Shinji Yokoyama

α-Helical requirements for free apolipoproteins to generate HDL and to induce cellular lipid efflux

The structural requirement has been studied for apolipoproteins in their free form to interact with cells, to generate high density lipoprotein (HDL), and to cause cellular lipid efflux (J. Biol. Chem. 266, 3080–3086, 1991). It is shown that human apolipoprotein (apo) A-IV and apolipophorin III ofManduca sexta cause cholesterol efflux from cholesterol-loaded mouse peritoneal macrophages and reduce intracellularly accumulated cholesteryl ester as a results of forming HDL-like particles with cellular lipids, as do apoA-I, A-II and E. On the other hand, similar to apoC-III, reduced-and-carboxymethylated human apoA-II had no such effect. Thus, apolipoproteins seem to require at least four amphiphilic helical segments per molecule to express this function.

Independent Regulation of Cholesterol Incorporation into Free Apolipoprotein-mediated Cellular Lipid Efflux in Rat Vascular Smooth Muscle Cells

Cholesterol was poorly available to free apolipoprotein (apo)A-I-mediated cellular lipid efflux from cholesterol-loaded rat vascular smooth muscle cells generating cholesterol-poorer pre-β-HDL particles than those generated from macrophages by the same reaction (Li, Q., Komaba, A., and Yokoyama, S. (1993) Biochemistry 32, 4597-4603). The factors known to induce transformation of the smooth muscle cells into a macrophage-like stage were used in order to modulate this reaction, such as human platelet-derived growth factor, macrophage colony-stimulating factor, and phorbol 12-myristate-13acetate (PMA). When the cells were stimulated by PMA following the pretreatment with platelet-derived growth factor plus macrophage colony-stimulating factor, cholesterol efflux mediated by free apoA-I increased 3-fold without changing phospholipid efflux, resulting in generation of pre-β-HDL particles more rich in cholesterol. This treatment had only a little or no effect on apparent cellular cholesterol efflux to HDL or lipid microemulsion, respectively. Overall cellular free cholesterol pool size was unaffected by the treatment, and probing by extracellular cholesterol oxidase did not detect gross change in the cellular surface cholesterol. This specific enrichment of cholesterol in the apoA-I-mediated cellular lipid efflux was reversed by protein kinase C inhibitors. Measurement of intracellular cholesterol esterification suggested that PMA induced translocation of intracellular cholesterol to a specific pool for apoA-I-mediated efflux, and a protein kinase C inhibitor reversed this effect.

Activation of human plasma cholestryl ester transfer protein by human apolipoprotein A-IV

Function of apolipoprotein (apo) A-IV was studied for its role in cholesteryl ester transfer protein (CETP; lipid transfer protein, LTP) reaction between lipid microemulsions having the diameter of low density lipoprotein, being compared to apoA-I. CETP hardly catalyzed lipid transfer without apolipoproteins. ApoA-IV bound to the surface of the microemulsion in equilibrium with a similar affinity to that of other helical apolipoproteins, and activated the transfer reaction by CETP of cholesteryl ester, triacylglycerol and phosphatidylcholine between the emulsions. The rate of the transfer reaction of cholesteryl ester and triacylglycerol was directly proportional to the amount of the bound apoA-IV to the surface of the emulsion. For phosphatidylcholine, activation was less effective until 40% of total binding capacity of lipid emulsion was occupied by the apolipoprotein. Cholesteryl ester was highly preferred by CETP over triacylglycerol when equal amount of these lipids was present in the core of the apoA-IV-activated emulsion, resulting in almost no triacylglycerol transfer. However, when the emulsion has the core exclusively of triacylglycerol, triacylglycerol was transferred by CETP with the rate in the same order as that of cholesteryl ester transfer. These findings were all comparable to the results with apoA-I, and also consistent with our previous observation for other amphiphilic helical apolipoproteins such as apoA-II, E and C-III.

Involvement of a cellular surface factor(s) in lipid-free apolipoprotein-mediated cellular cholesterol efflux

Involvement of cellular surface factors in cellular lipid efflux mediated by lipid-free apolipoprotein has been investigated. Lipid-free human apolipoprotein (apo) A-I generated net efflux of cholesterol and phospholipid from mouse peritoneal macrophages and rat aorta smooth muscle cells. Ratio of cholesterol to phospholipid was much lower in the lipid released by this mechanism from the smooth muscle cells than that from the macrophages, in agreement with our previous observation (Li, Q., Komaba, A. and Yokoyama, S. (1993) Biochemistry 32, 4597-4603). On the other hand, free apoA-I did not cause any lipid efflux from human erythrocytes. In contrast, apparent efflux of cellular cholesterol to HDL was similarly observed from all of these three cellular membranes. Trypsin treatment of the cultured macrophages completely inhibited apoA-I-mediated efflux of cholesterol and phospholipid. Smooth muscle cells also showed complete inhibition of the apoA-I-mediated cellular lipid efflux by trypsin treatment except that it required longer incubation with the enzyme. The same cellular treatment with trypsin even by prolonged incubation had only a limited effect on apparent cellular cholesterol efflux to HDL and apolipoprotein-free lipid microemulsions. Thus, free apolipoprotein-mediated cellular lipid efflux seems to depend on a trypsin-susceptible cellular surface factor(s) that erythrocytes may lack, being distinct from physicochemical cholesterol exchange reaction between cell and lipoprotein.

ENHANCEMENT OF THE HUMAN PLASMA LIPID TRANSFER PROTEIN REACTION BY APOLIPOPROTEINS

Transfer of cholesteryl ester between triacylglycerol/phospholipid microemulsions catalyzed by human plasma lipid transfer protein was investigated with a pyrene-containing analogue of which fluorescent properties depend on its concentration in the core of the microemulsions. The transfer of pyrene-cholesteryl ester between the emulsions was increased by the transfer protein linearly with its concentration, but maximally only to the extent of twice as much as spontaneous transfer in the given experimental conditions. When human apolipoproteins A-I or A-II are present in the reaction mixture enough to saturate the surface of the emulsion, the enhancement of the pyrene-cholesteryl ester transfer reaction by the transfer protein was 7.5-times more than in the absence of the apolipoproteins while the rate of spontaneous transfer was not affected significantly by the apolipoproteins. Bovine serum albumin did not have such an effect. Furthermore, the enhancement of the lipid transfer protein reaction by apolipoprotein A-I was linearly proportional to the percent saturation of the surface of the microemulsion with the apolipoprotein.

Regulation of the uptake of high-density lipoprotein-originated cholesteryl ester by HepG2 cells: Role of low-density lipoprotein and plasma lipid transfer protein

Cholesteryl ester uptake by the human hepatoma cell line HepG2 was studied in vitro by using radiolabeled cholesteryl ester as a tracer. After the cells were incubated in a lipoprotein deficient condition, the rate of radio labeled cholesteryl ester uptake from low-density lipoprotein (LDL) was estimated to be some 25-times higher than that from high-density lipoprotein (HDL). LDL-cholesteryl ester uptake was suppressed by preincubation of the cells with LDL, but pretreatment of the cells with HDL did not show significant effect. HDL-cholesteryl ester uptake was only slightly suppressed by pretreatment of the cells with LDL, and there was no effect with HDL pretreatment. HDL-cholesteryl ester uptake was not affected either by the presence of LDL or human plasma lipid transfer protein alone in the medium under our experimental conditions. Lipid transfer protein enhanced the uptake of radiolabeled cholesteryl ester originating from HDL by the cells only in the presence of LDL. Thus, lipid transfer protein catalyzes a bypass to LDL for the uptake by HepG2 cells of cholesteryl ester molecules which originate in HDL, and this pathway is much more efficient than direct uptake of cholesteryl ester originating in HDL by these cells.

Effect of serum lipid concentrations on restenosis after successful de novo percutaneous transluminal coronary angioplasty in patients with total cholesterol 160 to 240 mg/dl and triglycerides <350 mg/dl

The present study is the first to demonstrate a possible role of triglycerides and HDL cholesterol in the multifactorial process of restenosis using validated computerized quantitative angiographic analysis in a group of patients with normal or only mildly elevated total and LDL cholesterol. The results of this study require validation by a prospective study in unselected consecutive patients undergoing PTCA. If these results are confirmed, a prospective randomized trial would then be appropriate to test strategies to lower the total/HDL cholesterol ratio, possibly using combination therapy targeting triglycerides and HDL cholesterol as well as LDL cholesterol.

Modulation of substrate selectivity in plasma lipid transfer protein reaction over structural variation of lipid particle.

The modulation of substrate selectivity of human plasma LTP reaction is the subject of the present investigation. The moderate selectivity by a factor of 5 to 6 was observed in the LTP-catalyzed transfer of cholesteryl ester over triacylglycerol between plasma lipoproteins. On the other hand, the transfer of cholesteryl ester by LTP was highly selective over the negligible transfer of triacylglycerol, by a factor of 60 to 500, between the microemulsions with LDL size, regardless of the activators such as human and pig apolipoprotein (apo) A-I, human apo C-III and apo E that bound to the surface of the emulsion in equilibrium. The presence of free cholesterol in these microemulsions reduced slightly the rate of cholesteryl ester transfer but had no effect on triacylglycerol transfer. Other surface-active reagents such as cholic acid, Triton X-100 and Tween-20, did not have an effect on the triacylglycerol transfer either. Triacylglycerol transfer by LTP became measurable between such lipid particles as prepared by co-sonication of lipid with pig apo A-I and isolated as the mixed-microemulsions in the density of LDL and HDL. In these conditions, the substrate selectivity for cholesteryl ester over triacylglycerol was a factor of 6 to 16 mimicking the ratio in plasma lipoproteins. The conformation of pig apo A-I estimated by circular dichroism showed that its apparent helical content was further more induced when apo A-I was integrated into the mixed-microemulsion by co-sonication than the lipid-bound apo A-I in equilibrium. Apo A-I, thus integrated into lipid particles, was highly resistant to the denaturation by guanidine hydrochloride while the lipid-bound apo A-I in equilibrium was denatured as readily as the lipid-free protein. Thus, triacylglycerol transfer by LTP was induced by structural modulation of substrate-carrying lipid particles such as higher integration of apolipoproteins.