Background Alirocumab, a monoclonal antibody to proprotein convertase subtilisin/kexin type 9 (PCSK9), lowers plasma low density lipoprotein cholesterol (LDL-C) and apolipoprotein B100 (apoB). Although studies in mice and cells have identified increased hepatic LDL receptors as the basis for LDL lowering by PCSK9 inhibitors, there have been no human studies characterizing the effects of PCSK9 inhibitors on lipoprotein metabolism. In particular, it is not known if inhibition of PCSK9 has any effects on very low density lipoprotein (VLDL) or intermediate density lipoprotein (IDL) metabolism. Inhibition of PCSK9 also results in reductions of plasma Lp(a) levels. The regulation of plasma Lp(a) levels, including the role of LDL receptors (LDLRs) in the clearance of Lp(a), is poorly defined, and there have been no mechanistic studies of the Lp(a) lowering by alirocumab in humans. Methods Eighteen (10F, 8M) participants completed a placebo-controlled, two-period study. They received 2 doses of placebo, 2 weeks apart, followed by 5 doses of 150 mg of alirocumab, 2 weeks apart. At the end of each period, fractional clearance rates (FCR) and production rates (PR) of apoB and apo(a) were determined. In 10 participants, postprandial triglycerides (TG) and apoB48 levels were measured. Results Alirocumab reduced ultracentrifugally isolated LDL-C by 55.1%, LDL-apoB by 56.3%, and plasma Lp(a) by 18.7%. The fall in LDL-apoB was due to an 80.4% increase in LDL-apoB FCR and a 23.9% reduction in LDL-apoB PR. The latter was associated with a 46.1% increase in IDL-apoB FCR coupled with a 27.2% decrease in conversion of IDL to LDL. The FCR of apo(a) tended to increase (24.6%) without any change in apo(a) PR. Alirocumab had no effects on FCRs or PRs of VLDL-apoB and VLDL-TG, or on postprandial plasma TG or apoB48 concentrations. Conclusions Alirocumab decreased LDL-C and LDL-apoB by increasing IDL- and LDL-apoB FCRs, and decreasing LDL-apoB PR. These results are consistent with increases in LDLRs available to clear IDL and LDL from blood during PCSK9 inhibition. The possible increase in apo(a) FCR during alirocumab treatment suggests that increased LDLRs may also play a role in the reduction of plasma Lp(a). Clinical Trials Registration Clinical trials.gov # NCT01959971

Lipoprotein lipase (LPL), the rate limiting enzyme for hydrolysis of lipoprotein triglyceride, also mediates nonenzymatic interactions between lipoproteins and heparan sulfate proteoglycans. To determine whether cell surface LPL increases LDL binding to cells, bovine milk LPL was added to upregulated and nonupregulated human fibroblasts along with media containing LDL. LDL binding to cells was increased 2-10-fold, in a dosedependent manner, by the addition of 0.5-10 gg/ml of LPL.

Objective: Sodium glucose cotransporter 2 (SGLT2) inhibition in humans leads to increased levels of LDL cholesterol and decreased levels of plasma triglyceride. Recent studies however, have shown this therapy to lower cardiovascular mortality. In this study, we aimed to determine how SGLT2 inhibition alters circulating lipoproteins. Approach and Results: We used a mouse model expressing human cholesteryl ester transfer protein and human apolipoprotein B100 to determine how SGLT2 inhibition alters plasma lipoprotein metabolism. The mice were fed a high fat diet and then were made partially insulin deficient using streptozotocin. SGLT2 was inhibited using a specific anti-sense oligonucleotide or canagliflozin, a clinically available oral SGLT2 inhibitor. Inhibition of SGLT2 increased circulating levels of LDL cholesterol and reduced plasma triglyceride levels. SGLT2 inhibition was associated with increased lipoprotein lipase activity in the post heparin plasma, decreased postprandial lipemia and faster clearance of radiolabeled VLDL from circulation. Additionally, SGLT2 inhibition delayed turnover of labeled LDL from circulation. Conclusions: Our studies in diabetic CETP-Apolipoprotein B100 transgenic mice recapitulate many of the changes in circulating lipids found with SGLT2 inhibition therapy in humans and suggest that the increased LDL cholesterol found with this therapy is due to reduced clearance of LDL from the circulation and greater lipolysis of triglyceride-rich lipoproteins. Most prominent effects of SGLT2 inhibition in the current mouse model were seen with ASO mediated knockdown of SGLT2.

Cholesteryl ester transfer protein (CETP) is a hydrophobic plasma protein that promotes the bidirectional transfer of cholesteryl esters (CE) and triglycerides (TG) between and among HDL particles and atherogenic apolipoprotein B-containing (ApoB-containing) lipoproteins, including the predominantly TG-rich VLDL, intermediate-density lipoprotein (IDL), and LDL particles (1-3). Genetic deficiency of CETP is associated with elevated HDL cholesterol (HDL-C) and reduced LDL-C (1), and common variants at the CETP locus are associated with HDL-C and LDL-C in inverse directions (3). Pharmacologic inhibition of CETP activity in humans raises HDL-C levels and generally reduces LDL-C levels (4-7).The mechanism by which CETP inhibition reduces LDL-C remains unknown. A study of ApoB kinetics during administration of the CETP inhibitor torcetrapib (120 mg), with or without atorvastatin (ATV), to subjects with dyslipidemia (8) suggested that in dyslipidemic subjects, torcetrapib monotherapy reduced LDL ApoB by increasing the fractional catabolic rate (FCR) and that torcetrapib administered with ATV may have reduced production of LDL ApoB. However, none of these changes were statistically significant. Thus, the study was underpowered for detecting changes in many of the ApoB kinetic parameters and led to no firm conclusions regarding the mechanisms responsible for the lowering of ApoB.BACKGROUND. Individuals treated with the cholesteryl ester transfer protein (CETP) inhibitor anacetrapib exhibit a reduction in both LDL cholesterol and apolipoprotein B (ApoB) in response to monotherapy or combination therapy with a statin. It is not clear how anacetrapib exerts these effects; therefore, the goal of this study was to determine the kinetic mechanism responsible for the reduction in LDL and ApoB in response to anacetrapib. METHODS.We performed a trial of the effects of anacetrapib on ApoB kinetics. Mildly hypercholesterolemic subjects were randomized to background treatment of either placebo (n = 10) or 20 mg atorvastatin (ATV) (n = 29) for 4 weeks. All subjects then added 100 mg anacetrapib to background treatment for 8 weeks. Following each study period, subjects underwent a metabolic study to determine the LDL-ApoB-100 and proprotein convertase subtilisin/kexin type 9 (PCSK9) production rate (PR) and fractional catabolic rate (FCR). RESULTS.Anacetrapib markedly reduced the LDL-ApoB-100 pool size (PS) in both the placebo and ATV groups. These changes in PS resulted from substantial increases in LDL-ApoB-100 FCRs in both groups. Anacetrapib had no effect on LDL-ApoB-100 PRs in either treatment group. Moreover, there were no changes in the PCSK9 PS, FCR, or PR in either group. Anacetrapib treatment was associated with considerable increases in the LDL triglyceride/cholesterol ratio and LDL size by NMR. CONCLUSION.These data indicate that anacetrapib, given alone or in combination with a statin, reduces LDL-ApoB-100 levels by increasing the rate of ApoB-100 fractional clearance. TRIAL REGISTRATION. ClinicalTrials.gov NCT00990808.

Vessel wall subendothelial extracellular matrix, a dense mesh formed of collagens, fibronectin, laminin, and proteoglycans, has important roles in lipid and lipoprotein retention and cell adhesion. In atherosclerosis, vessel wall heparan sulfate proteoglycans (

Lipoprotein lipase (LpL), which facilitates lipoprotein uptake by macrophages, associates with the cell surface by binding to proteoglycans (PGs). Studies were designed to identify and characterize specific PGs that serve as receptors for LpL and to examine effects of cell differentiation on LpL binding. PG synthesis was examined by radiolabeling THP-1 monocytes and macrophages (a cell line originally derived from a patient with acute monocytic leukemia) with [35S]sodium sulfate and [3H]serine or [3H]glucosamine. Radiolabeled PGs isolated from the cell surface were purified by chromatography and identified as chondroitin-4-sulfate (CS) PG and heparan sulfate (HS) PG. A sixfold increase in CSPG and an 11-fold increase in HSPG accompanied cell differentiation. Whereas HS glycosaminoglycan chains from both monocytes and macrophages were 7.5 kD in size, CS chains increased in size from 17 kD to 36 kD with cell differentiation, and contained hexuronyl N-acetylgalactosamine-4,6-di-O sulfate disaccharides. LpL binding was sevenfold higher to differentiated cells, and affinity chromatography demonstrated that two cell surface PGs bound to LpL: HSPG and the oversulfated CSPG produced only by differentiated cells. We conclude that differentiation-associated changes in cell surface PG of human macrophages have functional consequences that could increase the atherogenic potential of the cells.

Lipoprotein lipase (LPL)1 is a 120-kDa dimeric protein that associates with the luminal surface of endothelial cells in multiple organs but especially in cardiac and skeletal muscle and in adipose tissue (1). This enzyme hydrolyzes the triglyceride in circulating lipoproteins such as chylomicrons and VLDL and produces free fatty acids that are used for metabolic energy or for fat storage. Endothelial cells do not synthesize LPL; rather myocytes and adipocytes produce it. Thus, it is a protein that requires transcytosis across the endothelial cell barrier, in this case from the interstitial fluid to the luminal side of the cells.There are several possible ways that LPL could cross the endothelial barrier. Nonspecific transport of molecules across endothelial monolayers occurs either via paracellular routes between the cells or via vesicular transit through cells (2). Alternatively, a specific transcytosis pathway could exist which requires LPL to associate with a cell surface receptor and then transports LPL through the cells. This process would be analogous to that which transfers IgA across epithelial cells (3). The first step in a specific LPL transcytosis pathway would involve LPL interaction with the basolateral side of endothelial cells. LPL binds to a number of cell surface molecules including heparan sulfate proteoglycans (HSPGs) and members of the LDL receptor family (4). In bovine endothelial cells the most highly expressed of these receptors is the VLDL receptor (VLDLr) (5). A previous study suggested that HSPGs are required for LPL transcytosis (6). It is, however, unclear whether HSPGs are sufficient for transport or whether HSPGs must operate in concert with receptors. The binding of LPL to several members of the LDL receptor family leads to uptake and degradation of LPL by cells. There are no data on whether these receptors participate in transendothelial movement of LPL or other ligands.In this report, we present data showing that LPL transcytosis across endothelial monolayers requires both HSPGs and the VLDLr. LPL transcytosis was diminished by removal of HSPGs and inhibition of receptors by RAP, a 39-kDa protein that was copurified with the LDL receptor-related protein (LRP) (7). This protein binds to members of the LDL receptor family and inhibits ligand binding and uptake by those receptors (8, 9). Furthermore, antibodies against the VLDLr blocked LPL translocation and increased expression of this receptor-increased transcytosis. Thus, LPL requires both HSPGs and receptors for translocation across endothelial cells. EXPERIMENTAL PROCEDURESPurification and Radioiodination of LPL-LPL was purified from unpasteurized bovine milk according to the method of Socorro et al. (10)

Objective Diabetic hypertriglyceridemia is thought to be primarily driven by increased hepatic de novo lipogenesis. However, experiments in animal models indicated that insulin deficiency should decrease hepatic de novo lipogenesis and reduce plasma triglyceride levels. Approach and Results To address the discrepancy between human data and genetically altered mouse models, we investigated whether insulin deficient diabetic mice had triglyceride changes that resemble those in diabetic humans. Streptozotocin (STZ)–induced insulin deficiency increased plasma triglyceride levels in mice. Contrary to the mouse models with impaired hepatic insulin receptor signalling, insulin deficiency did not reduce hepatic triglyceride secretion and de novo lipogenesis-related gene expression. Diabetic mice had a marked decrease in postprandial TG clearance, which was associated with decreased lipoprotein lipase (LpL) and PPARα mRNA levels in peripheral tissues and decreased LpL activity in skeletal muscle, heart and brown adipose tissue. Diabetic heterozygous LpL knockout mice had markedly elevated fasting plasma triglyceride levels and prolonged postprandial TG clearance. Conclusion Insulin deficiency causes hypertriglyceridemia by decreasing peripheral lipolysis and not by an increase in hepatic TG production and secretion.