L.M. Harada

The rise of the plasma lipid concentration elicited by dietary sodium chloride restriction in Wistar rats is due to an impairment of the plasma triacylglycerol removal rate

Studies in humans have indicated that dietary salt restriction raises plasma levels of total cholesterol (TC) and triacylglycerols (TAG). In order to explain the mechanisms involved, a rat experimental model was developed consisting of chronic feeding ad libitum isocaloric diets with variable sodium chloride contents. Rates of synthesis of plasma TAG were measured either as the increase of plasma TAG after blocking its removal from plasma by the intra-arterial pulse infusion of Triton-WR 1339, or as the plasma rate of incorporation of [(14)C]-oleic acid [(14)C]-TAG. Plasma TAG removal rate was determined by the intra-arterial pulse infusion of a lipid emulsion. Severe salt restriction increased the plasma concentrations of TAG (71%) and of TC (10%). This result was not due to modification of the rate of synthesis of plasma TAG but was attributed to a 55% slower rate of removal of the TAG-containing lipoproteins. An increased plasma non-esterified fatty acid concentration, probably due to a salt restriction-related insulin resistance, may have impaired the activity of the enzyme lipoprotein lipase.

Macrophages take up triacylglycerol-rich emulsions at a faster rate upon co-incubation with native and modified LDL: An investigation on the role of natural chylomicrons in atherosclerosis

Chylomicrons play a role in atherosclerosis, however, because the mechanisms involved in the cell uptake of these particles are not fully understood, investigations were carried out using a radioactively labeled protein‐free triacylglycerol‐rich emulsion incubated with peritoneal macrophages obtained from normal and apoE‐knockout mice. Experiments were done in the presence of substances that inhibit several endocytic processes: EDTA for low density lipoprotein receptor, fucoidan for scavenger receptor, cytochalasin B for phagocytosis, and a lipopolysaccharide for lipoprotein lipase. In addition, triacylglycerol‐rich emulsions were also prepared in the presence of native or modified radioactively labeled low density lipoprotein particles that are known to accumulate in the arterial intima. Probucol was also used to prevent the possible role played by an antioxidant in triacylglycerol‐rich emulsion uptake. We have shown that triacylglycerol‐rich emulsion alone is taken up by a coated‐pit‐dependent mechanism, mediated by macrophage secretion of apolipoprotein E. Furthermore, native, aggregated, acetylated, and moderately macrophage‐oxidized low density lipoprotein stimulate the uptake of a triacylglycerol‐rich emulsion through several mechanisms such as an actin‐dependent pathway, scavenger receptors, and lipolysis mediated by lipoprotein lipase. On the other hand, in spite of the interaction of low density lipoprotein forms with a triacylglycerol‐rich emulsion, the cellular triacylglycerol‐rich emulsion uptake is impaired by copper‐oxidized low density lipoprotein, possibly due to its diminished affinity towards lipoprotein lipase. We have also shown that macrophages take up aggregated low density lipoprotein better than the acetylated or oxidized forms of low density lipoprotein. J. Cell. Biochem. 84: 309–323, 2002.

Lipoprotein desialylation simultaneously enhances the cell cholesterol uptake and impairs the reverse cholesterol transport system: in vitro evidences utilizing neuraminidase-treated lipoproteins and mouse peritoneal macrophages

Desialylation of low density lipoprotein (LDL) brings about accumulation of cholesterol in cultured cells. The influence of the neuraminidase-treated lipoprotein (LP) on the reverse cholesterol transport system was investigated in vitro utilizing very low density lipoprotein (VLDL), LDL, total high density lipoprotein (HDL) and its subfractions, HDL2 and HDL3, isolated from healthy donor plasma and mouse peritoneal macrophages. It was found that LP desialylation significantly: (1) decreased the capacity of total HDL and of HDL2, but not of HDL3, to efflux cellular cholesterol; (2) lowered the cholesterol esterification rate by lecithin:cholesterol acyltransferase (LCAT) without modifying the intrinsic LCAT activity of HDL; (3) increased the cholesteryl ester transfer from HDL to apo B-containing LP mediated by cholesteryl ester transfer protein (CETP); (4) enhanced the uptake by macrophages of cholesterol from HDL and LDL, although the amount of cholesterol taken up by the cells was much greater from the desialylated LDL than from desialylated HDL. Taken together, these in vitro evidences indicate that, in addition to enhancing the cell cholesterol LP uptake, desialylation may contribute to the premature development of atherosclerosis by impairing the reverse cholesterol transport system.

Regulation of hepatic cholesterol metabolism in CETP/LDLr mice by cholesterol feeding and by drugs (cholestyramine and lovastatin) that lower plasma cholesterol.

1 The hepatic mechanisms involved in the simultaneous regulation of plasma cholesterol concentration and cholesteryl ester transfer protein (CETP) activity were investigated by sharply modifying the hepatic rates of cholesterol synthesis. This was accomplished by cholestyramine, lovastatin and cholesterol feeding in human CETP transgenic mice cross‐bred with low‐density lipoprotein receptor (LDLr)‐knockout mice, generating CETP+/–/LDLr+/– mice, which present a plasma lipoprotein profile resembling that of humans. 2 Analyses of pooled data showed that the plasma CETP activity correlated positively with plasma total cholesterol concentration, hepatic CETP mRNA and the liver microsomal cholesterol content; a negative correlation was found between plasma CETP activity and the liver 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase and LDLr mRNA levels. These coordinated events represent an efficient control system that stabilizes the cell cholesterol content. 3 Nonetheless, not all cholesterol metabolism regulatory systems seem to fit into a coherent pattern of responses, suggesting that other unknown cellular mechanisms play roles depending on the type of pharmacological intervention. 4 For example, microsomal cholesterol content was not affected by cholestyramine, but was increased on cholesterol feeding (as predicted), and, surprisingly, on lovastatin treatment. Furthermore, although both plasma cholesterol‐lowering drugs increased CYP7A1 mRNA and had no effect on CYP27 mRNA, other metabolic components were differentially modified. Cholestyramine and lovastatin, respectively, did not modify and increased both HMG‐CoA and sterol responsive element binding protein 1c mRNA, did not modify and lowered liver X receptor α mRNA, lowered and increased ATP binding cassette A1 mRNA and lowered and did not modify scavenger receptor B1 mRNA. 5 That is, different to unabsorbed cholestyramine, lovastatin, as an absorbed plasma cholesterol‐lowering drug, may have modified the activity of other unknown genes that play roles in the interaction of CETP with the metabolism of hepatic cholesterol.

Plasma lipases and lipid transfer proteins increase phospholipid but not free cholesterol transfer from lipid emulsion to high density lipoproteins.

BackgroundPlasma lipases and lipid transfer proteins are involved in the generation and speciation of high density lipoproteins. In this study we have examined the influence of plasma lipases and lipid transfer protein activities on the transfer of free cholesterol (FC) and phospholipids (PL) from lipid emulsion to human, rat and mouse lipoproteins. The effect of the lipases was verified by incubation of labeled (3H-FC,14C-PL) triglyceride rich emulsion with human plasma (control, post-heparin and post-heparin plus lipase inhibitor), rat plasma (control and post-heparin) and by the injection of the labeled lipid emulsion into control and heparinized functionally hepatectomized rats.ResultsIn vitro, the lipase enriched plasma stimulated significantly the transfer of 14C-PL from emulsion to high density lipoprotein (p<0.001) but did not modify the transfer of 3H-FC. In hepatectomized rats, heparin stimulation of intravascular lipolysis increased the plasma removal of 14C-PL and the amount of 14C-PL found in the low density lipoprotein density fraction but not in the high density lipoprotein density fraction. The in vitro and in vivo experiments showed that free cholesterol and phospholipids were transferred from lipid emulsion to plasma lipoproteins independently from each other. The incubation of human plasma, control and control plus monoclonal antibody anti-cholesteryl ester transfer protein (CETP), with 14C-PL emulsion showed that CETP increases 14C-PL transfer to human HDL, since its partial inhibition by the anti-CETP antibody reduced significantly the 14C-PL transfer (p<0.05). However, comparing the nontransgenic (no CETP activity) with the CETP transgenic mouse plasma, no effect of CETP on the 14C-PL distribution in mice lipoproteins was observed.ConclusionsIt is concluded that: 1-intravascular lipases stimulate phospholipid transfer protein mediated phospholipid transfer, but not free cholesterol, from triglyceride rich particles to human high density lipoproteins and rat low density lipoproteins and high density lipoproteins; 2-free cholesterol and phospholipids are transferred from triglyceride rich particles to plasma lipoproteins by distinct mechanisms, and 3 - CETP also contributes to phospholipid transfer activity in human plasma but not in transgenic mice plasma, a species which has high levels of the specific phospholipid transfer protein activity.

Cholesteryl ester transfer protein gene mutations in Brazilian hyperalphalipoproteinemia

To the Editor: Cholesteryl ester transfer protein (CETP) plays a central role in high-density lipoprotein (HDL) metabolism and is a key protein in the reverse cholesterol transport (1, 2). CETP facilitates the transfer of cholesteryl ester from HDL to apolipoprotein B-containing lipoproteins, and its deficiency is associated with hyperalphalipoproteinemia (HALP) (3, 4). Although the inverse association between HDL-cholesterol (HDL-C) concentrations and cardiovascular disease (CVD) is well established (5), the role of CETP in atherosclerosis remains controversial (6–8). Several mutations at the CETP gene locus have been described, which cause depletion of CETP activity and consequently high HDL-C in plasma (4, 9). HALP patients due to plasma CETP deficiency have been reported, mostly from Japan (3, 4, 8, 10), but there are some reports of CETP deficiency from German, Caucasian, and Asian populations (9). In this study, we investigated the prevalence of the most studied CETP gene mutations (intron 14 splicing defect, Int14A, and exon 15 missense mutation, D442G) in Brazilian HALP subjects (152 HALP and 139 controls, CTL). In addition, we evaluated the impact of each genetic mutation on the degree of carotid atherosclerosis, the concentrations of lipoproteins, the activities of CETP, phospholipid transfer protein (PLTP), and lipases in the plasma. For the identification of the CETP mutations, the genomic DNAs were extracted from peripheral leukocytes and analyzed by the polymerase chain reaction-restriction fragment length polymorphism method, as described previously (11–13). The Brazilian population is ethnically diverse, with a predominance of Afro-descendents. The frequency of the Int14A and D442G alleles in the HALP population was 0.023 and 0.0033, respectively. The prevalence of Int14A mutation was 4%, which was lower than that observed in the Japanese HALP population (32%) (14) but higher than that in the North-American HALP (0.7%) (9) and Japanese-American HALP (0.5%) subjects (6). The prevalence of the D442G mutation was far lower (0.7%) than that reported for the HALP (above 22%) and Japanese general population (4.5–7%) (4, 12) and for JapaneseAmerican subjects (5.1%) (6). Among the six Int14A mutation carriers (Table 1), we found one homozygote, a 61-yearold white woman, born from a non-consanguineous marriage, with family history of coronary artery disease (CAD), but no clinical cardiovascular damage. This is the first description of a homozygote Int14A CETP mutation outside Japan. Among the heterozygotes for Int14A, a 29-year-old male presented corneal arcus with established CAD and a 46-year-old female presented a carotid atheroma with no other manifestations of CVD. Both were whites, with no biochemical characteristics distinct from other mutation carriers. Three individuals had positive family histories of CAD. A 73-year-old male, from Asian origin and heterozygote for the D442G, presented results similar to the CTL group. Besides no personal or family register of CAD, he was the only one who presented increased intima-media thickness (IMT), possibly because he was the oldest. When we considered all mutation carriers together, higher HDL-C concentration (83%), lipoprotein lipase (LPL, 11%) and PLTP (60%) and lower CETP (36%) and hepatic lipase (HL, 26%) activities were observed. While the D442G carrier presented CETP, LPL, HL, and PLTP activities closer to the values from CTL group, the homozygote for Int14A mutation had an HL activity below the reference interval (2.5 and 97.5 percentiles of CTL), suggesting a double gene defect as described by Hirano et al. (7). The double deficiency of CETP and HL is Clin Genet 2006: 69: 455–457 # 2006 The Authors Printed in Singapore. All rights reserved Journal compilation # 2006 Blackwell Munksgaard

Moderate hyperalphalipoproteinaemia in a Brazilian population is related to lipoprotein lipase activity, apolipoprotein A-I concentration, age and body mass index

We investigated 95 Brazilian adults, aged 21-79 years, who were divided into two groups defined as having high-density lipoprotein (HDL)-cholesterol concentrations above [hyperalphalipoproteinaemia (HALP); n=48] or below (controls; n=47) the 90th percentile of a local population. The activities of lipid transfer proteins and enzymes involved in the plasma reverse cholesterol transport and the prevalence of factors that modulate HDL metabolism (alcohol consumption, ponderosity, physical exercise, menopause and use of hormone replacement treatment in women and smoking) were measured, as well as the prevalence of cardiovascular disease and of its various risk factors. The two groups showed no differences in their frequencies of cardiovascular disease. The HDL2/HDL3-cholesterol and triacylglycerol (triglyceride) ratios and the activities of the phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) were similar in both groups. Lipoprotein lipase (LPL) and hepatic lipase (HL) activities were 35% higher (P=0.0002) and 40% lower (P=0.0006) respectively, in HALP compared with control subjects. In a multivariate analysis, HDL-cholesterol and its subfractions were influenced by LPL, apolipoprotein A-I, age (negative relationship) and body mass index (negative relationship). Use of alcohol and ponderosity, as well as the interaction of these factors, explained the LPL activity. HL activity was modulated by smoking, and hormone-replacement therapy influenced the apolipoprotein A-I concentration. CETP activity was influenced by race and PLTP by age. The unique phenotype found in this Brazilian HALP population, namely low HL and high LPL activities, could be determined mostly by genetic components, on which future work will focus.

Soy protein containing isoflavones favorably influences macrophage lipoprotein metabolism but not the development of atherosclerosis in CETP transgenic mice

The possibility that soy protein containing isoflavones influences the development of experimental atherosclerosis has been investigated in ovariectomized mice heterozygous for the human CETP transgene and for the LDL-receptor null allele (LDLr+/− CETP+/−). After ovariectomy at 8 wk of age they were fed a fat/cholesterol-rich diet for 19 wk and divided into three experimental groups: dietary unmodified soy protein containing isoflavones (mg/g of diet), either at low-dose (Iso Low, 0.272, n=25), or at high-dose (Iso High, 0.535, n=28); and the atherogenic diet containing an isoflavone-depleted alcohol-washed soy protein as a control group (n=28). Aortic root lipid-stained lesion area (mean μm2×103±SD) did not differ among Iso Low (12.3±9.9), Iso High (7.4±6.4), and controls (10.7±12.8). Autoantibody titers against plasma oxidized LDL did not differ among the experimental groups. Using the control mice as the reference value (100%), in vitro mouse peritoneal macrophage uptake of labeled acetylated LDL-cholesterol was lower in the Iso High (68%) than in the Iso Low (85%) group. The in vitro percent removal by exogenous HDL of labeled unesterified cholesterol from macrophages previously enriched with human [4-14C]-cholesteryl oleate acetylated LDL was enhanced in the Iso High group (50%). In spite of these in vitro potentially antiatherogenic actions, soy protein containing isoflavones did not modify the average size of lipid-stained area in the aortic root.

Association of postalimentary lipemia with atherosclerotic manifestations

We identified different lipemic and metabolic responses after the ingestion of a standardized meal by healthy adults and related them to atherosclerotic markers. Samples from 60 normolipidemic adults were collected before and after a liquid meal (40 g fat/m2 body surface) at 0, 2, 4, 6, and 8 h for measurements of lipids, free fatty acids (FFA), insulin, cholesteryl ester transfer protein (CETP), autoantibodies to epitopes of oxidized LDL (oxLDL Ab), lipolytic activities, and apolipoprotein E polymorphism. Mean carotid intima-media thickness (cIMT) was determined by Doppler ultrasound. The volunteers were classified into early (N = 39) and late (N = 31) triacylglycerol (TAG) responders to the test meal. Late responders showed lower HDL cholesterol concentration at fasting and in the TAG peak, lower insulin and higher FFA concentrations compared to early responders. Multivariate regression analyses showed that mean cIMT was associated with gender (male) and age in early responders and by cholesterol levels at the 6th hour in late responders. oxLDL Ab were explained by lipoprotein lipase and negatively by hepatic lipase and oxLDL Ab (fasting period) by CETP (negative) and FFA (positive). This study is the first to identify a postalimentary insulin resistance state, combined with a reduced CETP response exclusively among late responders, and the identification of the regulators of postalimentary atherogenicity. Further research is required to determine the metabolic mechanisms described in the different postalimentary phenotypes observed in this study, as well as in different pathological states, as currently investigated in our laboratory.