Expression of different lipoprotein receptors in natural killer cells and their effect on natural killer proliferative and cytotoxic activity

Natural killer (NK) cells take up chylomicrons (CM), very low density (VLDL), low density (LDL), high density (HDL) and acetyl-modified low density (AcLDL) lipoproteins through different receptors, VLDL being the lipoprotein with the highest uptake and HDL the lowest. The uptake of LDL can be selectively blocked by the anti-LDL receptor, which does not affect the uptake of CM, VLDL, HDL and AcLDL. Although the uptake of lipoproteins assessed by flow cytometry using DiI is not very high, the lipoproteins are able to induce an increase in proliferative responses, VLDL, AcLDL and HDL being the most important ones with 12- and 17-fold increments, respectively. CM, VLDL and LDL at low concentrations increase NK cytotoxic activity, while HDL and AcLDL inhibit, in a dose-dependent fashion, the killing of NK cells against K562. These results suggest the presence of four different receptors that are responsible for the cytotoxic and proliferative responses observed.     

The genome organization of the broad bean necrosis virus (BBNV)

In this study the effect of lipoprotein lipase (LPL) on the selective uptake of high density lipoprotein (HDL) cholesteryl esters (CE) by hepatic cells was investigated. Human HDL3 (d 1.125-1.21 g/ml) was radiolabeled with 125I in the protein moiety and with 3H in the CE moiety. LPL was prepared from bovine milk. Human hepatocytes in primary culture and human Hep3B hepatoma cells were incubated in medium containing doubly radiolabeled HDL3 with or without LPL. Without LPL, apparent HDL3 particle uptake according to the lipid tracer (3H) was in excess of that due to the protein label (125I) indicating selective CE uptake from HDL3. Addition of LPL increased selective CE uptake up to 7-fold. This stimulation of HDL3 selective CE uptake was independent of the lipolytic activity of LPL as suggested by several experimental approaches. Cell surface heparan sulfate proteoglycan deficiency decreased the LPL-mediated increase in selective CE uptake suggesting an important role for these molecules. In low density lipoprotein (LDL) receptor- or LDL receptor-related protein-(LRP)-deficient cells, LPL increased selective CE uptake as it did in normal cells yielding no evidence that these receptors play a role in the LPL effect on selective CE uptake. In summary, lipoprotein lipase increases the selective uptake of high density lipoprotein-associated cholesteryl ester by hepatic cells in culture. This effect is dependent on cell surface heparan sulfate proteoglycans but independent of lipolysis and of endocytosis mediated by low density lipoprotein receptor-related or low density lipoprotein receptors.     

Responses of dorsal column nuclei neurons in rats with experimental mononeuropathy

During nerve cell degeneration, cholesterol released from the degrading cells is conserved through the formation of a cholesterol-apolipoprotein (apo) E complex which is subsequently taken up by regenerating nerve cells. The aim of the present project was to identify the physiologically relevant lipoprotein receptor for this lipoprotein complex which has remained elusive. HDL was separated into apo E-rich and apo E-poor subfractions and labelled with [14C]-sucrose. Labelled apo E-rich HDL bound to rat brain membranes in a time- and ligand concentration-dependent manner and was a saturable process. Essentially no binding occurred with [14C]-apo E-poor HDL or with free apo E. Binding was partially inhibited by low density lipoprotein (LDL) and by alpha 2-macroglobulin. These results provide new evidence that native apoE-rich HDL particles resembling those present in the brain bind to rat brain membranes and that the binding may be due, at least in part, to the LDL receptor and to the LDL-receptor related protein. Evidence was also provided for the presence of a receptor which binds [14C]-sucrose human apoE-rich HDL in human brain. Characterisation of the receptor which mediates the uptake of cholesterol from HDL-like complexes by brain cells is important in understanding the role of apoE in the central nervous system and of the alterations which occur in disorders such as Alzheimer's disease.     

Inhibition of LPL expression in human monocyte-derived macrophages is dependent on LDL oxidation state: a key role for lysophosphatidylcholine

The regulation of macrophage lipoprotein lipase (LPL) secretion and mRNA expression by atherogenic lipoproteins is of critical relevance to foam cell formation. LPL is present in arterial lesions and constitutes a bridging ligand between lipoproteins, proteoglycans, and cell receptors, thus favoring macrophage lipoprotein uptake and lipid accumulation. We investigated the effects of native and of oxidized lipoproteins on the expression of LPL in an in vitro human monocyte-macrophage system. Exposure of mature macrophages (day 12) to highly copper-oxidized human low density lipoprotein (LDL) (100 microg protein per milliliter) led to marked reduction in the expression of LPL activity (-62%, P<0.01) and mRNA level (-47%, P<0.05); native LDL, acetylated LDL, and LDL oxidized for <6 hours were without effect. The reduction in LPL activity became significant at a threshold of 6 hours of LDL oxidation (-31%, P<0.05). Among the biologically active sterols formed during LDL oxidation, only 7beta-hydroxycholesterol (5 microg/mL) induced a minor reduction in macrophage LPL activity, whereas 25-hydroxycholesterol was without effect. By contrast, lysophosphatidylcholine, whose LDL content increased in parallel with the degree of oxidation, induced significant reductions in LPL activity and mRNA levels at concentrations of 2 to 20 micromol/L (-34% to -53%, P<0.01). Our results demonstrate that highly oxidized LDL (>6-hour oxidation) exerts negative feedback on LPL secretion in human monocytes-macrophages via a reduction in mRNA levels. By contrast, native LDL and mildly oxidized LDL (<6-hour oxidation) did not exert a feedback effect on LPL expression. We speculate that the content of lysophosphatidylcholine and, to a lesser degree, of 7beta-hydroxycholesterol in oxidized LDLs is responsible for the downregulation of LPL activity and mRNA abundance in human monocyte-derived macrophages and may therefore modulate LPL-mediated pathways of lipoprotein uptake during conversion of macrophages to foam cells.     

Abnormal activation of lipoprotein lipase by non-equilibrating apoC-II: further evidence for the presence of non-equilibrating pools of apolipoproteins C-II and C-III in plasma lipoproteins

Using artificial triglyceride emulsions, we have demonstrated the presence of non-equilibrating pools of apolipoproteins C-II and C-III in human plasma lipoproteins. As the concentrations of acceptor triglycerides were increased, a greater fraction of both apoC-II and apoC-III shifted away from the native plasma lipoproteins to the artificial lipid emulsions. All of the apoC-II and apoC-III in very low density and high density lipoproteins (VLDL and HDL), however, could not be removed from native plasma lipoproteins. The percent of total plasma apoC-II and apoC-III that could be recovered in the VLDL and HDL density fractions varied when plasma from different individuals was used. When plasma samples from normotriglyceridemic subjects were used, HDL was the primary donor of apoCs. The percent of total plasma apoCs associated with HDL decreased from 60% to 25% for apoC-II and from 65% to 15% for apoC-III. When plasma samples from hypertriglyceridemic subjects were incubated with artificial lipid emulsions, VLDL was the primary donor of apoCs. HDL from hypertriglyceridemic subjects only accounted for 5-10% of total fasting plasma apoCs and did not contribute significantly to the final apoC contents of the artificial triglyceride emulsions. To evaluate the significance of the depletion of exchangeable apoCs from plasma HDL, we also examined the ability of control and apoC-depleted HDL to serve as activator for bovine milk lipoprotein lipase (LPL) in vitro. When HDL depleted of exchangeable apoCs were used as the source of plasma apolipoproteins for the activation of LPL in vitro, only 5-10% of the maximal activity obtained with native HDL was demonstrated. In fact, in the presence of comparable concentrations of HDL apoC-II, activation of LPL was the least with HDL which lacked exchangeable apoCs. Our data thus indicated that the presence of exchangeable apoC-II on HDL is necessary for the activation of LPL in vitro. This finding is consistent with our data that suggest that HDL from hypertriglyceridemic subjects do not stimulate LPL as well as HDL from normolipidemic subjects.     

Hepatic lipase facilitates the selective uptake of cholesteryl esters from remnant lipoproteins in apoE-deficient mice

We have investigated the role of hepatic lipase (HL) in remnant lipoprotein metabolism independent of lipolysis by using recombinant adenovirus to express native and catalytically inactive HL (HL-145G) in apolipoprotein (apo)E-deficient mice characterized by increased plasma concentrations of apoB-48-containing remnants. In the absence of apoE, the mechanisms by which apoB-48-containing remnants are taken up by either low density lipoprotein (LDL)-receptor or LDL-receptor-related protein (LRP) remain unclear. Overexpression of either native or catalytically inactive HL in apoE-deficient mice led to similar reductions (P > 0.5) in the plasma concentrations of cholesterol (41% and 53%) and non high density lipoprotein (HDL)-cholesterol (41% and 56%) indicating that even in the absence of lipolysis, HL can partially compensate for the absence of apoE in this animal model. Although the clearance of [3H]cholesteryl ether from VLDL was significantly increased (approximately 2-fold; P < 0. 02) in mice expressing native or inactive HL compared to luciferase controls, the fractional catabolic rates (FCR) of [125I-labeled] apoB- very low density lipoprotein (VLDL) in all three groups of mice were similar (P > 0.4, all) indicating selective cholesterol uptake. Hepatic uptake of [3H]cholesteryl ether from VLDL was greater in mice expressing either native HL (87%) or inactive HL-145G (72%) compared to luciferase controls (56%). Our combined findings are consistent with a role for HL in mediating the selective uptake of cholesterol from remnant lipoproteins in apoE-deficient mice, independent of lipolysis. These studies support the concept that hepatic lipase (HL) may serve as a ligand that mediates the interaction between remnant lipoproteins and cell surface receptors and/or proteoglycans. We hypothesize that one of these pathways may involve the interaction of HL with cell surface receptors, such as scavenger receptor (SR)-BI, that mediate the selective uptake of cholesteryl esters.     

Decreased atherosclerosis in heterozygous low density lipoprotein receptor-deficient mice expressing the scavenger receptor BI transgene

Scavenger receptor type B class I (SR-BI), initially identified as a receptor that recognizes low density lipoprotein (LDL), was recently shown to mediate the selective uptake of high density lipoprotein (HDL) cholesteryl esters in liver and steroidogenic tissues. To evaluate effects on atherosclerosis, transgenic mice with liver-specific overexpression of SR-BI (SR-BI Tg mice) have been crossed onto LDL receptor-deficient backgrounds. To induce atherosclerosis in a setting of moderate hypercholesterolemia, heterozygous LDL receptor-deficient mice (LDLR1) were fed a high fat/cholesterol/bile salt diet, and homozygous LDL receptor knock-outs (LDLR0) were fed a high fat/cholesterol diet. LDLR1/SR-BI Tg mice showed decreases in VLDL, LDL, and HDL cholesterol and a significant 80% decrease in mean lesion area in the aortic root compared with LDLR1 mice (female LDLR1 74, 120 micrometers(2) versus LDLR1/SR-BI Tg 12, 667 micrometers(2); male 25, 747 micrometers(2)++ versus 5, 448 micrometers(2), respectively). LDLR0/SR-BI Tg mice showed decreased LDL and HDL cholesterol but increased VLDL cholesterol and no significant difference in extent of atherosclerosis compared with LDLR0 mice. Combined data analysis showed a strong correlation between atherosclerotic lesion area and the VLDL+LDL cholesterol level but no correlation with HDL level. These studies demonstrate a strong anti-atherogenic potential of hepatic SR-BI overexpression. In mice with marked overexpression of SR-BI, the protective effect appears to be primarily related to the lowering of VLDL and LDL cholesterol levels.     

In vivo metabolism of alpha-tocopherol in lipoproteins and liver: studies on rabbits in response to acute cholesterol loading

OBJECTIVE: To investigate the transport of alpha-tocopherol in lipoproteins of rabbits under normal diet and under acute loading of cholesterol. DESIGN: Two New Zealand White rabbits were fed 14C-alpha-tocopherol acetate in a single oral dose and the recovery of radiolabel in lipoproteins and plasma was monitored. Low density lipoprotein (LDL) from these animals was obtained and labeled with [3H] cholesteryl ester. Three other rabbits were injected with this double-labeled LDL in the native form; while three other animals received this LDL in the acetylated form. RESULTS: Plasma clearance, liver uptake and levels of radiolabel in high density lipoprotein (HDL) of animals injected with 14C[3H]acetyl LDL were significantly higher than those in animals injected with 14C[3H]native LDL. Larger particles of HDL, rich in apolipoprotein E (apoE) carried significantly higher levels of both labels in rabbits injected with acetylated LDL. CONCLUSION: These results provide evidence for in vivo mechanisms of "reverse alpha-tocopherol transport", analogous to "reverse cholesterol transport".     

Neutral lipid mass transfer among lipoproteins in plasma from normolipidemic subjects is not an equimolar heteroexchange

To further characterize the cholesteryl ester transfer protein (CETP)-mediated distribution of neutral lipids that occurs among lipoproteins in plasma, the net mass transfer of core lipids between donor and acceptor lipoproteins in intact plasma was measured in ten healthy normolipidemic subjects. The rate of loss of cholesteryl ester (CE) from high density lipoprotein-3 (HDL3) (19.5 +/- 8.8 nmol/ml per h) was linear and increased significantly (P < 0.01) during the 6-h incubation. Approximately 50% of the CE transferred from HDL3 (118.7 +/- 54.3 nmol/ml) went to very low density lipoprotein (VLDL); the remainder was distributed to low density lipoprotein (LDL) (approximately 30%) and HDL2 (approximately 20%). The rate of loss of triglyceride (TG) from VLDL (14.5 +/- 6.6 nmol/ml per h) to the HDL subfractions and LDL also was linear and increased significantly with time (P < 0.01). About 50% of the TG mass lost from VLDL (85.2 +/- 38.4 nmol/ml) was transferred to LDL and the remainder was recovered in HDL2 (approximately 10%) and HDL3 (approximately 40%). As the number of nmoles of CE lost from HDL3 was almost three times greater than the nmoles of TG it acquired, these findings indicate that the exchange of core lipids in plasma that result from the interaction between CETP-VLDL-HDL3 is not equimolar. Even in the absence of VLDL, HDL3 continued to donate CE to LDL and HDL2 to almost the same degree as in intact plasma (plasma minus VLDL: 17.5 +/- 5.9 nmol/ml per h vs. intact plasma: 20.2 +/- 7.5 nmol/ml per h) without accepting any TG. Our findings demonstrate that independent pathways exist for the transfer of CE and TG among the plasma lipoproteins and, contrary to what is generally believed, a heteroexchange of TG for CE during cholesteryl ester transfer is not obligatory.     

Low- and high-density lipoproteins as mitogenic factors for vascular smooth muscle cells: individual, additive and synergistic effects

The mitogenic activities of low (LDL)- and high (HDL)-density lipoproteins have been examined in cultures of human vascular smooth muscle cells (VSMC). LDL and HDL3 dose-dependently (EC50 values approximately 50 micrograms/ml) stimulated DNA and protein synthesis ([3H]-thymidine and [3H]-leucine incorporation, respectively) in the absence of exogenously added mitogens. The synthetic responses of VSMC to combinations of LDL and HDL3 were additive, indicating that each lipoprotein mediates discrete effects. LDL or HDL3 promoted VSMC proliferation under strict mitogen-free conditions, but this growth response was not sustained. VSMC exposed to combinations of lipoproteins (either LDL or HDL3) and growth factors (either PDGF-BB, EGF, bFGF or IGF) exhibited synergistic DNA synthesis responses. In the combined presence of PDGF-BB and either LDL or HDL3, VSMC proliferation was sustained. Anionized lipoprotein preparations (oxidized, acetylated, carbamylated or malonimylated) also stimulated DNA and protein synthesis. Since the antioxidant beta-hydroxylated toluene did not block the effect of native LDL on DNA synthesis, and fucoidin, a specific competitor for the 'scavenger' receptor, did not inhibit oxidized LDL-induced DNA synthesis, activation of mitogenic signals by lipoproteins does not depend on lipid peroxidation. Rather, the apparent intrinsic mitogenic potential of lipoproteins may depend upon their direct activation of replication-coupled signal transduction systems.     

Multiple processes are involved in the uptake of chylomicron remnants by mouse peritoneal macrophages

The processes responsible for the uptake of chylomicron remnants by macrophages were investigated using freshly isolated cells from low density lipoprotein (LDL) receptor, very low density lipoprotein (VLDL) receptor and apolipoprotein E knockout mice. In peritoneal macrophages from normal mice, the metabolism of chylomicron remnants was inhibited 40% by anti-LDL receptor antibody and 60% by a high concentration of receptor-associated protein (RAP). Together they reduced the amount processed by 70%. Digestion of cell proteoglycans decreased remnant degradation by 20% while the addition of acetyl-LDL had no effect. When LDL receptors were absent, the absolute rates of metabolism were less than that of normal cells and were not inhibited by the anti-LDL receptor antibody; the rates, however, were reduced to less than half by RAP. These suggest that the LDL receptor-related protein (LRP) or another LDL receptor family member(s) contributes to chylomicron remnant uptake and becomes the major mechanism of uptake when LDL receptors are absent. In contrast, the VLDL receptor was not involved as its absence did not affect chylomicron remnant metabolism. Similarly, the absence of apoE production did not affect the amount of remnant uptake; however, the proportion that was sensitive to RAP was eliminated. The level of LRP expression was not altered in these cells whereas there was a decrease in LDL receptors. This suggests that the apoE content of chylomicron remnants is sufficient for its recognition by LDL receptors but additional apoE is required for its uptake by the LRP and that there is an up-regulation of a non-LDL receptor family mechanism in apoE deficiency. Together these studies suggest that even in the absence of LDL receptors or apoE secretion, chylomicron remnants could contribute to lipid accumulation in the artery wall during atherogenesis.     

Nutritional status and aminoglycoside dosing

Serum amyloid P component (SAP) is a glycoprotein in human plasma. We previously showed that SAP is specifically localized in human atherosclerotic lesions, suggesting that SAP may play a role in atherogenesis. In this study, the interactions between human SAP and high density lipoprotein (HDL), low density lipoprotein (LDL) and very low density lipoprotein (VLDL) were investigated by using a solid phase plate assay. Biotinylated SAP bound to immobilized HDL and VLDL in a calcium-dependent, saturable manner. The SAP-HDL and SAP-VLDL bindings reached saturation at 4 nM and 16 nM of SAP, respectively. The bindings were inhibited by native SAP in a dose-dependent manner. No binding between SAP and LDL was found in the presence of calcium or EDTA, which indicates the specificity of SAP-lipoproteins interactions. These results suggest that the function of SAP is related to its capability to interact with lipoproteins and this may have important implications in atherosclerosis and in amyloidosis.     

Relationships between infant temperament, demographic variables, and family dynamics of childrearing families

The prothrombinase complex (factor [F]Xa, FVa, calcium ions, and lipid membrane) converts prothrombin to thrombin (FIIa). To determine whether plasma lipoproteins could provide a physiologically relevant surface, we determined the rates of FIIa production by using purified human coagulation factors, and isolated fasting plasma lipoproteins from healthy donors. In the presence of 5 nmol/L FVa, 5 nmol/L FXa, and 1.4 micromol/L prothrombin, physiological levels of very low density lipoprotein (VLDL) (0.45 to 0.9 mmol/L triglyceride, or 100 to 200 micromol/L phospholipid) yielded rates of 2 to 8 nmol Flla x L(-1) x s(-1) in a donor-dependent manner. Low density lipoprotein (LDL) and high density lipoprotein (HDL) also supported prothrombinase but at much lower rates (< or =1.0 nmol FIIa x L(-1) x s[-1]). For comparison, VLDL at 2 mmol/L triglyceride yielded approximately 50% the activity of 2X10(8) thrombin-activated platelets per milliliter. Although the FIIa production rate was slower on VLDL than on synthetic phosphatidylcholine/phosphatidylsenne vesicles (approximately 50 nmol FIIa x L(-1) x s[-1]), the prothrombin Km values were similar, 0.8 and 0.5 micromol/L, respectively. Extracted VLDL lipids supported rates approaching those of phosphatidylcholine/phosphatidylserine vesicles, indicating the importance of the intact VLDL conformation. However, the presence of VLDL-associated, factor-specific inhibitors was ruled out by titration experiments, suggesting a key role for lipid organization. VLDL also supported FIIa generation in an assay system comprising 0.1 nmol/L FVIIa; 0.55 nmol/L tissue factor; physiological levels of FV, FVIII, FIX, and FX; and prothrombin (3 nmol/L FIIa x L(-1) x s[-1]). These results indicate that isolated human VLDL can support all the components of the extrinsic coagulation pathway, yielding physiologically relevant rates of thrombin generation in a donor-dependent manner. This support is dependent on the intact lipoprotein structure and does not appear to be regulated by specific VLDL-associated inhibitors. Further studies are needed to determine the extent of this activity in vivo.     

Effect of lipoproteins on cultured human mesangial cells

It was recently reported that low-density lipoprotein (LDL) promotes mesangial cell proliferation, and oxidized LDL is cytotoxic for mesangial cells. However, there have been few studies about the effects of other lipoproteins on mesangial cells. Accordingly, we investigated the effect of various lipoproteins on cultured human mesangial cells using 3H-thymidine (3H-TdR) incorporation and cell counting assays. We also investigated the levels of several cytokines in mesangial cell culture supernatants after stimulation by the lipoproteins. Addition of very-low-density lipoprotein (VLDL) at concentrations up to 100 micrograms/mL, intermediate-density lipoprotein (IDL) at up to 50 micrograms/mL, and LDL at up to 50 micrograms/mL induced the proliferation of cultured human mesangial cells, whereas cell growth was inhibited at higher concentrations. Oxidized LDL caused a concentration-dependent decrease of 3H-TdR incorporation. High-density lipoprotein (HDL) had no proliferative effective effect at any concentration. Exposure to VLDL, IDL, LDL, or a high concentration of HDL enhanced the secretion of interleukin-6, platelet-derived growth factor, and transforming growth factor-beta by mesangial cells, whereas tumor necrosis factor-alpha secretion was stimulated by oxidized LDL. These finding indicate that triglyceride (TG)-rich lipoproteins (VLDL and IDL) promote mesangial cell proliferation as well as LDL, whereas oxidized LDL has the reverse effect. These effects of lipoproteins may be related to modulation of various cytokines. Accordingly, TG-rich lipoproteins, LDL, and oxidized LDL may be involved in mesangial cell proliferation and injury in patients with mesangial proliferative glomerulonephritis.     

Cholesteryl ester transfer protein gene polymorphism is a determinant of HDL cholesterol and of the lipoprotein response to a lipid-lowering diet in type 1 diabetes

The TaqIB cholesteryl ester transfer protein (CETP) gene polymorphism (B1B2) is a determinant of HDL cholesterol in nondiabetic populations. Remarkably, this gene effect appears to be modified by environmental factors. We evaluated the effect of this polymorphism on HDL cholesterol levels and on the lipoprotein response to a linoleic acid-enriched, low-cholesterol diet in patients with type 1 diabetes. In 44 consecutive type 1 diabetic patients (35 men), CETP polymorphism, apolipoprotein (apo) E genotype, serum lipoproteins, serum CETP activity (measured with an exogenous substrate assay, n = 30), clinical variables, and a diet history were documented. The 1-year response to diet was assessed in 14 type 1 diabetic patients, including 6 B1B1 and 6 B1B2 individuals. HDL cholesterol was higher in 10 B2B2 than in 14 B1B1 homozygotes (1.63 +/- 0.38 vs. 1.24 +/- 0.23 mmol/l, P < 0.01). HDL cholesterol, adjusted for triglycerides and smoking, was 0.19 mmol/l higher for each B2 allele present. CETP activity levels were not significantly different between CETP genotypes. Multiple regression analysis showed that VLDL + LDL cholesterol was associated with dietary polyunsaturated:saturated fatty acids ratio (P < 0.02) and total fat intake (P < 0.05) in the B1B1 homozygotes only and tended to be related to the presence of the apo E4 allele (P < 0.10). In response to diet, VLDL + LDL cholesterol fell (P < 0.05) and HDL cholesterol remained unchanged in 6 B1B1 homozygotes. In contrast, VLDL + LDL cholesterol was unaltered and HDL cholesterol decreased (P < 0.05) in 6 B1B2 heterozygotes (P < 0.05 for difference in change in VLDL + LDL/HDL cholesterol ratio). This difference in response was unrelated to the apo E genotype. Thus, the TaqIB CETP gene polymorphism is a strong determinant of HDL cholesterol in type 1 diabetes. This gene effect is unlikely to be explained by a major influence on the serum level of CETP activity, as an indirect measure of CETP mass. Our preliminary data suggest that this polymorphism may be a marker of the lipoprotein response to dietary intervention.     

Catalytically inactive lipoprotein lipase expression in muscle of transgenic mice increases very low density lipoprotein uptake: direct evidence that lipoprotein lipase bridging occurs in vivo

Lipoprotein lipase (LPL) is the central enzyme in plasma triglyceride hydrolysis. In vitro studies have shown that LPL also can enhance lipoprotein uptake into cells via pathways that are independent of catalytic activity but require LPL as a molecular bridge between lipoproteins and proteoglycans or receptors. To investigate whether this bridging function occurs in vivo, two transgenic mouse lines were established expressing a muscle creatine kinase promoter-driven human LPL (hLPL) minigene mutated in the catalytic triad (Asp156 to Asn). Mutated hLPL was expressed only in muscle and led to 3,100 and 3,500 ng/ml homodimeric hLPL protein in post-heparin plasma but no hLPL catalytic activity. Less than 5 ng/ml hLPL was found in preheparin plasma, indicating that proteoglycan binding of mutated LPL was not impaired. Expression of inactive LPL did not rescue LPL knock-out mice from neonatal death. On the wild-type (LPL2) background, inactive LPL decreased very low density lipoprotein (VLDL)-triglycerides. On the heterozygote LPL knock-out background (LPL1) background, plasma triglyceride levels were lowered 22 and 33% in the two transgenic lines. After injection of radiolabeled VLDL, increased muscle uptake was observed for triglyceride-derived fatty acids (LPL2, 1.7x; LPL1, 1.8x), core cholesteryl ether (LPL2, 2.3x; LPL1, 2.7x), and apolipoprotein (LPL1, 1.8x; significantly less than cholesteryl ether). Skeletal muscle from transgenic lines had a mitochondriopathy with glycogen accumulation similar to mice expressing active hLPL in muscle. In conclusion, it appears that inactive LPL can act in vivo to mediate VLDL removal from plasma and uptake into tissues in which it is expressed.     

Apolipoprotein A-I complexed with phospholipid promotes hepatic lipoprotein and apolipoprotein secretion in the perfused hamster liver

BACKGROUND: Apolipoprotein A-I within high density lipoprotein (HDL) plays a significant role in the process of reverse cholesterol transport from peripheral tissues to the liver. However, additional roles are not well defined for it in hepatic cholesterol metabolism. We have previously shown in the hamster that dietary cholesterol supplementation resulted in enhancement of apolipoprotein A-I (Apo A-I) in secreted nascent hepatic very low density lipoprotein (VLDL), suggesting that apolipoprotein A-I itself may play a role in hepatic lipoprotein secretion. METHODS: Using the isolated hamster liver with Apolipoprotein A-I perfusion, we then examined the hypothesis that Apo A-I alone or in association with phosphotidylcholine (PC) i.e., Apo A-I/PC as a HDL-like particle, has effects upon hepatic lipoprotein and bile secretion. Ultracentrifugation was performed on perfusate samples at 3 hours on control vs treated livers (Apo A-I/PC, Apo A-I, or PC) to access lipid and protein concentration in VLDL, low density lipoprotein (LDL) and HDL. Four to thirty percent gradient SDS polyacrylamide electrophoresis (PAGE) and Western blot analysis were used on delipidated lipoprotein fractions and microsomes to assess apolipoproteins Apo B, A-I, II, and E. RESULTS: We found that perfusion of reconstituted HDL vesicles containing human apolipoprotein A-I and PC (Apo A-I/PC) 10 mg and 10 mg, respectively, in 22 mL for 3 hours into isolated hamster liver increased cholesterol (CH) and triglyceride (TG) components in secreted HDL; 45- and 6-fold, and in LDL; 15- and 2-fold, respectively. No significant changes occurred in VLDL or in biliary lipids. Concomitantly, Apo A-I/PC perfusion increased Apo E and Apo A-II and HDL and Apo B in LDL, while Apo E decreased in VLDL. Apo A-I/PC perfusion did not change the apolipoprotein content of hepatic microsomes of the perfused liver. Perfusion of apolipoprotein A-I (without PC) or PC (without apolipoprotein A-I) had none of these effects. CONCLUSION: These results indicate that the perfused discoidal apolipoprotein A-I/PC particle affects hepatic lipoprotein assembly and secretion, whereby both lipid and apolipoprotein components are enhanced in secreted HDL and LDL of hepatic origin.     

Influence of plasma cholesteryl ester transfer activity on the LDL and HDL distribution profiles in normolipidemic subjects

The relations of cholesteryl ester transfer protein (CETP) activity to the distribution of low density lipoproteins (LDLs) and high density lipoproteins (HDLs) were investigated in fasting plasma samples from 27 normolipidemic subjects. LDL and HDL subfractions were separated by electrophoresis on 20-160 g/L and 40-300 g/L polyacrylamide gradient gels, respectively. Subjects were subdivided into two groups according to their LDL pattern. Monodisperse patterns were characterized by the presence of a single LDL band, whereas polydisperse patterns were characterized by the presence of several LDL bands of different sizes. To investigate the influence of lipid transfers on LDL patterns, total plasma was incubated at 37 degrees C in the absence of lecithin:cholesterol acyltransferase (LCAT) activity. The incubation induced a progressive transformation of polydisperse patterns into monodisperse patterns. Under the same conditions, initially monodisperse patterns remained unchanged. Measurements of the rate of radiolabeled cholesteryl esters transferred from HDL3s to very low density lipoproteins (VLDLs) and LDLs revealed that subjects with a monodisperse LDL pattern presented a significantly higher plasma CETP activity than subjects with a polydisperse LDL pattern (301 +/- 85%/hr per milliliter versus 216 +/- 47%/hr per milliliter, respectively; p < 0.02). In addition, when total plasma was incubated for 24 hours at 37 degrees C in the absence of LCAT activity, the relative mass of cholesteryl esters transferred from HDLs to apolipoprotein B-containing lipoproteins was greater in plasma with monodisperse LDL than in plasma with polydisperse LDL (0.23 +/- 0.06 versus 0.17 +/- 0.06, respectively; p < 0.02). These results indicated that in normolipidemic plasma, CETP could play an important role in determining the size distribution of LDL particles. The analysis of lipoprotein cholesterol distribution in the two groups of subjects sustained this hypothesis. Indeed, HDL cholesterol levels, the HDL:VLDL+LDL cholesterol ratio, and the esterified cholesterol:triglyceride ratio in HDL were significantly lower in plasma with the monodisperse LDL pattern than in plasma with the polydisperse LDL pattern (p < 0.01, p < 0.01, and p < 0.02, respectively). Plasma LCAT activity did not differ in the two groups. Plasma CETP activity correlated positively with the level of HDL3b (r = 0.542, p < 0.01) in the entire study population. Whereas plasma LCAT activity correlated negatively with the level of HDL2b (r = -0.455, p < 0.05) and positively with the levels of HDL2a (r = 0.475, p < 0.05) and HDL3a (r = 0.485, p < 0.05), no significant relation was observed with the level of HDL3b.(ABSTRACT TRUNCATED AT 400 WORDS)     

Relationship between lipoprotein lipase and high density lipoprotein cholesterol in mice: modulation by cholesteryl ester transfer protein and dietary status

Plasma lipoprotein lipase (LPL) activity correlates with high density lipoprotein (HDL) cholesterol levels in humans. However, in several mouse models created either through transgenesis or targeted inactivation of LPL, no significant changes in HDL cholesterol values have been evident. One possible explanation for this species difference could be the absence of plasma cholesteryl ester transfer protein (CETP) activity in mice. To explore this possibility and further investigate interactions between LPL and CETP modulating HDL cholesterol levels in vivo, we examined the relationship between LPL activity and HDL levels in mice expressing the simian CETP transgene, compared with littermates not carrying the CETP gene. On a chow diet, increasing LPL activity was associated with a trend towards increased HDL levels (51 +/- 29 vs. 31 +/- 4 mg/dL highest vs. lowest tertiles of LPL activity, P = 0.07) in mice expressing CETP, while no such effects were seen in the absence of CETP (65 +/- 12 vs. 61 +/- 15 mg/ dL). Furthermore, in the presence of CETP, a significant positive correlation between LPL activity and HDL cholesterol was evident (r = 0.15, P = 0.006), while in the absence of CETP no such correlation was detected (r = 0.15, P = 0.36), highlighting the interactions between LPL and CETP in vivo. When mice were challenged with a high fat, high carbohydrate diet, strong correlations between LPL activity and HDL cholesterol were seen in both the presence (r = 0.45, P = 0.03) and absence (r = 0.73, P < 0.001) of CETP. Therefore, under altered metabolic contexts, such as those induced by dietary challenge, the relation between LPL activity and HDL cholesterol may also become evident. Here we have shown that both genetic and environmental factors may modulate the association between LPL activity and HDL cholesterol, and provide explanations for the absence of any changes in HDL values in mice either transgenic or with targeted disruption of the LPL gene.     

Increases in dietary cholesterol and fat raise levels of apoprotein E-containing lipoproteins in the plasma of man

Previous studies from this laboratory have determined that diets containing the usual amounts of fat to which are added 750-1500 mg/day cholesterol elevate the plasma cholesterol concentration by variable amounts, depending upon the ratio of polyunsaturated to saturated fatty acids (P/S ratio) of the diet. Diets with P/S ratios of 0.25-0.4 are accompanied by elevations of low density lipoprotein (LDL) cholesterol, whereas diets with a P/S ratio of 2.5 produce no significant changes in cholesterol levels. On the low P/S ratio diets, the structure, composition, and interaction with cultured fibroblasts of LDL are not significantly changed. Plasma high density lipoprotein (HDL) cholesterol levels remain constant, but HDL2 increase relative to HDL3. In the present study, not only dietary cholesterol but also total dietary fat was altered. Six normal young men were fed a basal diet consisting of 18% protein, 51% carbohydrate, and 30% fat, containing 250 mg/day cholesterol. After 2 weeks, an experimental diet consisting of 18% protein, 42% carbohydrate, and 39% fat, containing 1760 mg/day cholesterol, was fed for 4 weeks. The P/S ratios of both diets were about 0.4. Plasma samples were taken twice during each dietary period from 12- to 14-h-fasted subjects and analyzed for their contents of lipoprotein lipids. Plasma levels of LDL and HDL cholesterol increased by 30 and 13 mg/dl, respectively; total and very low density lipoprotein (VLDL) triglyceride concentrations were unaltered. The plasma concentrations of apoproteins (apo) B, E. and A-I, but not A-II, were elevated. Plasma samples also were studied by zonal ultracentrifugation, gel permeation column chromatography, and Pevikon electrophoresis. Although on zonal ultracentrifugation the total concentrations of LDL were increased, the flotation properties and chemical compositions of LDL were not changed. By contrast, HDL2 and HDL3L concentrations increased, and HDL2 became enriched with cholesteryl esters. On gel permeation chromatography, with the subjects on the basal diet, plasma cholesterol eluted in two peaks, corresponding to LDL and HDL. The sizes of the peaks increased on the experimental diet. ApoE eluted in two peaks: one at the leading edge of LDL (corresponding to VLDL or IDL) and the other in the area between LDL and HDL, corresponding to HDLC. On the experimental diet, the apoE peak between LDL and HDL increased. On Pevikon electrophoresis apoE migrated between the LDL and HDL bands. This apoE peak was increased on the experimental diet. These findings suggest that increasing the concentrations of both dietary cholesterol and total fat can increase the levels of plasma LDL, HDL2, and HDLC in fasting normal subjects. Thus, the concentrations of some putatively atherogenic as well as antiatherogenic lipoproteins increased in plasma, and the apparent paradox between the epidemiological and metabolic behaviors of some lipoproteins remains. Clearly, more work is needed to resolve the roles of various lipoproteins in plasma in atherosclerosis.