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Epidemiological studies have shown a relationship between low concentrations of high density lipoprotein (HDL) cholesterol and the incidence risk of cardiovascular diseases1,2. Recent pharmacological studies3 have clearly demonstrated the protective role of HDL and their involvment in reverse cholesterol transport in vivo 4,5. In that respect apo E-free HDL has been long known to bind to a variety of cells and to promote cholesterol efflux6. Among peripheral tissues, adipose tissue is recognized both in man and rodents for its ability to accumulate, store and, when needed, mobilize a large pool of unesterified cholesterol7,8. Thus adipose cells represent a cell type suitable to study the first step in reverse cholesterol transport, i.e. cholesterol efflux. Unfortunately adipocytes isolated from adipose tissue loose their viability within a few hours, preventing the analysis of middle-term and long-term responses. During the last decade have been established in our laboratory preadipocyte cell lines from adipose tissue of genetically obese ob/ob mice9 and their lean counterpart10. The validity of these cellular models is supported by i) the biochemical properties of differentiated cells which are similar, if not identical, to those of adipocytes isolated from fat tissue and ii) the ability of undifferentiated cells to differentiate in vivo within a few weeks into fully mature fat cells after their injection into athymic mice, under conditions where these cells could be unambiguously demonstrated not to be fat cells originating from the host animal11.

Binding of human lipoproteins to cultured mouse Ob17 preadipose and adipose cells was studied, using labeled VLDL, LDL and apoprotein E-free HDL. In each case, saturation curves were obtained, yielding linear Scatchard plots. The K d values were found to be respectively 6.4, 31 and 24 μg/ml for VLDL, LDL and apoprotein E-free HDL, whereas the maximal numbers of binding sites per cell were 4.2 · 104, 1.5 · 104 and 2.5 · 105. The binding of 125I-LDL was competitively inhibited by LDL > VLDL > total HDL ; human LDL and mouse LDL were equipotent in competition assays. Methylated LDL and apoprotein E-free HDL were not competitors. In contrast, the binding of 125I-apoprotein E-free HDL was competitively inhibited by apoprotein E-free HDL > total HDL and the binding of 125I-HDL3 by mouse HDL. Thus, mouse adipose cells possess distinct apoprotein B, E and apoprotein E-free HDL binding sites which can recognize heterologous or homologous lipoproteins. The cell surface receptor of LDL in mouse preadipose cells shows similarities with that described for human fibroblasts, since: (1) the LDL binding initiated the process of internalization and degradation of the apoprotein B and apoprotein E-containing lipoproteins; (2) receptor-mediated uptake of cholesterol LDL led to a parallel but incomplete decrease in the [14C]acetate incorporation into cholesterol and in the activity of HMG-CoA reductase. Growing (undifferentiated) or growth-arrested cells (differentiated or not) showed no significant changes in the K d values for lipoprotein binding. In contrast, the maximal number of binding sites correlated with the proliferative state of the cells and was independent of cell differentiation. The results are discussed with respect to cholesterol accumulation in adipose cells.

Confluent Ob1771 cells treated with an inhibitor of spermidine and spermine synthesis, methylglyoxyal bis(guanylhydrazone), were dependent on putrescine addition for the expression of glycerol-3-phosphate dehydrogenase and acyl-CoA synthetase, which behaved as late markers of adipose conversion. A similar dependence was observed with drug-treated Ob17MT18 and 3T3-F442A preadipocyte cells, but not with non-differentiating 3T3-C2 cells. Studies in drug-treated Ob1771 cells at the mRNA level showed that the parallel expression of mRNAs encoding for glycerol-3-phosphate dehydrogenase and an homologue of serine proteinases of Mr 28,000 [Cook, Groves, Min & Spiegelman (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6480-6484] was also dependent on putrescine addition. Double-isotope experiments with [14C]putrescine and [3H]spermidine, as well as analysis of the polyamine content in drug-treated Ob1771 cells under various conditions, demonstrate after putrescine addition that the expression of late markers of adipose conversion was highly correlated with a 2-fold increase in the intracellular concentration of spermidine. No correlation was observed with changes in the intracellular concentrations of putrescine and spermine. Long-term exposure of untreated Ob1771 cells to growth hormone, which led to the expression of late markers of adipose conversion [Doglio, Dani, Grimaldi & Ailhaud (1986) Biochem. J. 238, 123-129] was also accompanied by the same increase in spermidine concentration, which attained values identical with those determined in drug-treated cells supplemented with putrescine. This observation suggests that the permissive effect of growth hormone on the terminal differentiation of adipose cells might e related to changes in the intracellular concentration of spermidine.

Binding of high-density lipoproteins to cultured mouse Ob1771 adipose cells was studied, using labeled human HDL3, mouse HDL and apolipoprotein AI- or AII-containing liposomes. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 18, 42, 30 and 3.4 micrograms/ml, whereas the maximal binding capacities were found to be 160, 100, 90 and 21 ng/mg of cell protein. Apoprotein AI not inserted into liposomes did not bind. The binding of 125I-HDL3 was competitively inhibited by apolipoprotein AI-containing liposomes greater than mouse HDL greater than HDL3. The binding of 125I-labeled apolipoprotein AI- and 125I-labeled apolipoprotein AII-containing liposomes was competitively inhibited by HDL3, apolipoprotein AI- and apolipoprotein AII-containing liposomes. Dimyristoylphosphatidylcholine liposomes containing or not cholesterol did not interfere with the binding of labeled HDL3 or apolipoprotein-containing liposomes. Binding studies on crude membranes of Ob1771 adipose cells revealed the presence of intracellular binding sites for LDL and HDL3. Thus, adipose cells have specific binding sites for apolipoprotein E-free HDL and apolipoprotein AI (or AII) is the ligand for these binding sites. Long-term exposure of adipose cells to LDL cholesterol as a function of LDL concentration led to an accumulation of cellular unesterified cholesterol. This process was saturable and reversible as a function of time and concentration by exposure to HDL3 or apolipoprotein AI-containing liposomes, whereas apolipoprotein AII-containing liposomes did not promote any cholesterol efflux. Since long-term exposure of adipose cells to LDL and HDL3 did not affect the number of apolipoprotein B,E receptors and apolipoprotein E-free binding sites, respectively, it appears that adipose cells do not show efficient cholesterol homeostasis and thus could accumulate or mobilize unesterified cholesterol.

Background CER-001 comprises recombinant human apolipoprotein A-I complexed with phospholipids that mimics natural, nascent, pre-β high-density lipoprotein (HDL). We present animal model data showing dose-dependent increases in cholesterol efflux with CER-001 and its subsequent elimination by reverse lipid transport, together with inhibition of atherosclerotic plaque progression. We report the first phase I study results with CER-001 in humans, starting at 0.25 mg/kg, which is 1/80th of the safe dose (20 mg/kg) established in 4-week multiple-dose animal studies dosed every second day. Methods Healthy volunteers, 18–55 years old with a low-density lipoprotein-cholesterol:HDL-cholesterol ratio greater than 3.0, received single intravenous escalating doses of CER-001 (0.25–45.0 mg/kg) and placebo in a double-blind randomised cross-over fashion. Subjects were followed up for 3 weeks post-dose. Assessments included adverse event monitoring, blood sampling, and clinical laboratory measurements. Results Thirty-two subjects were enrolled. All CER-001 doses (0.25–45 mg/kg) were safe and well tolerated, with an adverse event profile similar to placebo. Effects on clinical chemistry, haematology and coagulation parameters were comparable to placebo. No adverse effects of CER-001 on electrocardiograms were observed. No antibodies to apolipoprotein A-I were detected following single-dose administration of CER-001. Plasma apolipoprotein A-I levels increased in a dose-related manner and returned to baseline by 24 h post-dose for doses up to 10 mg/kg but remained in circulation for >72 h post-dose for doses >10 mg/kg. CER-001 caused elevations in plasma cholesterol and total and unesterified cholesterol in the HDL fraction. Mobilisation of unesterified cholesterol in the HDL fraction was seen with CER-001 at doses as low as 2 mg/kg. Conclusion CER-001 is well tolerated when administered to humans as single doses up to 45 mg/kg and mobilises and eliminates cholesterol via reverse lipid transport. Electronic supplementary material The online version of this article (doi:10.1007/s40261-017-0506-3) contains supplementary material, which is available to authorized users.

The regulation of cholesterol synthesis has been studied using a rat epithelial intestinal cell line (IRD 98) as a cellular model. As observed in other cell types, mevinolin increases the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) and concomitantly reduces the incorporation of [14C]acetate into cholesterol. Free cholesterol is able to suppress reductase activity. In contrast, when cells are shifted from standard culture medium to lipoprotein-deficient medium, an increase in hydroxymethyIglutaryl-CoA reductase specific activity (2–5-fold) is observed. The possible regulatory roles of the different classes of human lipoproteins were thus compared. The effects of a long-term exposure to LDL and HDL vary according to cell density. In actively growing cells, VLDL and LDL cause a decrease in the level of hydroxymethylglutaryl-CoA reductase, whereas HDL do not have a significant effect. In contrast, in subconfluent preresting cells, HDL provoke large decreases in hydroxymethylglutaryl-CoA reductase activity as compared to VLDL and LDL. While LDL binding is constant, the maximal binding capacity of HDL in subconfluent cells is seven times that of actively growing cells. Altogether, these results suggest an important role for HDL in the regulation of intestinal cholesterol synthesis.

The role of a high-affinity receptor site for high-density lipoproteins (HDL) has been investigated in parental Ob1771 adipose cells and their transformed counterparts after transfer of the complete early region of polyoma virus (Ob17PY cells). Binding of ApoAI, ApoAII and HDL3 occurs in Ob1771 cells and derived membranes, whereas no binding is observed in Ob17PY cells and derived membranes. After thymidine block, growth-arrested Ob17PY cells become able to bind ApoAI, ApoAII and HDL3; this recovery is prevented in actinomycin D- or cycloheximide-treated cells. In contrast to ApoAI, ApoAII or HDL3 binding, both growing and growth-arrested Ob17PY cells do show receptor activities for low density lipoproteins and transferrin, respectively, which are similar in affinity and maximal capacity. Following cholesterol accumulation which takes place in the presence of LDL cholesterol, subsequent exposure to HDL3 or ApoAI promotes cholesterol efflux from Ob1771 cells and growth-arrested Ob17PY cells but not from growing Ob17PY cells. These results show that the presence of a high-affinity receptor site for HDL in intact adipose cells is required for the promotion of reverse cholesterol transport.

Cholesterol efflux was studied in cultured adipose cells after preloading with LDL cholesterol. Long-term exposure to LpAI and LpAI:AII particles isolated from the HDL fraction showed that LpAI particles only were able to promote cholesterol efflux. Liposomes containing different ApoAI/ApoAII molar ratios were tested: the larger the proportion of ApoAI, the faster the ability to remove cholesterol from Ob1771 cells. Dose-response curves showed that LpAI particles were active within a physiological range of concentrations, whereas LpAI:AII particles had no effect at all concentrations. The results are in favour of LpAI particles being the active components of the HDL fraction for the promotion of cholesterol efflux and suggest that LpAI particles and LpAI:AII particles represent distinct metabolic entities.

Epidemiological studies have shown a relationship between low concentrations of high density lipoprotein (HDL) cholesterol and the incidence risk of cardiovascular diseases1,2. Recent pharmacological studies3 have clearl demonstrated the protective role of HDL in that respect. In the last decade much attention has been paid to the significance of apolipoprotein determination4,5. Thus, ApoAI, the major protein of HDL, appears to be a better predictor of coronary artery disease (CAD) than HDL cholesterol6,7. Two main types of lipoprotein particles are identified within HDL : those which contain ApoAI and ApoAII (LpAI:AII) and those which contain ApoAI but not ApoAII (LpAI). It has been shown quite recently that CAD subjects are characterized by a different distribution of ApoAI between LpAI and LpAI:AII, the data supporting the view that LpAI might represent the “antiatherogenic” fraction of HDL8.