transport vesicles from intestinal endoplasmic 2002;80:753–69. reticulum. J Biol Chem 2007;282:17974–84. 75. Sukonina V, Lookene A, Olivecrona T, Olivecrona G. 57. Siddiqi S, Saleem U, Abumrad NA, Davidson NO, Storch J, Angiopoietin-like protein 4 converts lipoprotein lipase to Siddiqi SA, et al. A novel multiprotein complex is required to inactive monomers and modulates lipase activity in adipose generate the prechylomicron transport vesicles from intestinal tissue. Proc Natl Acad Sci 2006;103:17450–5. ER. J Lipid Res 2010;51:1918–28. 76. Romeo S, Yin W, Kozlitina J, Pennachio LA, Boerwinkle E, 58. Antonny B, Schekman R. ER export: public transportation by the Hobbs HH, et al. Rare loss-of-function mutations in ANGPTL COPII coach. Curr Opinion Cell Biol 2001;13:438–43. family members contribute to plasma triglyceride levels in 59. Levy E, Harmel E, Laville M, Sanchez R, Emonnot L, Sinnett D, humans. J Clin Invest 2009;119:70–9. et al. Expression of Sar1b enhances chylomicron assembly 77. Dallinga-Thie GM, Franssen R, Mooij HL, Visser ME, Hassing and key components of the coat protein complex II system HC, Peelman F, et al. The metabolism of triglyceride-rich driving vesicle budding. Arterioscler Thromb Vasc Biol lipoproteins revisited: new players, new insight. Athero- 2011;31:2692–9. sclerosis 2010;211:1–8. 60. Siddiqi SA, Siddiqi S, Mahan J, Peggs K, Gorelick FS, Mansbach 78. Davies SJ, Goulbourne CN, Barnes RH, Turlo KA, Gin P, CM. The identification of a novel endoplasmic reticulum to Golgi Vaughan S, et al. Assessing mechanisms of GPIHBP1 and SNARE complex used by the prechylomicron transport vesicle. lipoprotein lipase movement across endothelial cells. J Lipid J Biol Chem 2006;281:20974–82. Res 2012;53:2960–7. 61. Swift LL, Zhu M, Kakkad B, Jovanovska A, Neely MD, 79. Davies BS, Beigneux AP, Barnes RH, Tu Y, Gin P, Weinstein MM, Valyi-Nagy K, et al. Subcellular localization of microsomal et al. GPIHBP1 is responsible for the entry of lipoprotein lipase transfer protein. J Lipid Res 2003;44:1841–9. into capillaries. Cell Metab 2010;12:42–52. 62. Weinberg RA, Gallagher JW, Fabritius MA, Shelness GS. Apo-IV 80. Beigneux AP, Davies BS, Gin P, Weinstein MM, Farber E, Qiao X, modulates the secretory trafficking of apoB and the size of et al. Glycosylphospatidylinositol-anchored high-density triglyceride rich lipoproteins. J Lipid Res 2012;53:736–43. lipoprotein-binding protein 1 plays a critical role in the lipolytic 63. Hockey KJ, Anderson RA, Cook VR, Hantgan RR, Weinberg processing of chylomicrons. Cell Metab 2007;5:279–91. RB. Effect of the apolipoprotein A-IV Q360H polymorphism 81. Williams KJ. Molecular processes that handle and mishandle on postprandial plasma triglyceride clearance. J Lipid Res dietary lipids. J Clin Invest 2008;118:3247–59. 2001;42:211–7. 82. MacArthur JM, Bishop JR, Stanford KI, Wang L, Bensadoun A, 64. Marcais C, Veges B, Charriere S, Pruneta V, Merlin M, Billon S, Witzum JL, et al. Liver heparin sulphate proteoglycans mediate et al. Apoa5 Q139X truncation predisposes to late-onset clearance of triglyceride-rich lipoproteins independently of LDL hyperchylomicronemia due to lipoprotein lipase impairment. receptor family members. J Clin Invest 2007;117:153–64. J Clin Invest 2005;115:2862–9. 83. Fuki IV, Kuhn KM, Lomazov IR, Rothman VL, Tuszynski GP, 65. Hahne P, Krempler F, Schaap FG, Soyal SM, Höffinger H, Miller K, Lozzo RV, et al. The syndecan family of proteoglycans: novel et al. Determinants of plasma apolipoprotein A-V and APOA5 receptors mediating internalization of atherogenic lipoproteins gene transcripts in humans. J Intern Med 2008;264:452–62. in vitro. J Clin Invest 1997;100:1611–22. 66. Prieur X, Huby T, Rodriguez JC, Couvert P, Chapman MJ. 84. Williams KJ, Chen K. Recent insights into factors affecting Apolipoprotein AV: gene expression, physiological role in lipid remnant lipoprotein uptake. Curr Opin Lipidol 2010;21: metabolism and clinical relevance. Future Lipidol 2008;3:371–84. 218–28. 67. Lee JH, Giannikopoulos P, Duncan SA, Wang J, Johansen CT, 85. Zheng C, Murdoch SJ, Brunzell JD, Sacks FM. Lipoprotein lipase Brown JD, et al. The transcription factor cyclic AMP responsive bound to apolipoprotein B lipoproteins accelerates clearance of element binding protein H regulates triglyceride metabolism. postprandial lipoproteins in humans. Arterioscler Thromb Vasc Nat Med 2011;17:812–5. Biol 2006;26:891–6. 68. Ferre P. The biology of peroxisome proliferator-activated 86. Saito H, Dhanasekaran P, Nguyen D, Baldwin F, Weisgraber KH, receptors. Relationship with lipid metabolism and insulin Wehrli S, et al. Characterization of the heparin binding sites in sensitivity. Diabetes 2004;53:S43–50. human apolipoprotein E. J Biol Chem 2003;278:14782–7. 69. Ravel RJ. Triglyceride-rich lipoproteins and plasma lipid 87. Mamo JC, Smith D, Yu KC, Kawaguchi A, Shiba-Harada M, transport. Arterioscler Thromb Vasc Biol 2010;30:9–19. Yamamura T, et al. Accumulation of chylomicron remnants in 70. Mahmood HM, Kancha RK, Zhou Z, Luchoomun J, Zu H, homozygous subjects with familial hypercholesterolemia. Eur J Bakillah A. Chylomicron assembly and catabolism: role Clin Invest 1998;28:379–84. of apolipoproteins and receptors. Biochim Biophys Acta 88. Rubinaztein DC, Cohen JC, Berger GM, van der Westhuyzen DR, 1996;1300:151–70. Coetzee GA, Gevers W. Chylomicron remnants clearance 1720 Ramasamy: Lipoprotein metabolism

from the plasma is normal in familial hypercholesterolemic 105. Fisher EA, Khanna NA, McLeod RS. Ubiquitination homozygotes with defined receptor defects. J Clin Invest regulates the assembly of VLDL in HepG2 cells and is the 1990;86:1306–12. committing step of the apoB-100 ERAD pathway. J Lipid Res 89. Watts GF, Barrett PH, Marais AD, Dane-Stewart CA, Martins IJ, 2011;52:1170–80. Dimmitt SB, et al. Chylomicron remnant metabolism in familial 106. Rutledge AC, Qiu W, Zhang R, Kohen-Avramoglu R, hypercholesterolemia studied with the stable isotope breath Nemat-Gorgani N, Adeli K. Mechanisms targeting apolipo- test. Atherosclerosis 2001;157:519–27. protein B100 on proteosomal degradation: evidence that 90. Mortimer BC, Beveridge DJ, Martins IJ, Redgrave TG. Intracellular degradation is initiated by BiP binding at the N terminus and localisation and metabolism of chylomicron remnants in the the formation of a p97 complex at the c terminus. Arterioscler livers of low density lipoprotein receptor deficient mice and Thromb Vasc Biol 2009;29:579–85. apoE deficient mice. J Biol Chem 1995;270:28767–76. 107. Fisher EA, Pan M, Chen X, Wu X, Wang H, Jamil H, et al. 91. Martins IJ, Hone E, Chi C, Seydel U, Martins RN, Redgrave TG. The triple threat to nascent apolipoprotein B. J Biol Chem Relative roles LDLr and LRP in the metabolism of chylomicron 2001;276:27855–63. remnants in genetically manipulated mice. J Lip Res 108. Pan M, Maitin V, Parathath S, Andreo U, Lin SX, Germain C, 2000;41:205–13. et al. Presecretory oxidation, aggregation and autophagic 92. Herz J, Strickland DK. LRP: a multifunctional scavenger and destruction of apoprotein B: a process for late stage quality signaling receptor. J Clin Invest 2001;108:779–84. control. Proc Natl Acad Sci 2008;105:5862–7. 93. Beisiegel U, Weber W, Bengtsson-Olivecrona G. Lipoprotein 109. Brodsky JL, Fisher EA. The many interesting pathways lipase enhances the binding of chylomicrons to low underlying apolipoprotein B secretion and degradation. Trends density lipoprotein receptor protein. Proc Natl Acad Sci Endocrinol Metab 2008;19:254–9. 1991;88:8342–6. 110. Williams KJ, Brocia RW, Fisher EA. The unstirred water layer 94. Gordts PL, Bartelt A, Nilsson SK, Annaert W, Christoffersen C, as a site of control of apolipoprotein B secretion. J Biol Chem Nielsen LB, et al. Impaired LDL receptor related protein 1990;265:16741–4. 1 translocation correlates with improved dyslipidemia 111. Larsson SL, Skogsberg J, Bjorkesgren J. The low density and atherosclerosis in apoE deficient mice. PLos One lipoprotein receptor prevents secretion of dense apoB100- 2012;7:e38330. containing lipoproteins from the liver. J Biol Chem 95. Nilsson SK, Lookene A, Beckstead JA, Gliemann J, Ryan RO, 2004;279:831–6. Olivecrona G. Apolipoprotein A-V interaction with members 112. Blasiole DA, Oler AT, Attie AD. Regulation of apoB secretion of the lipoprotein receptor gene family. Biochemistry by the low density lipoprotein receptor requires exit from the 2007;46:3896–904. endoplasmic reticulum and interaction with apoE or apoB. 96. Lookene A, Beckstead JA, Nilsson S, Olivecrona G, Ryan RO. J Biol Chem 2008;283:11374–81. Apolipoprotein A-V heparin interactions. J Biol Chem 113. Sundaram M, Yao Z. Recent progress in understanding 2005;280:25383–7. protein and lipid factors affecting hepatic VLDL assembly and 97. Tiwari S, Siddiqi SA. Intracellular trafficking and secretion of secretion. Nutr Metab 2010;7:35–52. VLDL. Arterioscler Thromb Vasc Biol 2012;32:1079–86. 114. Walther TC, Farese RV. The life of lipid droplets. Biochim 98. Khalil MB, Sundaram M, Zhang HY, Links PH, Raven JF, Biophys Acta 2009;1791:459–66. Monmontri B, et al. The level and compartmentalization of 115. Lehner R, Lian J, Quiroga AD. Lumenal lipid metabolism. phosphatidate phosphatase-1 (lipin-1) control the assembly Implications for lipoprotein assembly. Arterioscler Thromb and secretion of VLDL. J Lipid Res 2009;50:47–58. Vasc Biol 2012;32:1087–93. 99. Segrest JP, Jones MK, De Loof H, Dashti N. Structure of 116. Siddiqi SA. VLDL exits from the endoplasmic reticulum in a apoliproteins B-100 in low density lipoproteins. J Lipid Res specialized vesicle. The VLDL transport vesicle in rat primary 2001;42:1346–67. hepatocytes. Biochem J 2008;413:333–42. 100. Yamaguchi J, Conlon DM, Liang JJ, Fisher EA, Ginsberg HN. 117. Gusarova V, Brodsky JL, Fisher EA. Apolipoprotein B100 exit Translocation efficiency of apolipoprotein B is determined by from the endoplasmic reticulum (ER) is COPII-dependent and the presence of -sheet domains, not pause transfer sequences. its lipidation to very low density lipoprotein occurs post-ER. J Biol Chem 2006;281:27063–71. J Biol Chem 2003;278:48051–8. 101. Chuck SL, Lingappa VR. Analysis of pause transfer sequence 118. Yamaguchi J, Gamble MV, Conlon D, Liang J, Ginsberg HN. The from apolipoprotein B. J Biol Chem 1993;268:22794–801. conversion of apoB100 low density lipoprotein/high density 102. Liao W, Yeung SJ, Chan L. Proteosome mediated degradation lipoprotein particles to apoB100 very low density lipoproteins of apolipoprotein targets both nascent peptides cotransla- in response to oleic acid occurs in the endoplasmic reticulum tionally before translocation and full length apolipoprotein B and not in the Golgi in McA RH7777 cells. J Biol Chem after translocation into the endoplasmic reticulum. J Biol Chem 2003;278:42643–51. 1998;273:27225–30. 119. Gusarova V, Seo J, Sullivan ML, Watkins SC, Brodsky JL, 103. Gusarova V, Caplan AJ, Brodsky JL, Fisher EA. Apoprotein B Fisher EA. Golgi-associated maturation of very low density degradation is promoted by the molecular chaperones hsp90 lipoproteins involves conformational change in apolipoprotein and hsp70. J Biol Chem 2001;276:24891–900. B, but is not dependent on apolipoprotein E. J Biol Chem 104. Hrizo SL, Gusarova V, Habie DM, Goeckeler JL, Fisher EA, 2007;282:19453–62. Brodsky JL. The Hsp110 molecular chaperone stabilizes 120. Siddiqi S, Mani AM, Siddiqi A. The identification of the SNARE apolipoprotein from B from endoplasmic reticulum-associated complex required for the fusion of VLDL-transport vesicle with degradation. J Biol Chem 2007;282:32665–75. hepatic cis-Golgi. Biochem J 2010;429:391–401. Ramasamy: Lipoprotein metabolism 1721

121. Mensenkamp AR, Jong MC, van Goor H, van Luyn MJ, Bloks V, 138. Abifadel M, Varret M, Rabes JP, Allard D, Ougerram K, Havinga R, et al. Apolipoprotein E participates in the regulation Devillers M, et al. Mutations in PCSK9 cause autosomal of very low density lipoprotein-triglyceride secretion by the dominant hypercholesterolemia. Nat Genet 2003;34:154–6. liver. J Biol Chem 1999;274:35711–8. 139. Benjannet S, Rhainds D, Hamelin J, Nassoury N, Seidah NG. 122. Sundaram M, Yao Z. Intrahepatic role of exchangeable apolipo- The proprotein convertase (PC) PCSK9 is inactivated by furin proteins in lipoprotein assembly and secretion. Arterioscler and/or PC5/6A. J Biol Chem 2006;281:30561–72. Thromb Vasc Biol 2012;32:1073–8. 140. Seidah NG, Benjannet S, Wickham L, Marcinkiewicz J, Jasmin 123. Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, SB, Stifani S, et al. The secretory proprotein convertase Herz J. Hypercholesterolemia in low density receptor knockout neural apoptosis-regulated convertase 1 (NARC-1): liver mice and its reversal by adenovirus-mediated gene delivery. regeneration and neuronal differentiation. Proc Natl Acad Sci J Clin Invest 1993;92:883–93. 2003;100:928–33. 124. Hauser PS, Narayanaswami V, Ryan RO. Apolipoprotein 141. Dong B, Wu M, Li H, Kraemer FB, Adeli K, Seidah NG, et al. E: from lipid transport to neurobiology. Prog Lipid Res Strong induction of PCSK9 gene expression through HNF1a 2011;50:62–74. and SREBP2: mechanism for resistance to LDL-cholesterol 125. Morrow JA, Arnold KS, Dong J, Balestra ME, Innerarity TL, lowering effect of statins in dyslipidemic hamsters. J Lipid Res Weisgraber KH. Effect of arginine 172 on the binding of 2010;51:1486–95. apolipoprotein E to the low density lipoprotein receptor. J Biol 142. Lagace TA, Curtis DE, Garuti R, McNutt MC, Park SW, Prather Chem 2000;275:2576–80. HB, et al. Secreted PCSK9 decreases the number of LDL 126. Beisiegel U, Weber W, Ihrke G, Herz J, Stanley KK. The receptors in hepatocytes and in livers of parabiotic mice. J Clin LDL-receptor protein, LRP, is an apolipoprotein E-binding Invest 2006;116:2995–3005. protein. Nature 1989;341:162–4. 143. McNutt MC, Lagace TA, Horton JD. Catalytic activity is not 127. Malby S, Pickering R, Saha S, Smallridge R, Linse S, required for secreted PCSK9 to reduce low density receptors in Downing AK. The first epidermal growth factor-like domain of HepG2 cell. J Biol Chem 2007;282:20799–803. the low density lipoprotein receptor contains a noncanonical 144. Kwon HJ, Lagace TA, McNutt MC, Horton JD, Deisenhofer J. calcium binding site. Biochemistry 2001;40:2555–63. Molecular basis for LDL receptor recognition by PCSK9. Proc 128. Jeon H, Meng W, Takagi J, Eck MJ, Springer TA, Blacklow SC. Natl Acad Sci 2008;105:1820–5. Implications for familial hypercholesterolemia from the 145. Zhang D, Lagace TA, Garuti R, Zhao Z, McDonald M, Horton JD, structure of the LDL receptor YWTD-EGF domain pair. Nat Struct et al. Binding of proprotein convertase subtilisin/kexin type Biol 2001;8:499–504. 9 to epidermal growth factor like repeat A of low density 129. Zhang L, Reue K, Fong LC, Young SG, Tontonoz P. Feedback lipoprotein receptor decreases receptor recycling and regulation of cholesterol uptake by the LXR-IDOL-LDLR axis. increases degradation. J Biol Chem 2007;282:18602–12. Arteriscler Thromb Vasc Biol 2012;32:2541–6. 146. Yamamoto T, Lu C, Ryan RO. A two step binding model of PCSK9 130. Zelcer N, Hong C, Boyadjian R, Tontonoz P. LXR regulates interaction with the low density lipoprotein receptor. J Biol cholesterol uptake through Idol-dependent ubiquitination of Chem 2011;286:5464–70. the LDL receptor. Science 2009;325:100–4. 147. Tveten K, Holla OL, Cameron J, Strom TB, Berge KE, Laerdahl JK, 131. Calkin AC, Goult BT, Zhang L, Fairall L, Hong C, Schwabe JW, et al. Interaction between the ligand-binding domain of the LDL et al. FERM dependent E3 ligase recognition is a conserved receptor and the C-terminal domain of PCSK9 is required for mechanism for targeted degradation of lipoprotein receptors. PCSK9 to remain bound to the LDL receptor during endosomal Proc Natl Acad Sci 2011;108:20107–12. acidification. Hum Mol Genet 2012;21:1402–9. 132. Rudenko G, Henry L, Henderson K, Ichtchenko K, Brown MS, 148. Kosenko T, Golder M, Leblond G, Weng W, Lagace TA. Low Goldstein JL, et al. Structure of the LDL receptor extracellular density lipoprotein binds to proprotein covertase subtilisin/ domain at endosomal pH. Science 2002;298:2353–8. kexin type 9 (PCSK9) in human plasma and inhibits PCSK9 133. Goldstein JL, Brown MS. The LDL receptor. Arterioscler Thromb mediated low density lipoprotein degradation. J Biol Chem Vasc Biol 2009;29:431–8. 2013;288:8279–88. 134. Motamed M, Zhang Y, Wang ML, Seeman J, Kwon HJ, 149. Mayer G, Poirier S, Seidah NG. Annexin A2 is a C-terminal Goldstein JL, et al. Identification of the luminal loop 1 of PCSK9-binding protein that regulates endogeneous low SCAP protein as the sterol sensor that maintains cholesterol density lipoprotein levels. J Biol Chem 2008;283:31791–801. homeostasis. J Biol Chem 2011;286:18002–12. 150. Wang Y, Huang Y, Hobbs HH, Cohen JC. Molecular character- 135. Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the isation of proprotein convertase subtilisin/kexin type 9-mediated complete program of cholesterol and fatty acid synthesis. J Clin degradation of the LDLR. J Lipid Res 2012;53:1932–43. Invest 2002;109:1125–31. 151. Lipari MT, Li W, Moran P, Kong-Beltran M, Sai T, Lai J, et al. 136. Scotti E, Hong C, Yoshinga Y, Tu Y, Hu Y, Zelcer N, et al. Targeted Furin-cleaved proprotein convertase subtilisin/kexin type disruption of the Idol gene alters cellular regulation of the low 9 (PCSK9) is active and modulates low density lipoprotein density lipoprotein receptor by sterols and liver X receptor receptor and serum cholesterol levels. J Biol Chem agonists. Mol Cell Biol 2011;31:1885–93. 2012;287:43482–91. 137. Gerin I, Clerbaux L, Haumont O, Lanthier N, Das AK, Burant 152. Garcia CK, Wilund K, Arca M, Zuliani G, Fellin R, Maioli M, CF, et al. Expression of mi33 from an SREBP2 intron inhibits et al. Autosomal recessive hypercholesterolemia caused by cholesterol export and fatty acid oxidation. J Biol Chem mutations in a putative LDL receptor adaptor protein. Science 2010;285:33652–61. 2001;292:1394–8. 1722 Ramasamy: Lipoprotein metabolism

153. Yamamoto T, Takahashi S, Sakai J, Kawarabayasi Y. The very 169. Clarke R, Peden JF, Hopewell JC, Kyriakou T, Goel A, Heath SC, low density lipoprotein receptor. Trends Cardiovasc Med et al. Genetic variants associated with Lp(a) lipoprotein level 1993;3:144–8. and coronary disease. New Engl J Med 2009;361: 154. Sakai J, Hoshino A, Takahashi S, Miura Y, Ishii H, Suzuki H, 2518–28. et al. Structure, chromosome location, and expression of the 170. Li Y, Luke MM, Shiffman D, Devlin JJ. Genetic variants in human very low density lipoprotein receptor gene. J Biol Chem the apolipoptotein(a) gene and coronary heart disease. 1994;269:2173–82. Circ Cardiovasc Genet 2011;4:565–73. 155. Takahashi S, Sakai J, Fujino T, Hattori H, Zenimaru Y, Suzuki J, 171. Kraft HG, Lingenhel A, Kochl S, Hoppichler F, Kronenberg F, et al. The very low density lipoprotein receptor: charac- Abe A, et al. Apolipoprotein(a) kringle IV repeat number terization and functions as a peripheral lipoprotein receptor. predicts risk for coronary heart disease. Arterioscler Thromb J Atheroscler Thromb 2004;11:200–8. Vasc Biol 1996;16:713–9. 156. Niemeier A, Gafvels M, Heeren J, Meyer N, Angelin B, 172. Hobbs HH, White AL. Lipoprotein(a): intrigues and insights. Beiseigel U. VLDL receptor mediates the uptake of human Curr Opin Lipidol 1999;10:225–36. chylomicron remnants in vitro. J Lipid Res 1996;37: 173. Lippi G, Franchini M, Targher G. Screening and therapeutic 1733–42. management of lipoprotein(a) excess: review of the epidemio- 157. Tao H, Aakula S, Abumrad NN, Hajri T. Peroxisome proliferator- logical evidence, guidelines and recommendations. Clin Chim activated receptor-? regulates the expression and function of Acta 2011;412:797–801. very low density lipoprotein receptor. Am J Physiol Endocrinol 174. Bennett A, Di Angelantonio E, Erquo S, Eiriksdottir G, Metab 2010;298:E68–79. Sigurdsson G, Woodward M, et al. Lipoprotein(a) levels and 158. Heeren J, Weber W, Beisiegel U. Intracellular processing of risk of future coronary heart disease: large scale prospective endocytosed triglyceride-rich lipoproteins comprises both data. Arch Intern Med 2008;168:598–608. recycling and degradation. J Cell Sci 1999;112:349–59. 175. The Emerging Risk Factors Collaboration. Lipoprotein(a) 159. Heeren J, Grewal T, Jackie S, Beisiegel U. Recycling of concentration and the risk of coronary heart disease, stroke apolipoprotein E and lipoprotein lipase through endosomal and nonvascular mortality. J Am Med Assoc 2009;302: compartments in vivo. J Biol Chem 2001;276:42333–8. 412–23. 160. Swift LL, Farkas MH, Major AS, Valyi-Nagy K, Linton MF, Fazio S. 176. Lamon-Fava S, Marcovina SM, Albers JJ, Kennedy H, De Luca C, A recycling pathway for resecretion of internalized apolipo- White CC, et al. Lipoprotein(a) levels, apo(a) isoform size, and protein E in liver cells. J Biol Chem 2001;276:22965–70. coronary heart disease risk in the Framingham Offspring Study. 161. Heeren J, Grewal T, Laatsch A, Rottke D, Rinninger F, Enrich C, J Lipid Res 2011;52:1181–7. et al. Recycling of apoprotein E is associated with cholesterol 177. Nordestgaard BG, Chapman MJ, Ray K, Boren J, Andreotti F, efflux and high density protein internalization. J Biol Chem Watts GF, et al. Lipoprotein(a) as a cardiovascular risk factor: 2003;278:14370–8. current status. Eur Heart J 2010;31:2844–53. 162. Heeren J, Grewal T, Laatsch A, Becker N, Rinninger F, Rye K, 178. Langlois MR. Laboratory approaches for predicting and et al. Impaired recycling of apolipoprotein E4 is associated managing the risk of cardiovascular disease: postanalytical with intracellular cholesterol accumulation. J Biol Chem opportunities of lipid and lipoprotein testing. Clin Chem Lab 2004;279:55483–92. Med 2012;50:1169–81. 163. Marcovina SM, Morrisett JD. Structure and metabolism of 179. Edelstein C, Davidson NO, Scanu AM. Oleate stimulates lipoprotein(a). Curr Opin Lipidol 1995;6:136–45. the formation of triglyceride rich particles containing 164. Rader DJ, Cain W, Ikewaki K, Talley G, Zech LA, Usher D, et al. apoB100-apo(a) in long term primary cultures of human The inverse association of plasma lipoprotein(a) concen- hepatocytes. Chem Phys Lipids 1994;67/68:135–43. trations with apolipoprotein(a) isoform size is not due to 180. Becker L, McLeod RS, Marcovina SM, Yao Z, Koschinsky ML. differences in Lp(a) catabolism but to differences in production Identification of a critical lysine residue in apolipo- rate. J Clin Invest 1994;93:2758–63. protein B-100 that mediates noncovalent interaction with 165. Valentini K, Aveynier E, Leaute S, Laporte F, Hadjian AJ. apolipoprotein(a). J Biol Chem 2001;276:36155–62. Contributions of apoprotein(a) size, pentanucleotide TTTTA 181. Callow MJ, Rubin EM. Site specific mutagenesis demonstrates repeat and C/T(+93) polymorphisms of the apo(a) gene to that cysteine 4326 of apolipoprotein B is required for regulation of lipoprotein(a) plasma levels in a population of covalent linkage with apoliporotein(a) in vivo. J Biol Chem young European Caucasians. Atherosclerosis 1999;147: 1995;270:23914–7. 17–24. 182. Cain WJ, Millar JS, Himebauch AS, Tietge UJ, Maugeais C, 166. Pati U, Pati N. Lipoprotein(a), atherosclerosis and Usher D, et al. Lipoprotein(a) is cleared from the apolipoprotein(a) gene polymorphisms. Mol Genet Metab plasma primarily by the liver in a process mediated by 2000;71:87–92. apolipoprotein(a). J Lipid Res 2005;46:2681–91. 167. Boerwinkle E, Leffert CC, Lin J, Lackner C, Chiesa G, Hobbs HH. 183. Tsimikas S, Hall JL. Lipoprotein(a) as a potential causal Apolipoprotein(a) gene accounts for greater than 90% of the genetic risk factor of cardiovascular disease. J Am Coll Cardiol variation in plasma lipoprotein(a) concentrations. J Clin Invest 2012;60:716–21. 1992;90:52–60. 184. Koschinsky ML, Marcovina S. Structure function relationships 168. Blanco-Vaca F, Gaubatz JW, Bren N, Kottke BA, Morrisett JD, in apolipoprotein(a): insights into lipoprotein(a) assembly and Guevara J. Identification and quantification of apoliprotein pathogenicity. Curr Opin Lipidol 2004;15:167–74. in addition to apo(a) and apoB100 in human lipoprotein (a). 185. Kang C, Dominguez M, Loyau S, Miyata T, Durlach V, Chem Phys Lipids 1994;67–68:35–42. Angles-Cano E. Lp(a) particles mold fibrin-binding properties Ramasamy: Lipoprotein metabolism 1723

of apo(a) in size dependent manner. Arterioscler Thromb Vasc 202. Kypreos KE, Zannis VI. Pathway of biogenesis of apolipoprotein Biol 2002;22:1232–8. E-containing HDL in vivo with the participation of ABCA1 and 186. Tsimikas S, Tsironis LD, Tselepsis AD. New insights into the LCAT. Biochem J 2007;403:359–67. role of lipoprotein (a) associated lipoprotein-associated 203. Saito H, Dhanasekaran P, Nguyen D, Deridder E, Holvoet P,

phospholipase A2 in atherosclerosis and cardiovascular Lund-Katz S, et al. a-Helix formation is required for high affinity disease. Arterioscler Thromb Vasc Biol 2007;27:2094–9. binding of human apolipoprotein A-I to lipids. J Biol Chem 187. Lippi G, Franchini M, Salvagno GL, Guidi GC. Lipoprotein(a) 2004;279:20974–81. and cancer: antineoplastic effect besides its cardiovascular 204. Castellini LW, Lusis AJ. ApoAII versus ApoAI. Two for one potency. Cancer Treat Rev 2007;33:427–36. is not always a good deal. Arterioscler Thromb Vasc Biol 188. Glomset JA. The plasma lecithin cholesterol transferase 2001;21:1870–2. reaction. J Lipid Res 1968;9:153–67. 205. Ajees AA, Anantharamaiah GM, Mishra VK, Hussain MM, 189. Timmins JM, Lee J, Boudyguina E, Kluckman KD, Brunham LR, Murthy HM. Crystal structure of human apolipoprotein A-I: Mulya A, et al. Targeted inactivation of hepatic Abca1 causes insights into its protective effect against cardiovascular profound hypoalphalipoproteinemia and kidney hyperca- disease. Proc Natl Acad Sci 2006;103:2126–31. tabolism of apoAI. J Clin Invest 2005;115:1333–42. 206. Frank PG, Marcel YL. Apolipoprotein A-I: structure-function 190. Vaisar T, Pennathur S, Green PS, Gharib SA, Hoofnagle AN, relationships. J Lipid Res 2000;41:853–72. Cheung MC, et al. Shotgun proteomics implicates protease 207. Chetty PS, Mayne L, Lund-Kutz S, Stranz D, Englander SW, inhibition and complement activation in the anti-inflammatory Phillips MC. Helical structure and stability in human apolipo- properties of HDL. J Clin Invest 2007;117:746–56. protein A-I by hydrogen exchange and mass spectrometry. Proc 191. Davidsson P, Hulthe J, Fagerberg B, Camejo G. Proteomics Natl Acad Sci 2009;106:19005–10. of apolipoproteins and associated proteins from plasma 208. Lagerstedt JO, Budamagunta MS, Oda MN, Voss JC. Electron high density lipoproteins. Arterioscler Thromb Vasc Biol paramagnetic resonance spectroscopy of site directed spin 2010;30:156–63. labels reveals the structural heterogeneity in the N-terminal 192. Cheung MC, Albers JJ. Characterization of lipoprotein particles domain of the apoAI in solution. J Biol Chem 2007;282:9143–9. isolated by immunoaffinity chromatography. Particles 209. Jayaraman S, Abe-Dohmae S, Yokoyama S, Cavigiolio G. Impact containing A-I and A-II and particles containing A-I but no A-II. of self association on function of apolipoprotein AI. J Biol Chem J Biol Chem 1984;259:12201–9. 2011;286:35610–23. 193. Brunham LR, Kruit JK, Iqbal J, Fievet C, Timmins JM, Pape TD, 210. Vedhachalam C, Ghering AB, Davidson WS, Lund-Katz S, et al. Intestinal ABCA1 directly contributes to HDL biogenesis in Rothblat GH, Phillips MC. ABCA1-induced cell surface vivo. J Clin Invest 2006;116:1052–62. binding sites for apoA-I. Arterioscler Thromb Vasc Biol 194. Oram JF, Vaughan AM. ATP-binding cassette cholesterol 2007;27:1603–9. transporters and cardiovascular disease. Circ Res 211. Hassan HH, Denis M, Lee DD, Iatan I, Nyholt D, Ruel I, et al. 2006;99:1031–43. Identification of an ABCA1-dependent phospholipid-rich 195. Mulligan JD, Flowers A, Tebon JJ, Bitgood C, Wellington C, plasma membrane apolipoprotein A-I binding site for nascent Hayden MR, et al. ABCA1 is essential for efficient basolateral HDL formation: implications for current models of HDL cholesterol efflux during the absorption of dietary cholesterol biogenesis. J Lipid Res 2007;48:2428–42. in chickens. J Biol Chem 2003;278:13356–66. 212. Mulya A, Seo J, Brown AL, Gebre AK, Boudyguina E, 196. Neufeld EB, Demosky SJ, Stonik JA, Combs C, Remaley AT, Shelness GS, et al. Apolipoprotein M expression increases Duverger N, et al. The ABCA1 transporter functions on the the size of nascent pre HDL formed by ATP binding cassette basolateral surface of hepatocytes. Biochem Biophys Res transporter A1. J Lipid Res 2010;51:514–21. Comm 2002;297:974–9. 213. Baldan A, Bojanic DD, Edwards PA. The ABC of sterol transport. 197. Field JF, Watt K, Mathur SN. Origins of intestinal J Lipid Res 2009;50:S80–5. ABCA1-mediated HDL cholesterol. J Lipid Res 2008;49: 214. Kielar D, Dietmaier W, Langmann T, Aslanidis C, Probst M, 2605–19. Naruszewicz M, et al. Rapid quantification of human ABCA1 198. Yamauchi Y, Chang CY, Hayashi M, Abe-Dohmae S, Reid PC, mRNA in various cell types and tissues by real time reverse Chang TY, et al. Intracellular cholesterol mobilisation involved transcriptase PCR. Clin Chem 2001;47:2089–97. in the ABCA1/apoplipoprotein-mediated assembly of high 215. Cavelier C, Lorenzi I, Rohrer L, von Eckardstein A. Lipid efflux density lipoprotein in fibroblasts. J Lipid Res 2004;45: by the ATP binding transportesr ABCA1 and ABCG1. Biochem 1943–51. Biophys Acta 2006;1761:655–66. 199. Hara H, Yokoyama S. Interaction of free apolipoproteins with 216. Zhao C, Dahlman-Wright K. Liver X receptor in cholesterol macrophages. Formation of high density lipoprotein like metabolism. J Endocrinol 2010;204:233–40. lipoproteins and reduction of cellular cholesterol. J Biol Chem 217. Santamarina-Fojo S, Peterson K, Knapper C, Qiu Y, Freeman L, 1991;266:3080–6. Cheng J, et al. Complete genomic sequence of the human 200. Remaley AT, Stonik SJ, Demosky EB, Neufeld EB, Bocharov TG, ABCA1 gene: analysis of human and mouse ATO binding Vishnyakova TL, et al. Apolipoprotein specificity for lipid efflux cassette A promoter. Proc Natl Acad Sci 2000;97:7987–92. by the human ABCA1 transporter. Biochem Biophys Res Comm 218. Costet P, Luo Y, Wang N, Tall AR. Sterol-dependent transac- 2001;280:818–23. tivation of ABC1 promoter by the liver X receptor/retinoid X 201. Mendez AJ, Anantharamaiah GM, Segrest JP, Oram JF. Synthetic receptor. J Biol Chem 2000;275:28240–5. amphipathic helical peptides that mimic apoplipoprotein A-I in 219. Chinetti G, Lestavel S, Bocher V, Remaley AT, Neve B, clearing cellular cholesterol. J Clin Invest 1994;94:1698–705. Torra IP, et al. PPAR-alpha and PPAR-gamma activators 1724 Ramasamy: Lipoprotein metabolism

induce cholesterol removal from human macrophage foam 236. Thomas MJ, Bhat S, Sorci-Thomas MG. Three dimensional cells through stimulation of the ABCA1 pathway. Nat Med models of HDL apoA-I: implications for its assembly and 2001;7:53–8. function. J Lipid Res 2008;49:1875–83. 220. Oram JF, Lawn RM, Garvin MR, Wade DP. ABCA1 is the cAMP 237. Wu Z, Gogonea V, Lee X, Wagner MA, Li X, Huang Y, et al. inducible apolipoprotein receptor that mediates cholesterol Double superhelix model of high density lipoprotein. J Biol secretion from macrophages. J Biol Chem 2000;275:34508–11. Chem 2009;284:36605–19. 221. Lu R, Arakawa R, Ito-Osumi C, Iwamoto N, Yokoyama S. ApoA-I 238. Chetty PS, Mayne L, Kan Z, Lund-Kutz S, Englander SW, facilitates ABCA1 recycle/accumulation to cell surface by Phillips MC. Apolipoprotein A-I helical structure and stability inhibiting its intracellular degradation and increases HDL in discoidal high-density lipoprotein (HDL) particles by generation. Arterioscler Thromb Vasc Biol 2008;28:1820–4. hydrogen exchange and mass spectrometry. Proc Natl Acad Sci 222. Roosbeek S, Peelman F, Verhee A, Labeur C, Caster H, 2012;109:11687–92. Lensink MF, et al. Phosphorylation by protein kinase CK2 239. Segrest JP, Jones MK, Catte A, Thirumuruganandham SP. modulates the activity of the ATP binding cassette A1 Validation of previous computer models and MD simulations of transporter. J Biol Chem 2004;279:37779–88. discoidal HDL by a recent crystal structure of apoAI. J Lipid Res 223. Lin Y, Ma C, Hsu W, Lo H, Yang VC. Molecular interaction 2012;53:1851–63. between caveolin-1 and ABCA1 on high density lipoprotein 240. Sorci-Thomas MG, Owen JS, Fulp B, Bhat S, Zhu X, Parks JS, mediated cholesterol efflux in aortic endothelial cells. et al. Nascent lipoproteins formed by ABCA1 resemble lipid Cardiovasc Res 2007;75:575–83. rafts and are structurally organized by three apoAI monomers. 224. Fu Y, Hoang A, Escher G, Parton RG, Krozowski Z, Sviridov D. J Lipid Res 2012;53:1890–909. Expression of caveolin-1 enhances cholesterol efflux in hepatic 241. Tall AR. Plasma high density lipoproteins. Metabolism and cells. J Biol Chem 2004;279:14140–6. relationship to atherogenesis. J Clin Invest 1990;86:379–84. 225. Kiss RS, McManus DC, Franklin V, Tan WL, McKenzie A, 242. Francone OL, Gurakar A, Fielding C. Distributions and functions Chimini G, et al. The lipidation by hepatocytes of human of lecithin: cholesterol acyltransferase and cholesteryl apolipoprotein A-I occurs by both ABCA1-dependent and ester transfer protein in plasma lipoproteins. J Biol Chem independent pathways. J Biol Chem 2003;278:10119–27. 1989;264:7066–72. 226. Neufeld EB, Remaley AT, Demosky SJ, Stonik JA, Cooney AM, 243. Lund-Katz S, Phillips MC. High density lipoprotein structure- Comly M, et al. Cellular localization and trafficking of human function and the role in reverse cholesterol transport. Subcell ABCA1 transporter. J Biol Chem 2001;276:27584–90. Biochem 2010;51:183–227. 227. Oram JF. The ins and outs of ABCA. J Lipid Res 2008;49:1150–1. 244. Silva RA, Huang R, Morris J, Fang J, Gracheva EO, Ren G, 228. Faulkner LE, Panagotopulos SE, Johnson JD, Woollett LA, et al. Structure of apolipoprotein AI in spherical high Hui DY, Witting SR, et al. An analysis of the role of retroendo- density lipoproteins of different sizes. Proc Natl Acad Sci cytosis pathway in ABCA1-mediated cholesterol efflux from 2008;105:12176–81. macrophages. J Lipid Res 2008;49:1322–32. 245. Wu Z, Gogonea V, Lee X, May RP, Pipich V, Wagner MA, et al. 229. Vedhachalam C, Duong PT, Nickel M, Nguyen D, The low resolution structure of apoAI in spherical high density Dhanasekaran P, Saito H, et al. Mechanism of ATP-binding lipoprotein revealed by small angle neutron scattering. J Biol cassette transporter A1-mediated cellular lipid efflux to Chem 2011;286:12495–508. apolipoprotein AI and formation of high density lipoprotein 246. Vuorela T, Catte A, Niemela PS, Hall A, Hyvonen MT, Marrink SJ, particles. J Biol Chem 2007;282:25123–30. et al. Role of lipids in spheroidal high density lipoproteins. 230. Wang N, Silver DL, Thiele C, Tall AR. ATP-binding cassette PLoS Comput Biol 2010;6:e1000964. transporter A1 (ABCA1) functions as a cholesterol efflux 247. Shih AY, Sligar SG, Schulten K. Maturation of high density regulatory protein. J Biol Chem 2001;276:23742–7. lipoproteins. J R Soc Interface 2010;6:863–71. 231. Vedhachalam C, Sevugun C, Nickel M, Dhanasekaran P, 248. Gu F, Jones MK, Chen J, Patterson JC, Catte A, Jerome WG, Lund-Katz S, Rothblat GH, et al. Influence of apolipoprotein et al. Structures of discoidal high density lipoproteins. A (apo) A-I structure on nascent high density lipoprotein (HDL) combined computational experimental approach. J Biol Chem particle size distribution. J Biol Chem 2010;285:31965–73. 2010;285:4652–65. 232. Duong PT, Weibel GL, Lund-Katz S, Rothblat GH, Phillips MC. 249. Wang L, Atkinson D, Small DM. The interfacial properties Characterization and properties of pre1-HDL particles formed of ApoAI and an amphipathic peptide of a-Helix consensus by ABCA1-mediated cellular lipid efflux to apoA-I. J Lipid Res peptide of exchangeable apolipoproteins at the triolein/water 2008;49:1006–14. interface. J Biol Chem 2005;280:4154–65. 233. Segrest JP, Jones MK, Klon AE, Sheldah CJ, Hellinger M, De 250. Chapman MJ, Le Goff W, Guerin M, Kontush A. Cholesteryl Loof H, et al. A detailed molecular belt model for apolipo- ester transfer protein: at the heart of the action of lipid- protein A-I in discoidal high density lipoprotein. J Biol Chem modulating therapy with statins, fibrates, niacin and 1999;274:31755–8. cholesteryl ester transfer protein inhibitors. Eur Heart J 234. Borhani DW, Rogers DP, Engler JA, Brouillette CG. Crystal 2010;31:149–64. structure of truncated human apolipoprotein A-I suggests a 251. Rashid S, Barrett HR, Uffelman KD, Watanabe T, Adeli K, lipid bound conformation. Proc Natl Acad Sci 1997;94:12291–6. Lewis GF. Lipolytically modified triglyceride-enriched HDLs are 235. Mei X, Atkinson D. Crystal structure of C-terminal rapidly cleared from circulation. Arterioscler Thromb Vasc Biol truncated apolipoprotein A-I reveals the assembly of high 2002;22:483–7. density lipoprotein (HDL) by dimerization. J Biol Chem 252. Lamarche B, Uffelman KD, Carpentier A, Cohn JS, Steiner G, 2011;286:38570–82. Barrett PH, et al. Triglyceride enrichment of HDL enhances in Ramasamy: Lipoprotein metabolism 1725

vivo metabolic clearance of HDL apoAI in healthy men. J Clin 270. Zannis VI, Chroni A, Krieger M. Role of Apo-I, ABCA1, LCAT and Invest 1999;103:1191–9. SR-BI in the biogenesis of HDL. J Mol Med 2006;84:276–94. 253. Morton RE. Cholesteryl ester transfer protein and its plasma 271. Sankaranarayanan S, Oram JF, Asztalos BF, Vaughan AM, regulator: lipid transfer inhibitor protein. Current Opin Lipidol Lund-Katz S, Adorni MP, et al. Effects of acceptor composition 1999;10:321–7. and mechanism of ABCG1-mediated cellular free cholesterol 254. Morton RE, Greene DJ. Conversion of lipid transfer inhibitor efflux. J Lipid Res 2009;50:275–84. protein (apolipoprotein F) to its active form depends on LDL 272. Tarling EJ, Edwards PA. ATP binding cassette transporter G1 composition. J Lipid Res 2011;52:2262–71. (ABCG1) is an intracellular sterol transporter. Proc Natl Acad Sci 255. Zhang L, Yan F, Zhang S, Lei D, Charles MA, Cavigiollo G, et al. 2011;108:19719–24. Structural basis of transfer between lipoproteins by cholesteryl 273. Weisgraber KH, Mahey RW. Subfractionation of human high ester transfer protein. Nat Chem Biol 2012;8:342–9. density lipoproteins by heparin-Sepharose affinity chroma- 256. Charles MA, Kane JP. New molecular insights into CETP tography. J Lipid Res 1980;21:316–25. structure and function: a review. J Lipid Res 2012;53:1451–8. 274. Matsuura F, Wang N, Chen W, Jiang X, Tall AR. HDL from 257. Liang H, Rye KA, Barter PJ. Dissociation of lipid free apolipo- CETP-deficient subjects shows enhanced ability to promote protein A-I from high density lipoproteins. J Lipid Res cholesterol efflux from macrophages in an apoE and ABCG1 1994;35:1187–99. dependent pathway. J Clin Invest 2006;116:1435–42. 258. Albers JJ, Vuletic S, Cheung MC. Role of plasma phospholipid 275. Zhao Y, Thorngate PE, Weisgraber KH, Williams DL, Parks JS. transfer protein in lipid and lipoprotein metabolism. Biochim Apolipoprotein E is the major physiological activator of Biophys Acta 2012;1821:345–57. lecithin-cholesteryl ester acyltransferase (LCAT) on lipoprotein 259. Rye K, Jauhiainen M, Barter PJ, Ehnholm C. Triglyceride- B lipoproteins. Biochemistry 2005;44:1013–25. enrichment of high density lipoproteins enhances their 276. Davidson WS, Silva RA. Apolipoprotein structural organization remodelling by phospholipid transfer protein. J Lipid Res in high density lipoproteins: belts, bundles, hinges and 1998;39:613–22 hairpins. Curr Opin Lipidol 2005;16:295–390. 260. Jiang X, Bruce C, Mar J, Lin M, Ji Y, Francone OL, et al. Targeted 277. Krieger M. Charting the fate of “good cholesterol” identi- mutation of plasma phospholipid transfer protein gene fication and characterisation of the high density lipoprotein markedly reduces high density lipoprotein levels. J Clin Invest receptor SR-BI. Ann Rev Bioch 1999;68:523–58. 1999;103:907–14. 278. Murao K, Terpstra V, Green SB, Kondratenko N, Steinberg D, 261. Settasatian N, Duong M, Curtiss LK, Ehnholm C, Jauhiainen M, Quehenberger O. Characterisation of CLA-1, a human Huuskonen J, et al. The mechanism of the remodeling of high homologue of rodent scavenger receptor BI as a receptor for density lipoproteins by phospholipid transfer protein. J Biol high density lipoprotein and apoptotic thymocytes. J Biol Chem Chem 2001;276:26898–905. 1997;272:17551–7. 262. Oram JF, Wolfbauer G, Vaughan AM, Tang C, Albers JJ. 279. Rigotti A, Miettinen HE, Krieger M. The role of high density Phospholipid transfer protein interacts with and stabilizes lipoprotein receptor SR-BI in the lipid metabolism of endocrine ATP-binding Cassette Transporter A1 and enhances cholesterol and other tissues. End Rev 2003;24:357–87. efflux from cells. J Biol Chem 2003;278:52379–85. 280. Cao G, Garcia CK, Wyne KL, Schultz RA, Parker KL, Hobbs HH. 263. Kawano K, Qin S, Lin M, Tall AR, Jiang X. Cholesteryl Structure and localisation of the human gene encoding SR-BI/ ester transfer protein and phospholipid transfer protein CLA-1. Evidence for the transcriptional control by steroidogenic have nonoverlapping functions in vivo. J Biol Chem factor 1. J Biol Chem 1997;272:33068–76. 2000;275:29477–81. 281. Lopez D, McLean MP. Sterol regulatory element-binding 264. Favari E, Calabresi L, Adorni MP, Jessup W, Simonelli S, protein 1a binds to cis elements in the promoter of the rat Franceschini G, et al. Small discoidal pre-beta1 HDL particles high density lipoprotein receptor SR-BI gene. Endocrinology are efficient acceptors of cell cholesterol via ABCA1 and ABCG1. 1999;140:5669–81. Biochemistry 2009;48:11067–74. 282. Malerod L, Juvet JK, Hanssen-Bauer A, Eskild W, Berg T. 265. Vaughan AM, Oram JF. ABCG1 redistributes cell cholesterol to Oxysterol activated LXRalpha/RXR induces hSRBI promoter domains removable by high density lipoprotein but not by lipid activity in hepatoma cells and preadipocytes. Biochem depleted apolipoproteins. J Biol Chem 2005;250:30150–7. Biophys Res Comm 2002;299:916–23. 266. Gelissen IC, Harris M, Rye KA, Quinn C, Brown AJ, Kockx M, 283. Schoonjans K, Annicoote JS, Huby T, Botrugno OA, Fayard E, et al. ABCA1 and ABCG1 synergize to mediate cholesterol export Ueda Y, et al. Liver receptor homolg 1 controls the expression to Apo A-I. Arterioscler Thromb Vasc Biol 2006;26:534–40. of the scavenger receptor class B type 1. Embo Rep 267. Kennedy MA, Barrera GC, Nakamura K, Baldan A, Tarr PT, 2002;3:1181–7. Fishbein MC, et al. ABCG1 has a critical role in mediating 284. Chinetti G, Gbaguidi FG, Griglio S, Mallat Z, Antonucci M, cholesterol efflux to HDL and preventing cellular lipid Poulain P, et al. CLA-1/SR-BI is expressed in atherosclerotic accumulation. Cell Metab 2005;1:121–31. lesion macrophages and regulated by activators of peroxisome 268. Wang N, Ranalletta M, Matsura F, Peng F, Tall AR. LXR induced proliferator-activated receptors. Circulation 2000;101:2411–7. redistribution of ABCG1 to plasma membrane in macrophages 285. Jian B, Llera-Moya M, Yong J, Wang N, Phillips MC, Swaney JB, enhances cholesterol mass efflux to HDL. Arterioscler Thromb et al. Scavenger Receptor Class B type I as a mediator of Vasc Biol 2006;26:1310–6. cellular cholesterol efflux to lipoproteins and phospholipid 269. Gelissen IC, Sharpe LJ, Sandoval C, Rao G, Kockx M, acceptors. J Biol Chem 1998;273:5599–606. Krithrides L, et al. Protein kinase A modulates the activity of a 286. Rigotti A, Trigatti BL, Penman M, Rayburn H, Herz J, Krieger M. major isoform of ABCG1. J Lipid Res 2012;53:2133–40. A targeted mutation in the murine gene encoding the high 1726 Ramasamy: Lipoprotein metabolism

density lipoprotein (HDL) receptor scavenger receptor class modified (OxLDL and AcLDL) lipoproteins. Arteriscler Thromb B I reveals its key role in HDL metabolism. Proc Natl Acad Sci Vasc Biol 1997;17:2341–9. 1997;94:12610–5. 302. Out R, Kruijt JK, Rensen PC, Hildebrand RB, de Vos P, Van 287. Huang ZH, Mazzone T. ApoE-dependent sterol efflux from Eck M, et al. Scavenger receptor B1 plays a role in facilitating macrophages is modulated by scavenger receptor class B type chylomicron metabolism. J Biol Chem 2004;279:18401–6. I expression. J Lipid Res 2002;43:375–82. 303. Van Eck M, Hoekstra M, Out R, Bos ST, Kruijt BJ, Hildebrand RB, 288. Thuanhai ST, Lund-Katz S, Anantharamaiah GM, Williams DL, et al. Scavenger receptor BI facilitates the metabolism of VLDL Phillips MC. A quantitative analysis of apolipoprotein binding lipoproteins in vivo. J Lip Res 2008;49:136–46. to SR-BI. J Lipid Res 2003;44:1132–42. 304. Saddar S, Mineo C, Shaul PW. Signaling by the high affinity 289. Yancey PG, Llera-Moya M, Swarnakar S, Monzo P, Klein SM, HDL receptor scavenger receptor B type I. Arterioscler Thromb Connelly MA, et al. High density lipoprotein phospholipid Vasc Biol 2010;30:144–50. composition is a major determinant of the bi-directional flux 305. Badellino KO, Wolf ML, Reilly MP, Rader DJ. Endothelial and net movement of cellular free cholesterol mediated by lipase concentrations are increased in metabolic syndrome scavenger receptor BI. J Biol Chem 2000;275:36596–604. and associated with coronary atherosclerosis. PLoS Med 290. Thuahnai ST, Lund-Katz S, Williams DL, Phillips MC. Scavenger 2006;3:e22. receptor class B, type I-mediated uptake of various lipids into 306. Connelly PW. The role of hepatic lipase in lipoprotein cells. J Biol Chem 2001;276:43801–8. metabolism. Clin Chim Acta 1999;286:243–55. 291. Gu X, Kozarsky K, Krieger M. Scavenger receptor class B, type 307. Saltiel AR, Kahn CR. Insulin signalling and the regulation of I-mediated [3H] cholesterol efflux to high and low density glucose and lipid metabolism. Nature 2001;414:799–806. lipoproteins is dependent on lipoprotein binding to the 308. Schade DS, Woodside W, Eaton RP. The role of glucagon in the receptor. J Biol Chem 2000;275:29993–30001. regulation of plasma lipids. Metabolism 1979;28:874–86. 292. Liu T, Krieger M, Kan H, Zannis VI. The effects of mutations in 309. Xiao C, Pavlic M, Szeto L, Patterson BW, Lewis GF. Effects of helices 4 and 6 of apoA-I on scavenger receptor class B type acute hyperglucagonemia on hepatic and intestinal lipoprotein I (SR-BI) mediated cholesterol efflux suggest that formation production and clearance in healthy humans. Diabetes of a productive complex between reconstituted high density 2011;60:383–90. lipoprotein and SR-BI is required for efficient transport. J Biol 310. Kamagate A, Qu S, Perdomo G, Su D, Kim DH, Slisher S, et al. Chem 2002;277:21576–84. FoxO1 mediates insulin dependent regulation of hepatic VLDL 293. Llera-Moya de la M, Rothblat GH, Connelly MA, Kellner- production in mice. J Clin Invest 2008;118:2347–64. Weibel G, Sakr SW, Phillips MC, et al. Scavenger receptor BI 311. Sparks JD, Chamberlain J, O’Dell C, Khatun I, Hussain MM, (SR-BI) mediates free cholesterol flux independently of HDL Sparks CE. Acute suppression of apoB secretion by insulin tethering to the cell surface. J Lipid Res 1999;40:575–80. occurs independently of MTP. Biochem Biophys Res Comm 294. Kellner-Weibel G, de La Llera-Moya M, Connelly MA, Stoudt G, 2011;406:252–6. Christian AE, Haynes MP, et al. Expression of scavenger 312. Pavlic M, Xiao C, Szeto L, Patterson BW, Lewis GF. Insulin receptor BI in COS-7 cells alters cholesterol content and acutely inhibits intestinal lipoprotein secretion in humans distribution. Biochemistry 2000;39:221–9. in part by suppressing plasma free fatty acids. Diabetes 295. Pagler TA, Rhode S, Neuhofer A, Laggner H, Strobi W, 2010;59:580–7. Hinterndorfer C, et al. SR-BI mediated high density lipoprotein 313. Rizos CV, Elisaf MS, Liberopoulos EN. Effects of thyroid (HDL) endocytosis leads to HDL resecretion facilitating dysfunction on lipid profile. Open Cadiovasc Med J 2011;5: cholesterol efflux. J Biol Chem 2006;281:11193–204. 76–84. 296. Chen W, Sliver DL, Smith JD, Tall AR. Scavenger receptor 314. Shin D, Osbourne TF. Thyroid hormone regulation and BI inhibits ATP-binding cassette transporter 1-mediated cholesterol metabolism are connected through sterol cholesterol efflux in macrophages. J Biol Chem regulatory element-binding protein-2 (SREBP-2). J Biol Chem 2000;275:30794–800. 2003;278:34114–8. 297. Yvan-Charvet L, Pagler TA, Wang N, Senokuchi T, Brundert M, 315. Tan KC, Shiu SW, Kung AW. Plasma cholesterol ester transfer Li H, et al. SR-BI inhibits ABCG1-stimulated net cholesterol protein activity in hyper- and hypothyroidism. J Clin Endo efflux from cells to plasma HDL. J Lipid Res 2008;49:107–14. Metab 1998;83:140–3. 298. Ji A, Wroblewski JM, Cai L, de Beer MC, Webb NR, van der 316. Prieur X, Huby T, Coste H, Schaap FG, Chapman MJ, Westhuyzen DR. Nascent HDL formation in hepatocytes and Rodriguez JC. Thyroid hormone regulates the hypoglyceridemic role of ABCA1, ABCG1 and SR-BI. J Lipid Res 2012;53:446–55. gene APOA5. J Biol Chem 2005;280:27533–43. 299. Larrede S, Quinn CM, Jessup W, Frisdal E, Olivier M, Hsieh V, 317. Fugier C, Tousaint J, Prieur X, Plateroti M, Samarut J, Delerive P. et al. Stimulation of cholesterol efflux by LXR agonists in The lipoprotein lipase inhibitor ANGPTL3 is negatively cholesterol-loaded human macrophages is ABCA1 dependent regulated by thyroid hormones. J Biol Chem 2006;281:11553–9. but ABCG1 independent. Arterioscler Thromb Vasc Biol 318. Cordeiro A, Souza LL, Einecker-Lamas M, Pazos-Moura CC. Non 2009;29:1930–6. classic thyroid hormone signalling involved in hepatic lipid 300. Adorni MP, Zimetti F, Billheimer JT, Wang N, Rader DJ, metabolism. J Endocrinol 2013;216:R47–57. Phillips MC, et al. The roles of different pathways in the release 319. Carmen GY, Victor SM. Signalling mechanisms in regulating of cholesterol from macrophages. J Lipid Res 2007;48:2453–62. lipolysis. Cell Signal 2006;18:401–8. 301. Calvo D, Coronado-Gomez D, Lasuncion MA, Vega MA. CLA-1 320. Villena JA, Roy S, Sarkadi-Nagy E, Kim K, Sui H. Desnutrin, an is an 85-kD plasma membrane glycoprotein that acts as a adipocyte gene encoding a novel patatin domain containing high affinity receptor for both native (HDL, LDL and VLDL) and protein, is induced by fasting and glucocorticoids. Ectopic Ramasamy: Lipoprotein metabolism 1727

expression of desnutrin increases triglyceride hydrolysis. J Biol and familial defective apolipoprotein B100. Proc Natl Acad Sci Chem 2004;279:47066–75. 1989;86:587–91. 321. Jaworski K, Sarkadi-Nagy E, Duncan RE, Ahmadian M, Sul HS. 328. Schonfeld G. Familial hypobetalipoproteinemia: a review. Regulation of triglyceride metabolism IV. Hormonal regulation J Lipid Res 2003;44:878–83. of lipolysis in adipose tissue. Am J Physiol Gastrointest Liver 329. Zhao ZY, Tuakli-Wosornu Y, Lagace TA, Kinch L, Grishin NV, Physiol 2007;293:G1–4. Horton JD, et al. Molecular characterization of loss of function 322. Barrows BR, Parks EJ. Contributions of different fatty acid mutations in PCSK9 and identification of a compound sources to very low density lipoprotein triacyl glycerol in the heterozygote. Am J Hum Genet 2006;79:514–23. fasted and fed states. J Clin Endo Metab 2006;91:1446–52. 330. Soufi M, Rust S, Walter M, Schaefer JR. A combined 323. Ruge T, Hodson L, Cheeseman J, Dennis AL, Fielding BA, LDLreceptor/LDL receptor adapter protein 1 mutation as the Humphreys SM, et al. Fasted to fed trafficking of fatty acids cause of familial hypercholesterolemia. Gene 2013;521:200–3. in human adipose tissue reveals a novel regulatory step for 331. Wierzbicki AS, Viljoen A, Hardman TC, Mikhailidis DP. New enhanced fat storage. J Clin Endo Metab 2009;94:1781–8. therapies to reduce low density lipoprotein cholesterol. Curr 324. Xiao C, Dash S, Morgantini C, Lewis GF. Novel role of enteral Opin Cardiol 2013;28:452–7. monosaccharides in intestinal lipoprotein production in healthy 332. Soufi M, Ruppert V, Kurt B, Schaefer JR. The impact of severe humans. Arterioscl Thromb Vasc Biol 2013;33:1056–62. LDL receptor mutations on SREBP-pathway regulation 325. Moller N, Jorgensen JO. Effects of growth hormone on glucose, in homozygous familial hypercholesterolemia. Gene lipid and protein metabolism in human subjects. Endo Rev 2012;499:218–22. 2009;30:152–77. 333. Uehara Y, Ando S, Yahiro E, Oniki K, Ayaori M, Abe S, et al. 326. Xiao C, Hsieh J, Adeli K, Lewis GF. Gut-liver interaction in FAMP, a novel ApoA-I mimetic peptide, suppresses aortic triglyceride-rich lipoprotein metabolism. Am J Physiol Endo plaque formation through promotion of biological HDL function Metab 2011;301:E429–46. in ApoE-deficient mice. J Am Heart Assoc 2013;2:e000048. 327. Soria LF, Ludwig EH, Clarke HR, Vega GL, Grundy SM, McCarthy 334. Johansen CT, Hegele RA. Genetic basis of hypertriglyceridemic BJ. Association between a specific apolipoprotein B mutation phenotypes. Curr Opin Lipidol 2011;22:247–53.

Indra Ramasamy obtained her BSc degree in biochemistry (First Class Hons) from the University of London and continued to do her PhD in enzymology at the same university. She trained as a clinical biochemist at the University of Surrey and South Wales, UK. She has worked in clinical laboratories in the UK, USA and Australia. She is currently Principal Biochemist at the Worcester Royal Hospital, UK. She is author and co-author of publications in the fields of enzymology, protein chemistry, molecular biology and hematology.