Aerobic exercise improves reverse cholesterol transport in cholesteryl ester transfer protein transgenic mice.

We analyzed the effect of a 6-week aerobic exercise training program on the in vivo macrophage reverse cholesterol transport (RCT) in human cholesteryl ester transfer protein (CETP) transgenic (CETP-tg) mice. Male CETP-tg mice were randomly assigned to a sedentary group or a carefully supervised exercise training group (treadmill 15 m/min, 30 min sessions, five sessions per week). The levels of plasma lipids were determined by enzymatic methods, and the lipoprotein profile was determined by fast protein liquid chromatography (FPLC). CETP activity was determined by measuring the transfer rate of ¹4C-cholesterol from HDL to apo-B containing lipoproteins, using plasma from CETP-tg mice as a source of CETP. The reverse cholesterol transport was determined in vivo by measuring the [³H]-cholesterol recovery in plasma and feces (24 and 48 h) and in the liver (48 h) following a peritoneal injection of [³H]-cholesterol labeled J774-macrophages into both sedentary and exercise trained mice. The protein levels of liver receptors were determined by immunoblot, and the mRNA levels for liver enzymes were measured using RT-PCR. Exercise training did not significantly affect the levels of plasma lipids or CETP activity. The HDL fraction assessed by FPLC was higher in exercise-trained compared to sedentary mice. In comparison to the sedentary group, a greater recovery of [³H]-cholesterol from the injected macrophages was found in the plasma, liver and feces of exercise-trained animals. The latter occurred even with a reduction in the liver CYP7A1 mRNA level in exercised trained animals. Exercise training increased the liver LDL receptor and ABCA-1 protein levels, although the SR-BI protein content was unchanged. The RCT benefit in CETP-tg mice elicited by exercise training helps to elucidate the role of exercise in the prevention of atherosclerosis in humans.

Pitfalls in the assessment of murine atherosclerosis: [review]

This review provides examples of the fact that different procedures for the measurement of atherosclerosis in mice may lead to interpretation of the extent of atherosclerosis having markedly different biological and clinical significance for humans: 1) aortic cholesterol measurement is highly sensitive for the detection of early and advanced atherosclerosis lesions, but misses the identification of the location and complexity of these lesions that are so critical for humans; 2) the histological analysis of the aortic root lesions in simvastatin-treated and control mice reveals similar lesion morphology in spite of the remarkable simvastatin-induced reduction of the aortic cholesteryl ester content; 3) in histological analyses, chemical fixation and inclusion may extract the tissue fat and also shrink and distort tissue structures. Thus, the method may be less sensitive for the detection of slight differences among the experimental groups, unless a more suitable procedure employing physical fixation with histological sample freezing using optimal cutting temperature and liquid nitrogen is employed. Thus, when measuring experimental atherosclerosis in mice, investigators should be aware of several previously unreported pitfalls regarding the extent, location and complexity of the arterial lesion that may not be suitable for extrapolation to human pathology.

Pitfalls in the assessment of murine atherosclerosis.

This review provides examples of the fact that different procedures for the measurement of atherosclerosis in mice may lead to interpretation of the extent of atherosclerosis having markedly different biological and clinical significance for humans: 1) aortic cholesterol measurement is highly sensitive for the detection of early and advanced atherosclerosis lesions, but misses the identification of the location and complexity of these lesions that are so critical for humans; 2) the histological analysis of the aortic root lesions in simvastatin-treated and control mice reveals similar lesion morphology in spite of the remarkable simvastatin-induced reduction of the aortic cholesteryl ester content; 3) in histological analyses, chemical fixation and inclusion may extract the tissue fat and also shrink and distort tissue structures. Thus, the method may be less sensitive for the detection of slight differences among the experimental groups, unless a more suitable procedure employing physical fixation with histological sample freezing using optimal cutting temperature and liquid nitrogen is employed. Thus, when measuring experimental atherosclerosis in mice, investigators should be aware of several previously unreported pitfalls regarding the extent, location and complexity of the arterial lesion that may not be suitable for extrapolation to human pathology.

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.

Diminished rate of mouse peritoneal macrophage cholesterol efflux is not related to the degree of HDL glycation in diabetes mellitus.

The efflux of (14)C-cholesterol from mouse peritoneal macrophages mediated by in vivo and in vitro glycation of intact HDL(3) and by HDL(3) apolipoproteins was investigated. Cholesterol-laden cells were incubated a long time with HDL(3) from control subjects (C), poorly controlled diabetes mellitus patients (D) and with HDL C submitted to in vitro glycation (G), as well as with all their respectively isolated apolipoproteins. A diminished cholesterol efflux rate occurred in incubations with intact HDL(3) D but not with intact HDL(3)G or with apoHDL(3)C, G or D. The specific binding of (125)I-HDL(3)G to the cell receptor, obtained upon incubation in the absence and in the presence of excess unlabelled HDL(3), was lower than the control. The role of apoE secretion by cholesterol-laden macrophages on cholesterol efflux was analyzed by incubating apoE knockout and control mice macrophages with HDL C or HDL G: a lower cholesterol efflux was observed from apoE knockout macrophages but glycation of HDL(3) did not influence this process either. The diminished capacity to remove cholesterol by the HDL drawn from diabetic subjects must be attributed to other modifications of the lipoproteins, except for non enzymatic glycation. Thus, events that impair the cell cholesterol removal in diabetes mellitus are multifaceted.

Plasma lipoproteins from patients with poorly controlled diabetes mellitus and "in vitro" glycation of lipoproteins enhance the transfer rate of cholesteryl ester from HDL to apo-B-containing lipoproteins.

Alterations in the reverse cholesterol transport system have been described in diabetic mellitus patients in several but not all studies. Furthermore, recently published investigations suggest that a faster "in vitro" transfer rate of cholesteryl ester from high density lipoproteins to apoB-containing lipoproteins could be solely ascribed to variation of the plasma lipoprotein composition and concentration in the diabetic state. The present study analysed the influence of lipoprotein glycation on the cholesteryl ester transfer protein-mediated transfer of esterified cholesterol from high density lipoprotein and its subfractions to lighter density lipoproteins. For this purpose two sets of "in vitro" experiments were carried out utilizing:1) plasma lipoproteins drawn from diabetic and from normal subjects and; 2) normal lipoproteins or partially purified cholesteryl ester transfer protein submitted to "in vitro" glycation. The transfer rate of 14C-cholesteryl ester labelled HDL subfractions to low or very low density lipoproteins was measured in all experiments. After incubations with plasma d > 1.21 g/ml or with purified cholesteryl ester transfer protein, apoB-containing lipoproteins were precipitated with a dextran sulfate/MgCl2 solution. The "in vitro" glycation of the partially purified cholesteryl ester transfer protein, markedly impaired its activity. However, greater transfer rates were observed when lipoproteins from diabetic individuals or the "in vitro" glycated lipoproteins were utilized. This effect was attributed to glycation of the protein component of HDL. In conclusion, lipoprotein glycation elicits an enrichment of the apoB-containing lipoproteins with cholesteryl ester that is likely related to the premature atherosclerosis in patients with poorly controlled diabetes.