Compared effect of immunosuppressive drugs cyclosporine A and rapamycin on cholesterol homeostasis key enzymes CYP27A1 and HMG-CoA reductase.

Hyperlipidaemia, i.e. increase in total cholesterol and triglycerides, is a common side-effect of the immunosuppressive drugs rapamycin (RAPA) and cyclosporine A (CsA), and is probably related to inhibition of the 27-hydroxylation of cholesterol (acid pathway of bile acid biosynthesis). This might be one of the causes for the increase in plasma cholesterol, as 27-hydroxycholesterol is a potent suppressor of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), a key enzyme of cholesterol synthesis. As the sterol 27-hydroxylase (CYP27A1) inhibition by CsA is well known, we evaluated the effect of another immunosuppressive drug, RAPA, on this enzyme in HepG2 mitochondria, which confirmed the dose-dependent inhibition of mitochondrial CYP27A1 by cyclosporine (10-20 microM), while the inhibition by RAPA required a higher dose (50-100 microM). Corresponding K(i) was 10 microM for CsA (non-competitive inhibition) and 110 microM for RAPA (competitive inhibition). Cotreatment with both immunosuppressive drugs showed an additive inhibitory effect on CYP27A1 activity. Later, we analysed the effect of these immunosuppressants on HMGR expression in HepG2 cells, and a dose-dependent up-regulation of HMGR gene expression was observed. The results suggest that RAPA and CsA are both inhibitors of CYP27A1 activity with slightly different mechanisms and that they may accordingly increase HMGR expression.

Effects of dietary alpha linolenic acid on cholesterol metabolism in male and female hamsters of the LPN strain.

N-3 polyunsaturated fatty acids and estrogens are recognized as protective factors of atherosclerosis, however their interactions on cholesterol metabolism remain unclear. Male and female hamsters were fed for 9 weeks diets containing 12.5% lipids and rich in either alpha-linolenic acid ("linseed" diet) or saturated fatty acids ("butter" diet). Hamsters fed the "linseed" diet exhibited lower plasma concentrations of cholesterol (-29%), total LDL (-35%) and HDL (-17%), glucose (-20%), insulin (-40%) and of the LDL-cholesterol/HDL-cholesterol ratio (-27%) than those fed the "butter" diet. In the liver, cholesterol content was 2.7-fold lower in response to the "linseed" diet, whereas the concentration of HDL receptor (SR-BI) and the activities of HMGCoA reductase and cholesterol 7alpha-hydroxylase were 30 to 50% higher than with the "butter" diet. By contrast, the LDL receptor concentration did not vary with the diet. Females exhibited higher concentration of LDL (+24%), lower concentration of plasma triglycerides (-34%), total VLDL (-46%) and VLDL-cholesterol (-37%) and of biliary phospholipids (-19%). Besides, there was also an interaction between gender and diet: in males fed the "butter" diet, plasma triglycerides and VLDL concentration, were 2 to 4 fold higher than in the other groups. These data suggest that gene and/or metabolic regulations by fatty acids could interact with that of sex hormones and explain why males are more sensitive to dietary fatty acids.

Effects of dietary 27-hydroxycholesterol on cholesterol metabolism and bile acid biosynthesis in the hamster.

27-hydroxycholesterol (27OH-Chol) is an important endogenous oxysterol resulting from the action of sterol 27-hydroxylase (CYP27A1) on cholesterol in the liver and numerous extrahepatic tissues. It may act as a modulator of cholesterol and bile acid metabolism. The effects of 27OH-Chol on the main enzymes and receptors of cholesterol metabolism were investigated by feeding male hamsters a diet supplemented with 27OH-Chol (0.1% w/w) for 1 week. Intestinal scavenger class B, type I (SR-BI) protein level was decreased (-65%), but hepatic expression was increased (+34%). Liver 3beta-hydroxy-3beta-methyl glutaryl coenzyme A reductase (-58%), cholesterol 7alpha-hydroxylase (-54%), oxysterol 7alpha-hydroxylase (-44%), and sterol 12alpha-hydroxylase (-70%) activities were all decreased. Bile acid composition was changed (fourfold increase in the chenodeoxycholic/cholic acid ratio). This study demonstrates that dietary 27OH-Chol modulates major enzymes of cholesterol metabolism and alters the biliary bile acid profile, making it more hydrophobic, at least at this level of intake. Its effects on SR-BI protein levels are organ dependent. The properties of 27OH-Chol or its metabolites on cholesterol metabolism probably result from the activation of specific transcription factors.

A CLA mixture prevents body triglyceride accumulation without affecting energy expenditure in Syrian hamsters.

We examined the effects of feeding conjugated linoleic acids (CLA) to adult male hamsters on several components of energy metabolism and body composition. Hamsters (n = 54) were assigned for 6-8 wk to one of three diets: 1) a standard diet (in percentage energy: lipids, 33, carbohydrates, 49, and proteins, 18); 2) to the standard diet augmented with the 9c,11t-isomer of CLA to 1.6% of energy (R group); or 3) the standard diet augmented with the 9c,11t-isomer and the 10t,12c-CLA isomer to 3.2 (1.6 + 1.6) % of energy (CLA mix group). (15)N uniformly labeled milk-protein was included in the diet to measure the incorporation of dietary protein into liver and muscle. Basal metabolic rate, thermogenic response to feeding and energy expenditure during spontaneous activity or during an exercise at approximately 60% of VO(2max) were measured. Carnitine palmitoyltransferase-I (CPT-I), leptin, insulin and triiodothyronine concentrations, as well as the in vivo overall adiposity changes were also determined. After 6 wk, the whole-body triglyceride content determined in vivo by NMR was significantly higher in the R group than in the control and CLA mix groups. The CLA mix group differed from the others in the lack of body triglyceride accumulation between d 21 and d 45 of the study, and the appearance of a slight insulin-resistance (homeostatic model assessment index, P < 0.05). Paradoxically, the lack of effect on whole-body lipid oxidation was associated with a greater CPT-I-specific activity in tissues of both CLA-fed groups (P < 0.05). No other major effects of CLA feeding were detected. In conclusion, CLA supplementation in hamsters did not affect adipose weight or the components of energy expenditure despite a theoretically higher capacity of red muscle to oxidize lipids. Only a CLA mixture prevented whole-body triglyceride accumulation over time.

Increasing amounts of dietary myristic acid modify the plasma cholesterol level and hepatic mass of scavenger receptor BI without affecting bile acid biosynthesis in hamsters.

The purpose of this study was to analyze the effects of increasing amounts of dietary myristic acid (0.03 to 4.2% of the total dietary energy) on the plasma and hepatic cholesterol metabolism. Six groups of hamsters received semi-purified diets containing 0.05% cholesterol and 12.5% lipids and differing only by the nature of the triglycerides (Safflower oil, lard, lard/coconut oil (1:1), milk fat, milk fat/coconut oil (1:1), coconut oil) for 3 weeks. A positive regression between the plasma cholesterol level and the dietary myristic acid level was observed (r = 0.60, P < 0.0001). However, it is noteworthy that the increase in plasma total cholesterol only reflects an increase in the level of HDL-cholesterol. In parallel, the mass SR-BI decreased linearly with the increased level of myristic acid in the diet, whereas the LDL-R did not change. This study shows that increasing amounts of myristic acid (0.03 to 4.2%) do not alter the cholesterol or bile acid metabolism and increase only the HDL-C.

Dietary myristic acid modifies the HDL-cholesterol concentration and liver scavenger receptor BI expression in the hamster.

The influence of myristic acid in a narrow physiological range (0.5 to 2.4% of total dietary energy) on the plasma and hepatic cholesterol metabolism was investigated in the hamster. The hamsters were fed on a diet containing 12.5 g fat/100 g and 0.05 g cholesterol/100 g with 0.5% myristic acid (LA diet) for 3 weeks (pre-period). During the following 3 weeks (test period), they were divided into four dietary groups with 0.5% (LA), 1.2% (LM), 1.8% (ML) or 2.4% (M) myristic acid. Finally, half the hamsters in each group were again fed the LA diet for another 3 weeks (post-period). At the end of the test period, the hepatic expression of the scavenger receptor BI (SR-BI) was lower in the LM, ML and M groups than in the LA group whereas the hepatic cholesteryl ester concentration was higher. Cholesterol 7alpha hydroxylase activity was lower in the ML and M groups than in the LA and LM groups while the sterol 27 hydroxylase and 3-hydroxy-3-methyl glutaryl coenzyme A reductase activities were not modulated by dietary myristic acid. This is the first time a negative correlation has been observed between the HDL-cholesterol concentration and the hepatic mass of SR-BI (r -0.69; P<0.0001) under physiological conditions. An inverse linear regression was also shown between SR-BI and the percentage of myristic acid in the diet (r -0.75; P<0.0001). The hepatic mass of SR-BI in the M group had increased at the end of the post-period compared with the test-period values. The present investigation shows that myristic acid modulates HDL-cholesterol via a regulation of the SR-BI expression.

The Syrian golden hamster strain LPN: a useful animal model for human cholelithiasis.

The purpose of this study was to specify the main mechanisms at the origin of gallstone formation in very young (5-week old) or young adult (9-week old) LPN hamsters fed a sucrose-rich (normal lipid) lithogenic diet for one and four weeks, respectively. It was also to compare these mechanisms in the two strains of hamsters (LPN and Janvier) or when an anti-lithiasic diet was given by substituting 10% of the sucrose by beta cyclodextrin. The LPN strain of hamsters showed a very high incidence of cholesterol gallstones (73%) after receiving the lithogenic diet. The gallstone formation is very rapid and occurs in less than one week in very young hamsters which show a high cholesterol synthesis rate in the liver. The cholesterol and phospholipid concentrations in the bile, cholesterol saturation index (CSI) and hydrophobic index (HI) increased significantly, concomitantly with a higher liver cholesterol synthesis in very young hamsters and with a lower bile acid synthesis (neutral pathway: cholesterol 7alpha-hydroxylase, CYP7A1 and acidic pathway: sterol 27 hydroxylase, CYP27A1) in young adult hamsters. No significant changes in the lipoprotein receptor expression (LDLr, SR-BI) were observed after feeding the lithogenic diet. Adding ten per cent beta-cyclodextrin, a cyclic oligosaccharide that binds cholesterol and bile acids to the lithogenic diet at the expense of sucrose, induced a decrease in cholesterol bile secretion and in the CSI and HI and prevented cholesterol gallstone formation. Similarly, another strain of Syrian Golden hamsters (" Janvier ") which originally exhibited a smaller bile cholesterol concentration, lower liver cholesterol synthesis and higher CYP7A1/CYP27A1 activity ratio did not carry cholesterol gallstones when fed the lithogenic diet. The main parameters always found at the origin of cholelithiasis in the Hamster are discussed: a higher hepatic cholesterogenesis (HMGCoAR), a higher HMGCoAR/CYP7A1 activity ratio, a lower cholesterol ester storage capacity, a higher CYP27A1/CYP7A1 activity ratio correlated to a higher cholesterol secretion in the bile and higher CSI and HI. In LPN hamsters, the incidence of cholesterol gallstones is nil when CSI + HI < 0.8 and positive for CSI + HI > 0.9. An overall comparison of the data obtained in LPN Hamsters and in Man suggests that this hamster strain appears to be an interesting model for human cholelithiasis.

Dietary L-carnitine supplementation in obese cats alters carnitine metabolism and decreases ketosis during fasting and induced hepatic lipidosis.

This study was designed to determine whether dietary carnitine supplement could protect cats from ketosis and improve carnitine and lipid metabolism in experimental feline hepatic lipidosis (FHL). Lean spayed queens received a diet containing 40 (CL group, n = 7) or 1000 (CH group, n = 4) mg/kg of L-carnitine during obesity development. Plasma fatty acid, beta-hydroxybutyrate and carnitine, and liver and muscle carnitine concentrations were measured during experimental induction of FHL and after treatment. In control cats (CL group), fasting and FHL increased the plasma concentrations of fatty acids two- to threefold (P < 0.0001) and beta-hydroxybutyrate > 10-fold (from a basal 0.22 +/- 0.03 to 1.70 +/- 0.73 after 3 wk fasting and 3.13 +/- 0.49 mmol/L during FHL). In carnitine-supplemented cats, these variables increased significantly (P < 0.0001) only during FHL (beta-hydroxybutyrate, 1.42 +/- 0.17 mmol/L). L-Carnitine supplementation significantly increased plasma, muscle and liver carnitine concentrations. Liver carnitine concentration increased dramatically from the obese state to FHL in nonsupplemented cats, but not in supplemented cats, which suggests de novo synthesis of carnitine from endogenous amino acids in control cats and reversible storage in supplemented cats. These results demonstrate the protective effect of a dietary L-carnitine supplement against fasting ketosis during obesity induction. Increasing the L-carnitine level of diets in cats with low energy requirements, such as after neutering, and a high risk of obesity could therefore be recommended.