Objective-Angiopoietin-like 3 (Angptl3) is a regulator of lipoprotein metabolism at least by inhibiting lipoprotein lipase activity. Loss-of-function mutations in ANGPTL3 cause familial combined hypolipidemia through an unknown mechanism. Approach and Results-We compared lipolytic activities, lipoprotein composition, and other lipid-related enzyme/lipid transfer proteins in carriers of the S17X loss-of-function mutation in ANGPTL3 and in age-and sex-matched noncarrier controls. Gel filtration analysis revealed a severely disturbed lipoprotein profile and a reduction in size and triglyceride content of very low density lipoprotein in homozygotes as compared with heterozygotes and noncarriers. S17X homozygotes had significantly higher lipoprotein lipase activity and mass in postheparin plasma, whereas heterozygotes showed no difference in these parameters when compared with noncarriers. No changes in hepatic lipase, endothelial lipase, paraoxonase 1, phospholipid transfer protein, and cholesterol ester transfer protein activities were associated with the S17X mutation. Plasma free fatty acid, insulin, glucose, and homeostatic model assessment of insulin resistance were significantly lower in homozygous subjects compared with heterozygotes and noncarriers subjects. Conclusions-These results indicate that, although partial Angptl3 deficiency did not affect the activities of lipolytic enzymes, the complete absence of Angptl3 results in an increased lipoprotein lipase activity and mass and low circulating free fatty acid levels. This latter effect is probably because of decreased mobilization of free fatty acid from fat stores in human adipose tissue and may result in reduced hepatic very low density lipoprotein synthesis and secretion via attenuated hepatic free fatty acid supply. Altogether, Angptl3 may affect insulin sensitivity and play a role in modulating both lipid and glucose metabolism. Key Words: ANGPTL3 protein, human ◼ ANGPTL4 protein, human ◼ endothelial lipase, human ◼ familial combined hypolipidemia ◼ fatty acids, nonesterified ◼ hepatic lipase, human ◼ lipoprotein lipase

1. Phospholipid transfer protein (PLTP) mediates conversion of high-density lipoprotein (HDL3) to large particles, with concomitant release of apolipoprotein A-I (apoA-I). To study the mechanisms involved in this conversion, reconstituted HDL (rHDL) particles containing either fluorescent pyrenylacyl cholesterol ester (PyrCE) in their core (PyrCE-rHDL) or pyrenylacyl phosphatidylcholine (PysPC) in their surface lipid layer (PyrPC-rHDL) were prepared. Upon incubation with PLTP they behaved as native HDL3, in that their size increased considerably. 2. When PyrPC-rHDL was incubated with HDL3 in the presence of PLTP, a rapid decline of the pyrene excimer/monomer fluorescence ratio (E/M) occurred, demonstrating that PLTP induced mixing of the surface lipids of PyrPC-rHDL and HDL3. As this mixing was almost complete before any significant increase in HDL particle size was observed, it represents PLTP-mediated phospholipid transfer or exchange that is not directly coupled to the formation of large HDL particles. 3. When core-labelled PyrCE-rHDL was incubated in the presence of PLTP, a much slower, time-dependent decrease of E/M was observed, demonstrating that PLTP also promotes mixing of the core lipids. The rate and extent of mixing of core lipids correlated with the amount of PLTP added and with the increase in particle size. The enlarged particles formed could be visualized as discrete, non-aggregated particles by electron microscopy. Concomitantly with the appearance of enlarged particles, lipid-poor apoA-I molecules were released. These data, together with the fact that PLTP has been shown not to mediate transfer of cholesterol esters, strongly suggest that particle fusion rather than (net) lipid transfer or particle aggregation is responsible for the enlargement of HDL particles observed upon incubation with PLTP.4.ApoA-I rHDL, but not apoA-II rHDL, were converted into large particles, suggesting that the presence of apoA-I is required for PLTP-mediated HDL fusion. A model for PLTP-mediated enlargement of HDL particles is presented.

Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. Two forms of PLTP exist in human plasma, one catalytically active (high activity form, HA-PLTP) and the other inactive (low activity form, LA-PLTP) (Oka, T., Kujiraoka, T., Ito, M., Egashira, T., Takahashi, S., Nanjee, N. M., Miller, N. E., Metso, J., Olkkonen, V. M., Ehnholm, C., Jauhiainen, M., and Hattori, H. (2000) J. Lipid Res. 41, 1651-1657). The two forms are associated with macromolecular complexes of different size. The apparent size of LA-PLTP is 520 kDa and that of HA-PLTP is 160 kDa. Of the circulating PLTP mass only a minor portion is in the HA-PLTP form in normolipidemic subjects. In the present study we have isolated and partially characterized the LA and HA forms of PLTP. Both LA-and HA-PLTP bind to heparin-Sepharose and can be separated by elution with 0 -0.5 M NaCl gradient, with HA-PLTP displaying higher affinity for the matrix. LA-PLTP was further purified using hydrophobic butyl-Sepharose and anti-PLTP immunoaffinity chromatography steps. HA-PLTP was subjected to a second heparin-Sepharose step and hydroxylapatite chromatography. Analysis of the two forms of PLTP by SDS-PAGE, Western blotting, immunoprecipitation, and gel filtration demonstrates that LA-PLTP is complexed with apoA-I whereas HA-PLTP is not. Instead, HA-PLTP copurified with apoE. Based on these findings we suggest a model in which nascent PLTP enters the circulation as a high specific activity form not associated with apoA-I. During or after the transfer of lipolytic surface remnants to HDL, PLTP is transferred to apoA-I-containing HDL particles and thereby becomes part of the low activity complex.Both epidemiological and clinical studies provide strong evidence that low levels of high density lipoproteins (HDL) 1 is a major risk factor for the development of coronary heart disease (1-5). The ability of HDL to protect against atherosclerotic coronary artery disease is well documented, and although the exact molecular mechanism(s) behind this finding is still unsolved, it is thought to be due to the role of HDL in reverse cholesterol transport (6). The HDL in human plasma consist of several subpopulations of particles of distinct structure, function, and composition. This heterogeneity, which is the result of continuous remodeling of HDL by plasma factors, has important implications in terms of the cardioprotective functions of HDL (7). Plasma phospholipid transfer protein (PLTP) plays an essential role in the metabolism of HDL. Its role in the transfer of surface remnants from triglyceride-rich particles, very low density lipoproteins, and chylomicrons, to HDL during lipolysis is of importance for the maintenance of HDL levels (8 -10). It also modulates the size and composition of HDL particles (11, 12), a function important for the reverse cholesterol transport process. We recently reported the presence of two forms of PLTP in plasma (13), one catalytically active and the other inactive. Size-exclusion chromatography demonstrates th...

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