acyl-CoA:retinol Acyltransferase Research Articles

Radiation inactivation analysis of acyl-CoA:retinol acyltransferase and lecithin:retinol acyltransferase in rat liver

Microsomes from liver and several other tissues esterify retinol through both fatty acyl-CoA-dependent and -independent reactions. Two activities, acyl-CoA:retinol acyltransferase (ARAT) and lecithin:retinol acyltransferase (LRAT) activities, have been characterized enzymatically but neither has yet been purified and characterized biochemically. We have used the method of radiation inactivation to determine the target sizes of ARAT and LRAT in intact microsomal membranes from rat liver. After exposure of frozen liver microsomes to ionizing radiation, the activity of ARAT decayed exponentially yielding a target size of 73 +/- 18 kDa (mean +/- SD, n = 6). The activity of LRAT was assayed both by monitoring the esterification of retinol bound to the cellular retinol-binding protein (CRBP) and of solvent-dispersed retinol. With both assays a single exponential was observed with radiation doses of 9 to 150 Mrads. The slopes obtained with both LRAT assays were similar, yielding target sizes of 52 +/- 10 kDa (n = 10) for the LRAT assay with CRBP-retinol and 56 +/- 7 kDa (n = 6) for the LRAT assay with dispersed retinol. These target sizes did not differ from each other but were significantly smaller than that of ARAT. These data provide the first physical evidence of the independent entities which catalyze the ARAT and LRAT reactions of liver microsomes.

Fatty acyl CoA-dependent and -independent retinol esterification by rat liver and lactating mammary gland microsomes

Retinol esterification was examined in microsomes from rat liver and lactating mammary gland as a function of the form of retinol substrate, dependence on fatty acyl CoA, and sensitivity to phenylmethylsulfonyl fluoride (PMSF). Retinol bound to cellular retinol-binding protein (CRBP) or dispersed in solvent was esterified in a fatty acyl CoA-independent, PMSF-sensitive reaction, consistent with lecithin:retinol acyltransferase (LRAT) activity. LRAT activity exhibited the same K m (2 μ m retinol) between tissues but a higher V max in liver as compared to that in mammary gland (47 vs 8 pmol/min/mg microsome protein, respectively). Solvent-dispersed retinol was also esterifled in a fatty acyl CoA-dependent, PMSF-resistant reaction, consistent with acyl CoA:retinol acyltransferase (ARAT) activity. Retinol bound to CRBP was not a good substrate for this reaction. ARAT activity displayed a similar V max (300 pmol/min/mg microsome protein) between tissues but K m values of 15 and 5 μ m for retinol and fatty acyl CoA in mammary gland as compared to 30 and 25 μ m, respectively, in the liver. Thus, when substrate was near or below K m , retinol esterification occurred predominantly by LRAT in the liver and ARAT in the mammary gland, respectively. The concentration of CRBP in the cytosol, determined by Western blotting, was approximately 2 μ m in the liver but was almost nondetectable in the mammary gland. These data suggest that retinol esteriflcation is regulated via different mechanisms in liver and mammary gland and support a specific role for CRBP in the liver.

The effect of TCDD on Acyl CoA: Retinol acyltransferase activity and vitamin a accumulation in the kidney of male sprague‐dawley rats

Previous studies have shown that rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) show signs of toxicity that are similar to the responses of animals to a vitamin A-deficient diet. These include hypophagia, loss of body weight, loss of hepatic vitamin A, and accumulation of renal retinoids. Male Sprague-Dawley rats treated with 10, 30, or 100 nmol/kg of TCDD accumulated renal vitamin A, with retinyl palmitate concentrations reaching 8 times those of control animals, similar to that of male rats fed a vitamin A-free diet for 26 days. Acyl CoA:retinol acyltransferase (ACARAT) activities in both TCDD-treated rats and rats fed a vitamin A-free diet for 26 days were similarly elevated, and were strongly and positively correlated with the renal retinyl palmitate concentrations. Retinol concentrations in the kidneys of rats treated with TCDD or fed a vitamin A-free diet were only slightly elevated when compared to control rats. We suggest that accumulation of retinyl esters in the kidneys of rats treated with TCDD or fed a vitamin A-free diet occurs as a result of increased rates of retinol esterification.

CL 277,082: a novel inhibitor of ACAT-catalyzed cholesterol esterification and cholesterol absorption.

CL 277,082 (I) was found to be a potent inhibitor of acyl CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) in microsomes from a variety of tissues with IC50 values of 0.14 microM for intestinal mucosal microsomes, 0.74 microM for liver, and 1.18 microM for rat adrenal. I was also shown to inhibit ACAT in cultured smooth muscle cells (IC50 = 0.8 microM) and was found to be specific in inhibiting cholesterol esterification since it did not inhibit fatty acid incorporation into triglycerides or phospholipids. Also, other cholesterol esterifying enzymes such as lecithin:cholesterol acyltransferase (LCAT) and pancreatic cholesterol esterase were not inhibited by I, nor was esterification of retinol by acyl CoA:retinol acyltransferase (ARAT) from intestinal mucosal microsomes inhibited. I was a potent inhibitor of cholesterol absorption in cholesterol-fed rats by markedly inhibiting increases in liver and serum cholesterol concentration (ED50 = 5.2 mg/kg per day) while increasing the excretion of neutral 14C-labeled sterol in the feces.

Effect of cytostatics on liver retinol store in rat.

The effect of the cytostatics doxorubicin, 6-thioguanine and cytarabine on retinol store in rat liver was examined. When rats were treated with pharmacological doses of the combination of doxorubicin and 6-thioguanine for 10 days, the content of retinol in the liver was reduced by about 33%. In a longer term experiment, doxorubicin and cytarabine given separately reduced the retinol store by 33% and 11%, respectively, while doxorubicin and 6-thioguanine given in combination reduced retinol in liver by 31%. For one of the cytostatics (doxorubicin) the effects on plasma retinol and on acyl CoA:retinol acyltransferase (ARAT) activity in small intestine were also examined. Both were transiently reduced during the experiment.

Subcellular localization of retinoids, retinoid-binding proteins, and acyl-CoA:retinol acyltransferase in rat liver

Studies were conducted to define the subcellular localization of endogenous retinoids (vitamin A), retinoid-binding proteins, and acyl-CoA:retinol acyltransferase (ARAT) in liver and to determine whether their distributions were affected by hepatic vitamin A content. Quantitative subcellular fractionation techniques were used. Rats were fed purified diets either containing or lacking vitamin A to obtain animals with total retinoid stores ranging from 0.5 to 172 micrograms of retinol equivalent per gram of liver. Liver homogenates were fractionated by differential centrifugation to yield nuclear (N), mitochondrial-lysosomal (ML), microsomal (P), and high-speed supernatant (S) fractions. N, ML, and P were washed two more times by resuspension and centrifugation to remove constituents bound nonspecifically. S was further resolved into "floating lipid" and underlying "cytosol" by prolonged ultracentrifugation. The distributions of marker constituents were not affected by vitamin A status. Most of the retinyl ester in the liver was recovered in the S fraction where it was entirely (greater than 95%) associated with floating lipid. About half of the total free retinol was also recovered in the S fraction, but it was mostly (2/3) associated with cytosol per se. A substantial portion (30%) of the free retinol was recovered in the 3 X -washed microsomal (P) fraction. Sufficient binding capacity for retinol was present in both P (as retinol-binding protein) and S (as cellular retinol-binding protein) to quantitatively account for the amounts of free retinol present in the two fractions. ARAT activity in the liver was distributed among the subcellular fractions in a manner identical with an endoplasmic reticulum marker enzyme (NADPH-cytochrome C reductase).(ABSTRACT TRUNCATED AT 250 WORDS)

Intestinal Retinol Esterification and Serum Retinol in Children with Cystic Fibrosis

Summary: Children with cystic fibrosis (CF) often have a poor vitamin A status. We found in the present work a reduced level of serum retinol in older children with this disease. As retinol enters intestinal lymph as retinyl esters, the enzyme acyl‐CoA: retinol acyltransferase (ARAT) may be of importance for retinol absorption. We have assayed ARAT activity in duodenal mucosal homogenate from children with CF. There was a large variation within the group. However, mean ARAT activity was not significantly decreased, as compared with controls. Thus, the lack of pancreatic enzymes probably is the main reason for reduced vitamin A absorption in CF. In celiac disease and in lactose intolerance—both mucosal disorders — a significant reduction of mean ARAT activity was found.

Intestinal Retinol Esterification and Serum Retinol in Children with Cystic Fibrosis

Children with cystic fibrosis (CF) often have a poor vitamin A status. We found in the present work a reduced level of serum retinol in older children with this disease. As retinol enters intestinal lymph as retinyl esters, the enzyme acyl-CoA:retinol acyltransferase (ARAT) may be of importance for retinol absorption. We have assayed ARAT activity in duodenal mucosal homogenate from children with CF. There was a large variation within the group. However, mean ARAT activity was not significantly decreased, as compared with controls. Thus, the lack of pancreatic enzymes probably is the main reason for reduced vitamin A absorption in CF. In celiac disease and in lactose intolerance--both mucosal disorders--a significant reduction of mean ARAT activity was found.

The activity of acyl CoA: retinol acyltransferase in the rat: variation with vitamin A status.

Retinol esterification in the small intestine, liver and kidney of rats given a normal diet or a vitamin-A-free diet and of rats given large doses of vitamin A was studied. The active enzyme is a microsomal acyl CoA:retinol acyl transferase (ARAT). In the small intestine ARAT activity was 0.37 nmol ester/mg microsomal protein per min. Large doses of vitamin A increased the activity significantly, while the enzyme activity in the vitamin-A-deficient rats was in the range of that of the controls. Retinoic acid in physiological doses (0.064 mg three times per week) had no influence on ARAT activity. In the liver, ARAT activity of the controls was 0.58 nmol ester/mg microsomal protein per min. The activity was increased after large doses of vitamin A. It was not significantly reduced in vitamin-A-deficient animals. The kidney had a low, but significant ARAT activity, both in normal and vitamin-A-deficient animals and after large doses of vitamin A (range 0.08-0.14 nmol ester/mg microsomal protein per min). The vitamin-A-esterifying enzyme in the small intestine and liver of the rat seems to be influenced by the amount of retinol in the diet.

Microsomal esterification of retinol in human liver.

Recent work has shown that esterification of retinol in microsomes from rat liver, mammary gland and small intestine and from human small intestine is catalyzed by an acyl CoA: retinol acyl transferase (ARAT). The current study demonstrates ARAT activity in human liver microsomes. At optimal incubation conditions the rate of retinyl ester formation due to ARAT (0.37 +/- 0.31 nmole ester formed X mg microsomal protein-1 X minute-1, mean +/- SD, n = 6) suggests that the enzyme is of physiological importance.