Jari Metso

Phospholipid transfer protein mediated conversion of high density lipoproteins generates preβ1-HDL

High density lipoproteins (HDL) subclasses can be differentiated by two-dimensional non-denaturing polyacrylamide gradient gel electrophoresis (2D-PAGGE) and subsequent immunoblotting. The quantitatively minor HDL-subclasses pre beta 1-LpA-I and gamma-LpE are initial acceptors of cell-derived cholesterol into the plasma compartment. In this study we analysed the effect of phospholipid transfer protein (PLTP) on the electrophoretic distribution of HDL-subclasses in plasma as well as the ability of plasma, pre beta 1-LpA-I, and gamma-LpE to take up [3H]cholesterol from labeled fibroblasts. Pre beta 1-LpA-I but not gamma-LpE disappeared during a 16 hours incubation in the absence of PLTP. During a one minute incubation pre beta 1-LpA-I of pre-incubated plasma released 75% less [3H]cholesterol from radiolabeled fibroblasts than pre beta 1-LpA-I of control plasma. Pre-incubation of plasma reduced the uptake of [3H]cholesterol by gamma-LpE by 40%. Totally, the cholesterol efflux capacity of plasma decreased by 10% compared to the original sample. The amount of immunodetectable pre beta 1-LpA-I increased when plasma was incubated in the presence of PLTP while the amount of immunodetectable gamma-LpE did not change. After one minute incubation of PLTP-conditioned plasma with [3H]cholesterol-labeled fibroblasts, the amount of radioactive cholesterol taken up by pre beta 1-LpA-I was twice as high as in control plasma whereas the amount of [3H]cholesterol taken up by gamma-LpE remained unchanged. As a net result, treatment with PLTP increased the cholesterol efflux into total plasma by 40%. Together with results of previous studies our data suggest that the conversion of alpha-LpA-I3 into alpha-LpA-I2 by PLTP generates pre beta 1-LpA-I but not gamma-LpE. PLTP helps to enhance the uptake of cell-derived cholesterol by pre beta 1-LpA-I and, thereby, the cholesterol efflux capacity of normal plasma.

Apolipoprotein A-IV Polymorphism in the Finnish Population: Gene Frequencies and Description of a Rare Allele

The apolipoprotein A-IV (apoA-IV) allele frequencies were determined in 387 adult Finns by immunoblotting after isoelectric focusing of serum. The gene frequencies were: A-IV1 = 0.942 and A-IV2 = 0.058. The phenotypes of 147 mother-child pairs studied were in accordance with the two allelic modes of inheritance. In 2 subjects, a rare apoA-IV variant was found.

Oxysterol Binding Protein Induces Upregulation of SREBP-1c and Enhances Hepatic Lipogenesis

Background—Oxysterol binding protein (OSBP) has previously been implicated as a sterol sensor that regulates sphingomyelin synthesis and the activity of extracellular signal-regulated kinases (ERK). Methods and Results—We determined the effects of adenovirus-mediated hepatic overexpression of OSBP and its homologues ORP1L and ORP3 on mouse serum lipids. Whereas ORP1L and ORP3 had no effect on serum lipids, OSBP induced a marked increase of VLDL triglycerides (TG). Also, the liver tissue TG were elevated in the AdOSBP-injected mice, and their TG secretion rate was increased by 70%. The messenger RNAs for enzymes of fatty acid synthesis and their transcriptional regulator, SREBP-1c, as well as the Insig-1 mRNA, were upregulated two-fold in the OSBP-expressing livers. No change occurred in the messages of liver X receptor target genes ABCA1, ABCG5, and CYP7A1, and the Insig-2a mRNA was reduced. The phosphorylation of ERK was decreased in AdOSBP-infected liver and cultured hepatocytes. Importantly, silencing of OSBP in hepatocytes suppressed the induction of SREBP1-c by insulin and resulted in a reduction of TG synthesis. Conclusion—Our results demonstrate that OSBP regulates hepatic TG metabolism and suggest the involvement of OSBP in the insulin signaling pathways that control hepatic lipogenesis.

Angptl3 Deficiency Is Associated With Increased Insulin Sensitivity, Lipoprotein Lipase Activity, and Decreased Serum Free Fatty Acids

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.

Quantification of human plasma phospholipid transfer protein (PLTP): relationship between PLTP mass and phospholipid transfer activity

A sensitive sandwich-type enzyme-linked immunosorbent assay (ELISA) for human plasma phospholipid transfer protein (PLTP) has been developed using a monoclonal capture antibody and a polyclonal detection antibody. The ELISA allows for the accurate quantification of PLTP in the range of 25-250 ng PLTP/assay. Using the ELISA, the mean plasma PLTP concentration in a Finnish population sample (n = 159) was determined to be 15.6 +/- 5.1 mg/l, the values ranging from 2.30 to 33.4 mg/l. PLTP mass correlated positively with HDL-cholesterol (r = 0.36, P < 0.001), apoA-I (r = 0.37, P < 0.001), apoA-II (r = 0.20, P < 0.05), Lp(A-I) (r=0.26, P=0.001) and Lp(A-I/A-II) particles (r=0.34, P<0.001), and negatively with body mass index (BMI) (r = -0.28, P < 0.001) and serum triacylglycerol (TG) concentration (r = -0.34, P < 0.001). PLTP mass did not correlate with phospholipid transfer activity as measured with a radiometric assay. The specific activity of PLTP, i.e. phospholipid transfer activity divided by PLTP mass, correlated positively with plasma TG concentration (r=0.568, P<0.001), BMI (r=0.45, P<0.001), apoB (r = 0.45, P < 0.001). total cholesterol (r=0.42, P < 0.001), LDL-cholesterol (r = 0.34, P < 0.001) and age (r = 0.36, P < 0.001), and negatively with HDL-cholesterol (r= -0.33, P < 0.001), Lp(A-I) (r= -0.21, P < 0.01) as well as Lp(A-I/A-II) particles (r = -0.32, P < 0.001). When both PLTP mass and phospholipid transfer activity were adjusted for plasma TG concentration, a significant positive correlation was revealed (partial correlation, r = 0.31, P < 0.001). The results suggest that PLTP mass and phospholipid transfer activity are strongly modulated by plasma lipoprotein composition: PLTP mass correlates positively with parameters reflecting plasma high density lipoprotein (HDL) levels, but the protein appears to be most active in subjects displaying high TG concentration.

Macrophage Phospholipid Transfer Protein Contributes Significantly to Total Plasma Phospholipid Transfer Activity and Its Deficiency Leads to Diminished Atherosclerotic Lesion Development

Objective—Systemic phospholipid transfer protein (PLTP) deficiency in mice is associated with a decreased susceptibility to atherosclerosis, whereas overexpression of human PLTP in mice increases atherosclerotic lesion development. PLTP is also expressed by macrophage-derived foam cells in human atherosclerotic lesions, but the exact role of macrophage PLTP in atherosclerosis is unknown. Methods and Results—To clarify the role of macrophage PLTP in atherogenesis, PLTP was selectively disrupted in hematopoietic cells, including macrophages, by transplantation of bone marrow from PLTP knockout (PLTP−/−) mice into irradiated low-density lipoprotein receptor knockout mice. Selective deficiency of macrophage PLTP (PLTP−M/−M) resulted in a 29% (P<0.01 for difference in lesion area) reduction in aortic root lesion area as compared with mice possessing functional macrophage PLTP (384±36*103 &mgr;m2 in the PLTP−M/−M group (n=10), as compared with 539±35*103 &mgr;m2 in the PLTP+M/+M group (n=14)) after 9 weeks of Western-type diet feeding. The decreased lesion size in the PLTP−M/−M group coincided with significantly lower serum total cholesterol, free cholesterol, and triglyceride levels in these mice. Furthermore, plasma PLTP activity in the PLTP−M/−M group was 2-fold (P<0.001) lower than that in the PLTP+M/+M group. Conclusion—Macrophage PLTP is a significant contributor to plasma PLTP activity and deficiency of PLTP in macrophages leads to lowered atherosclerotic lesion development in low-density lipoprotein receptor knockout mice on Western-type diet.

Transformation of high density lipoprotein 2 particles by hepatic lipase and phospholipid transfer protein

High density lipoprotein 2 (HDL2) was incubated with phospholipid transfer protein (PLTP) or with hepatic lipase (H-TGL), and the incubation products were separated into a d < 1.22 g/ml and a d > 1.22 g/ml fractions. The d < 1.22 g/ml fraction produced by PLTP was larger, had lower apolipoprotein A-I and higher lipid and apolipoprotein A-II content than native HDL2. The d > 1.22 g/ml fraction represented 30% of the initial HDL2 protein and consisted of small, apolipoprotein A-I and phospholipid-rich particles, with a high sphingomyelin:phosphatidylcholine ratio. Incubation with H-TGL led to a d < 1.22 g/ml fraction which was comparable to native HDL2 regarding size and chemical composition. The d > 1.22 g/ml particles represented only 5% of the initial HDL2 protein and had slightly higher diameter and sphingomyelin:phosphatidylcholine ratio than those produced by PLTP. Enrichment of HDL2 with triglyceride prior to incubation increased the amount of protein released into the d > 1.22 g/ml fraction (20%) but had no effect on size and chemical composition of the particles. We conclude that PLTP and H-TGL promote the formation of small, pre-beta-like HDL particles from HDL2.

Acute-phase HDL in phospholipid transfer protein (PLTP)-mediated HDL conversion

In reverse cholesterol transport, plasma phospholipid transfer protein (PLTP) converts high density lipoprotein(3) (HDL(3)) into two new subpopulations, HDL(2)-like particles and prebeta-HDL. During the acute-phase reaction (APR), serum amyloid A (SAA) becomes the predominant apolipoprotein on HDL. Displacement of apo A-I by SAA and subsequent remodeling of HDL during the APR impairs cholesterol efflux from peripheral tissues, and might thereby change substrate properties of HDL for lipid transfer proteins. Therefore, the aim of this work was to study the properties of SAA-containing HDL in PLTP-mediated conversion. Enrichment of HDL by SAA was performed in vitro and in vivo and the SAA content in HDL varied between 32 and 58 mass%. These HDLs were incubated with PLTP, and the conversion products were analyzed for their size, composition, mobility in agarose gels, and apo A-I degradation. Despite decreased apo A-I concentrations, PLTP facilitated the conversion of acute-phase HDL (AP-HDL) more effectively than the conversion of native HDL(3), and large fusion particles with diameters of 10.5, 12.0, and 13.8 nm were generated. The ability of PLTP to release prebeta from AP-HDL was more profound than from native HDL(3). Prebeta-HDL formed contained fragmented apo A-I with a molecular mass of about 23 kDa. The present findings suggest that PLTP-mediated conversion of AP-HDL is not impaired, indicating that the production of prebeta-HDL is functional during the ARP. However, PLTP-mediated in vitro degradation of apo A-I in AP-HDL was more effective than that of native HDL, which may be associated with a faster catabolism of inflammatory HDL.

Absence of endogenous phospholipid transfer protein impairs ABCA1-dependent efflux of cholesterol from macrophage foam cells

In vitro experiments have demonstrated that exogenous phospholipid transfer protein (PLTP), i.e. purified PLTP added to macrophage cultures, influences ABCA1-mediated cholesterol efflux from macrophages to HDL. To investigate whether PLTP produced by the macrophages (i.e., endogenous PLTP) is also part of this process, we used peritoneal macrophages derived from PLTP-knockout (KO) and wild-type (WT) mice. The macrophages were transformed to foam cells by cholesterol loading, and this resulted in the upregulation of ABCA1. Such macrophage foam cells from PLTP-KO mice released less cholesterol to lipid-free apolipoprotein A-I (apoA-I) and to HDL than did the corresponding WT foam cells. Also, when plasma from either WT or PLTP-KO mice was used as an acceptor, cholesterol efflux from PLTP-KO foam cells was less efficient than that from WT foam cells. After cAMP treatment, which upregulated the expression of ABCA1, cholesterol efflux from PLTP-KO foam cells to apoA-I increased markedly and reached a level similar to that observed in cAMP-treated WT foam cells, restoring the decreased cholesterol efflux associated with PLTP deficiency. These results indicate that endogenous PLTP produced by macrophages contributes to the optimal function of the ABCA1-mediated cholesterol efflux-promoting machinery in these cells. Whether macrophage PLTP acts at the plasma membrane or intracellularly or shuttles between these compartments needs further study.

Mast cells promote atherosclerosis by inducing both an atherogenic lipid profile and vascular inflammation

Accumulating in vitro and in vivo studies have proposed a role for mast cells in the pathogenesis of atherosclerosis. Here, we studied the role of mast cells in lipoprotein metabolism, a key element in the atherosclerotic disease. Male mice deficient in low‐density lipoprotein receptors and mast cells on a Western diet for 26 weeks had significantly less atherosclerotic changes both in aortic sinus (55%, P = 0.0009) and in aorta (31%, P = 0.049), as compared to mast cell‐competent littermates. Mast cell‐deficient female mice had significantly less atherosclerotic changes in aortic sinus (43%, P = 0.011). Furthermore, we found a significant positive correlation between the extent of atherosclerosis and the number of adventitial/perivascular mast cells in aortic sinus of mast cell‐competent mice (r = 0.615, P = 0.015). Serum cholesterol and triglyceride levels were significantly lower in both male (63%, P = 0.0005 and 57%, P = 0.004) and female (73%, P = 0.00009 and 54%, P = 0.007) mast cell‐deficient mice, with a concomitant decrease in atherogenic apoB‐containing particles and serum preβ‐high‐density lipoprotein and phospholipid transfer protein activity in both male (69% and 24%) and female (74% and 54%) mast cell‐deficient mice. Serum soluble intercellular adhesion molecule was decreased in both male (32%, P = 0.004) and female (28%, P = 0.003) mast cell‐deficient mice, whereas serum amyloid A was similar between mast cell‐deficient and competent mice. In conclusion, mast cells participate in the pathogenesis of atherosclerosis in ldlr−/− mice by inducing both an atherogenic lipid profile and vascular inflammation. J. Cell. Biochem. 109: 615–623, 2010.