Decreased membrane fluidity and unsaturated fatty acids in Niemann-Pick disease type C fibroblasts.

Niemann-Pick disease type C (NP-C) is an autosomal recessive disorder characterized by the sequestration and trapping of endocytosed cholesterol in lysosomes. The NPC1 gene on chromosome 18 was recently identified but its physiological function remains unknown. We have studied the lipid compositions of cultured human NP-C fibroblasts and mouse SPM-3T3 cell line derived from the C57BL/KsJ NP-C model mouse, which belongs to the same complementation group. Fibroblasts derived from apparently normal age-matched individuals and a subline of SPM-3T3 cells which restores cholesterol metabolism by transfer of human chromosome 18 were used as controls. Levels of free cholesterol in whole cell homogenates increased about 1.5-fold in human NP-C fibroblasts and mouse SPM-3T3 cells, while in the plasma membrane, cholesterol content did not significantly change in NP-C fibroblasts but rather decreased in SPM-3T3 cells. The total phospholipid content did not significantly change; however, among phospholipid head groups, increases in sphingomyelin and decreases in other classes were observed in human NP-C fibroblasts and mouse SPM-3T3 cells. The ratios of saturated fatty acids to unsaturated fatty acids increased in both human and mouse cells. The increase was also confirmed in the plasma membrane fraction of SPM-3T3 cells. Membrane fluidity was examined using a 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescent probe. The DPH anisotropy values were markedly increased in NP-C fibroblasts and in SPM-3T3 cells. The results suggest that a NP-C mutation causes complex alterations in cellular lipid contents and biophysical properties of the membrane.

Effect of chitosan feeding on intestinal bile acid metabolism in rats.

The effect of chitosan feeding (for 21 days) on intestinal bile acids was studied in male rats. Serum cholesterol levels in rats fed a commercial diet low in cholesterol were decreased by chitosan supplementation. Chitosan inhibited the transformation of cholesterol to coprostanol without causing a qualitative change in fecal excretion of these neutral sterols. Increased fiber consumption did not increase fecal excretion of bile acids, but caused a marked change in fecal bile acid composition. Lithocholic acid increased significantly, deoxycholic acid increased to a lesser extent, whereas hyodeoxycholic acid and the 6 beta-isomer and 5-epimeric 3 alpha-hydroxy-6-keto-cholanoic acid(s) decreased. The pH in the cecum and colon became elevated by chitosan feeding which affected the conversion of primary bile acids to secondary bile acids in the large intestine. In the cecum, chitosan feeding increased the concentration of alpha-,beta-, and omega-muricholic acids, and lithocholic acid. However, the levels of hyodeoxycholic acid and its 6 beta-isomer, of monohydroxy-monoketo-cholanoic acids, and of 3 alpha, 6 xi, 7 xi-trihydroxy-cholanoic acid decreased. The data suggest that chitosan feeding affects the metabolism of intestinal bile acids in rats.

Metabolism of 3 beta-hydroxycholest-5-en-26-oic acid in hamsters.

Metabolism of 26-hydroxycholesterol to 3 beta-hydroxychol-5-en-24-oic acid and other C24-bile acids has been expected to occur by way of 3 beta-hydroxycholest-5-en-26-oic acid in studies in vitro. 3 beta-Hydroxycholest-5-en-26-oic acid was infused intravenously into bile fistula hamsters and the following C24-bile acids were identified: 3 beta-hydroxychol-5-en-24-oic acid, lithocholic acid, chenodeoxycholic acid, and a small amount of cholic acid.

Cholic acid synthesis from 26-hydroxycholesterol and 3-hydroxy-5-cholestenoic acid in the rabbit.

Intravenous administration of 26-hydroxycholesterol to the rabbit with a bile fistula yielded cholic acid in proportions (84 and 86%) not significantly different from that derived from cholesterol. By contrast, the naturally occurring C27 bile acid 3 beta-hydroxy-5-cholestenoic acid yielded not more than 8% cholic acid. Thus initial 26-hydroxylation of cholesterol followed by 7-alpha-hydroxylation can provide sufficient amounts of cholic acid to be considered a quantitatively significant pathway for bile acid synthesis, and in addition it is the only pathway that can be the source of the circulating levels of C24 and C27 monohydroxy bile acids.

Effects of cholesterol feeding to maternal rats on metabolism of cholesterol and bile acids in the dams and their offspring.

The influence of feeding cholesterol to rats during pregnancy and postpartum (from the 11th day of gestation to the third day after delivery) on the serum and hepatic cholesterol levels and on the bile acid composition in the pool and in the liver in relationship to the dams and their pups was examined. The hepatic content of cholesterol in both dam and offspring increased during cholesterol feeding without any changes in serum cholesterol level. In the dams, mainly the esterified cholesterol was increased; in the pups, mainly the free cholesterol was increased. Cholesterol feeding led to a pronounced increase in the pool of beta-muricholic acid and a relative decrease in the lithocholic acid concentration in pregnant rats. In fetal rats, the chenodeoxycholic acid pool was increased by cholesterol intake. The lithocholic acid pool was larger in the postpartum rats fed cholesterol than in the controls, while the concentration of alpha- and beta-muricholic acids was decreased. The neonates of cholesterol-fed dams had a larger pool of chenodeoxycholic acid but a smaller pool of beta-muricholic acid. These results suggest that the metabolism of cholesterol and of bile acids in dams and their offspring respond differently to cholesterol intake.

Effect of diabetes on the metabolism of chenodeoxycholic acid in isolated perfused rat liver.

The formation of alpha-muricholic acid and beta-muricholic acid from chenodeoxycholic acid was comparatively investigated in livers isolated from normal, streptozotocin-diabetic, and insulin-treated diabetic rats. [24-14C]Chenodeoxycholic acid or [24-14C]alpha-muricholic acid was infused into the perfused livers. There was no difference in biliary excretion of 14C among the different groups of rats after the infusion of each 14C-labelled bile acid. Biliary [14C]bile acids were chromatographed on a thin-layer plate and the distribution of radioactivity on the plate was measured by radioscanning. In the diabetic group, the formation ratio of alpha-muricholic acid and beta-muricholic acid from [24-14C]chenodeoxycholic acid and also that of beta-muricholic acid from [24-14C]alpha-muricholic acid were much smaller than in the normal group. Treatment of the diabetic group with insulin cancelled the difference in the infusion of each [24-14C]bile acid. The results indicate that not only 6 beta-hydroxylation of chenodeoxycholic acid to alpha-muricholic acid but also 7-epimerization of the latter acid to beta-muricholic acid is suppressed in an insulin-deficient state in rats.

Difference between cholic acid and chenodeoxycholic acid in dependence upon cholesterol of hepatic and plasmatic sources as the precursor in rats.

Some difference in functional pool of cholesterol acting as the precursor of bile acids is pointed out between cholic acid and chenodeoxycholic acid. In order to elucidate this problem further, some experiments were performed with rats equilibrated with [7(n)-3H, 4-(14)C] cholesterol by subcutaneous implantation. The bile duct was cannulated in one series of experiments and ligated in another. After the operation 14C-specific radioactivity of serum cholesterol fell, but reached practically a new equilibrium within three days. 14C-Specific radioactivity of serum cholesterol as well as of biliary bile acids in bile-fistula rats and urinary bile acids in bile duct-ligated rats was determined during a three days-period in the new equilibrated state. The results were as follows: (1) 14C-Specific radioactivity of cholic acid and chenodeoxycholic acid in bile was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was clearly lower than that of chenodeoxycholic acid. (2) 14C-Specific radioactivity of cholic acid and beta-muricholic acid in urine was lower than that of serum cholesterol, and 14C-specific radioactivity of cholic acid was lower than that of beta-muricholic acid. (3) Biliary as well as urinary beta-muricholic acid lost tritium label at 7-position entirely during the course of formation from [7(n)-3H, 4-(14)C]cholesterol.

Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats.

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-14C] acetate and of [1,2-3H] cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-14C] acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-3H) cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.

Effect of estradiol-17beta on the metabolism of bile acids in the rat.

The effect of estradiol-17beta on the metabolism of bile acids has been studied in male rats. 1. A 3-day acute treatment with 100 microgram/100 g body weight of the hormone caused a decrease in serum cholesterole and an increase in hepatic cholesterol. A chronic treatment with the same quantity twice a week for 3 weeks induced hypercholesteremia, which continued for 10 days after the final dosage, but there was no change in hepatic total cholesterol. 2. Fistula bile of both groups of treated rats collected during 30 min was analyzed for bile acid components. Hyodeoxycholic acid and/or chenodeoxycholic acid increased relative to cholic acid in both groups, as compared with the controls. 3. Kinetic study of the metabolism of bile acids in the chronically treated rats revealed a more than twofold enlargement of the pool size but an almost unchanged half-life of chenodeoxycholic acid. As for cholic acid, essentially no change in pool size or half-life was observed. 4. These results indicate that chronic treatment of male rats with estradiol-17beta causes an increase in elimination of hepatic cholesterol via the bile acid pathway.

Further studies on the possible compartmentation of the precursor pool of cholesterol for the biosynthesis of cholic acid in the rat.

In vivo and in vitro experiments with rats were carried out to investie precursor for the biosynthesis of cholic acid. When rats with a bile-fistula were given a mixture of [2-14C]mevalonate and [1,2-3H]cholesterol intravenously, the 14C:3H ratio in cholic acid in both whole homogenate and cytosol prepared from their lives was higher than that in free cholesterol in any subcellular fraction of the livers. When [2-14C] mevalonate was administered intravenously to bile-fistula rats, the specific radioactivity of free cholesterol in the hepatic microsomal fraction exceeded that in any other fraction, and the specific radioactivity of biliary cholic acid was remarkably high, exceeding that of microsomal free cholesterol. In similar experiments with [4-14C] cholesterol, the specific radioactivity of free cholesterol in the hepatic microsomal fraction exceeded that in any other subcellular fraction and the specific radioactivity of biliary cholic acid was lower than that of free cholesterol in any hepatic subcellular fraction. Tissue suspensions of rat livers in Krebs-Ringer bicarbonate (pH 7.4)-5.5 mM glucose were incubated with [2-14C]mevalonate in O2-CO2 (95:5, v/v) at 37 degrees. The specific radioactivity of free cholesterol in the microsomal fraction prepared from the incubated tissue exceeded the specific radioactivities of free cholesterol in the other subcellular fractions. The estimated specific radioactivity of taurocholate formed during the incubation was far higher than that of microsomal free cholesterol. These data indicate that hepatic microsomal free cholesterol which was newly synthesized in situ was preferentially incorporated into cholic acid.