Intestinal mucins and cholesterol uptake in vitro.

A mucus material, secreted by intestinal segments, with a high affinity for cholesterol, has been isolated and chemically characterized. The mucin contained 11% carbohydrate, largely as glucosamine, galactose and N-acetylneuraminic acid, and 19% lipid, of which 86% was unesterified fatty acid. The isolated material readily bound cholesterol in a stoichiometric manner. Conditions known to enhance cholesterol absorption in vivo also decreased mucin complexing to cholesterol in vitro. This association of cholesterol and intestinal surface mucin also occurred during incubations of intestinal segments with dispersed cholesterol, resulting in a high level of intestinal adsorption, with little or no cellular absorption of the sterol. However, when cholesterol was solubilized in simple or complex micelles containing bile salts, surface adsorption of cholesterol was reduced and net absorption was increased. The results suggest that surface mucin binding of cholesterol may represent at least one major diffusion limitation to cholesterol absorption in the intestine.

Dietary fiber and lymphatic absorption of cholesterol in the rat.

The indirect effects of short-term (3-day) feeding of several types of dietary fiber and nonnutritive materials on the subsequent absorption of cholesterol has been investigated in thoracic duct cannulated rats. Absorption was studied at timed intervals over 24 hr after duodenal introduction of a tracer dose of cholesterol at least 20 hr after the last feeding. In animals fed for 3 days with diets containing cholestryamine, bran, or cellulose, cholesterol absorption was significantly less than in control animals maintained on rat chow. Rats fed for 3 days with an alfalfa-containing diet showed large variations in cholesterol absorption that were not significantly different from controls. However, after 5 weeks, rats on the alfalfa diet showed a marked reduction in lymphatic absorption of the tracer sterol. These indirect effects of cholestryamine and fibers on cholesterol absorption were not attributable to a common mechanism; i.e., differences in transit times that were not significant, or dirrect binding of bile acids and cholesterol by the test materials.

Comparative intestinal and colonic absorption of [4-14C] cholesterol in the rat.

[4(-14)C] Cholesterol was administered as an aqueous emulsion with triolein and dry non-fat milk either directly into the upper duodenum or into the ileocaecal junction of lymph duct cannulated rats. Lymph flow rates were similar in the two groups of animals. Whereas ca. 53% of the administered tracer dose of cholesterol was absorbed when introduced into the upper small intestine, only 0.06% appeared in lymph when administered into the caecum. Furthermore, less than 0.01% of the administered isotope was detected in urine and blood. The data demonstrate that the large intestine does not contribute significantly to the absorption of exogenous cholesterol in the rat.

Activation of sterol ester hydrolase of bovine corpus luteum by N6,O2'-dibutyryl cyclic adenosine 3':5'-phosphate.

The direct activation of sterol ester hydrolase (E.C. 3.1.1.13) in homogenates of bovine corpus luteum by N6O2'-dibutyryl cyclic adenosine 3':5'-phosphate, (dibutyryl cAMP), adenosine triphosphate (ATP), and Mg2+ has been demonstrated. Variability in the extent of activation by the additions was minimized by homogenization of the tissue in 5 mM Mg2+'. Baseline sterol ester hydrolase activity was primarily associated with the 105,000 X g soluble fraction, and significant activation of the enzyme preparation preincubated with dibutyryl cAMP, ATP and Mg2+ occurred within the first 15 min, prior to addition of substrate. A requirement for protein kinase in the system was demonstrated by blocking the cofactor-dependent enzyme activation with commercial protein kinase inhibitor.

Essential fatty acid deficiency and adrenal cortical function in vitro.

Adrenocortical cells were prepared from rats maintained on essential fatty acid-deficient diets and control litter mates. Cells from control rats had high concentrations of essential fatty acids in the cholesteryl ester fraction of which approximately 22% was arachidonate. In contrast, cells from EFA-deficient rats had only 2.5% arachidonate in the cholesteryl esters, even though the total esterified cholesterol level was comparable to that of controls. In place of the essential fatty acids, the cholesteryl esters of these cells were rich in 20:3(n--9) and 22:3(n--9). When cells from EFA-deficient rats were incubated with ACTH or dibutyryl cyclic AMP, the output of corticosterone was the same as in controls. Also sterol esters were hydrolyzed to the same extent as in controls despite the unusual composition of the fatty acid esters. The phospholipids in both control and EFA-deficient cells contained high levels of arachidonate but were not hydrolyzed in either type of cell during incubation with ACTH or dibutyryl cyclic AMP. The results indicate that high levels of the prostaglandin precursors, namely linoleate and arachidonate, are not a sine qua non for the steroidogenic action of ACTH or cyclic AMP.

Protein kinase-mediated phosphorylation of a purified sterol ester hydrolase from bovine adrenal cortex.

Sterol ester hydrolase (cholesterol esterase, E.C. 3.1.1.13) of bovine adrenal cortex has been extensively purified by ammonium sulfate fractionation, acid precipitation, hydroxylapatite chromatography, and Sephadex G-200 chromatography. During the purification sequence, the hydrolase activity was purified free of endogenous protein kinase. With this purified preparation, activation by cyclic AMP and ATP-Mg2+ did not occur unless exogenous protein kinase was included in the activating system. Using [gamma-32P]ATP, the transfer of the terminal phosphate to the enzyme protein was demonstrated by three separate experimental approaches. With pooled fractions from Sephadex G-200 chromatography, significant binding of 32P by the enzyme protein was observed only in the presence of exogenous protein kinase. Time course studies disclosed a close concurrence between the extent of activation of the purified enzyme by cyclic AMP-dependent protein kinase and the level of 32P transfer from [gamma-32P]ATP to the enzyme protein. Finally, assays carried out during Sephadex G-200 chromatography showed a correspondence in the peaks for activated sterol ester hydrolase and for 32P binding by protein. The data confirm that activation of adrenal sterol ester hydrolase by cyclic AMP and ATP-Mg2+ involves protein kinase-catalyzed phosphorylation of the enzyme protein.

ACTH-induced hydrolysis of cholesteryl esters in rat adrenal cells.

Rat adrenal cortical cells have been prepared by collagenase dissociation of trypsin-treated adrenal tissue. The content and compositions of cholesteryl ester, phospholipid, and triglyceride fatty acids compare favorably with those of undissociated rat adrenal tissue. During 2-hour control incubations of adrenal cortical cells, steroidogenesis was not detected, and the levels of sterol ester, phospholipid, and triglyceride fatty acids were not significantly altered. Incubations with adrenocorticotropic hormone (ACTH) resulted in coricosterone production and significant depletions of sterol ester and triglyceride fatty acids, but not of phospholipid fatty acids. Although all fatty acid esters of cholesterol were hydrolyzed under these conditions, the greatest contributions to the net decrease in sterol esters were by oleate, arachidonate, and adrenate. Incubations with dibutyryl cyclic adenosine monophosphate (0.5 mM) resulted in significantly greater levels of corticosterone production than did ACTH (250 muunits), but the effects on cellular lipids were comparable to those seen with the tropic hormone. This study represents the first demonstration of hormone-induced hydrolysis of sterol esters in an in vitro cell suspension system. The results are discussed with respect to hormone-sensitive sterol ester hydrolase of adrenal cortex, and to the role of endogenous cholesteryl esters in the steroidogenic pathway.

Localization and origin at rat intestinal cholesterol esterase determined by immunocytochemistry.

Monospecific rabbit antisera to rat pancreas cholesterol esterase were employed in the unlabeled antibody enzyme method of immunocytochemistry in combination with the horseradish peroxidase--antihorseradish peroxidase complex to localize this pancreatic enzyme within the wall of rat small intestine. Intestinal rings were fixed in paraformaldehyde with satisfactory preservation of structure and retention of cholesterol esterase antigenic determinants. Fixed sections, 6 micrometers thick, were stained. In the light microscope, specific reaction product, represented by intense brown areas, was uniformly distributed within the absorptive cells but was notably absent from the microvillar membrane. Reaction product was also seen within the laminapropria and submucosa. In contrast, reaction product was absent from sections of proximal intestine surgically deprived of pancreatic juice for 72 hours. Furthermore, the intensity of staining in sections of normal intestine decreased with increasing distance from the pancreatic duct. These observations support the concept that "intestinal" cholesterol esterase is of pancreatic origin. This enzyme is localized within the cells as opposed to the absorptive surface.

Prostaglandin synthesis in rat adrenocortical cells.

The biosynthesis of prostaglandins by isolated rat adrenocortical cells has been studied by determinations of products formed during incubations with labeled arachidonic acid and by radioimmunoassays. Analysis by thin-layer chromatographic separation of silicic acid column fractions indicated that PGE2, PGA2, (B2) and PGF2 alpha were the predominant prostaglandins formed by rat adrenocortical cells. Approximately 75% of the incorporated isotope was associated with the prostaglandins of the PGE pathway [PGE2 + PGA2 (B2)]. This was a consistent finding whether cells were incubated directly with arachidonic acid or with cells prelabeled with the substrate prior to study. ACTH did not affect the uptake or oxidation of [1-14C]-arachidonate, but did significantly increase incorporation of labeled substrate into [14C]prostaglandins. Of the ACTH-induced increase, 92% was accounted for by an increase in prostaglandins of the E pathway. Studies with prelabeled cells indicated that 77% of the prostaglandins synthesized in both control and ACTH-stimulated adrenocortical cells was released into the incubation medium during the 2-hr study. These had the same composition [88% PGE2 + PGA2 (B2)] as did the intracellular prostaglandins. Analysis by radioimmunoassays gave comparable data on the distribution of E- and F-type prostaglandins in control cells and cells incubated with ACTH or dibutyryl cyclic AMP. Thus, with these techniques, 88-92% of the increased prostaglandin synthesis due to ACTH or cyclic AMP was produced by the PGE2 rather than the PGF2 alpha pathway.

Puromycin inhibition of cholesterol absorption in the rat.

The effect of puromycin on the intestinal absorption of cholesterol has been studied in rats with indwelling catheters in the left thoracic lymphatic duct. Puromycin administration to female rats produced a marked depression of cholesterol absorption under conditions where the absorption of simultaneously administered fatty acid was also dramatically inhibited. The same treatment of male rats also produced a significant depression in cholesterol absorption, but was without effect on absorption of the fatty acid. Despite the depressions of lipid absorption in puromycin-treated animals, there was no accumlation of either cholesterol or fatty acid in the intestinal mucosa of either sex. Actinomycin D treatment of fasting male and female rats, receiving constant infusions of saline, had no effect on the rate of lymph production. This suggest that altered lymph production was not responsible for the depressed lipid absorption observed in fed animals treated with protein synthesis inhibitors. The selective inhibition of cholesterol absorption in male rats also precludes the possibility that the major effect of the inhibitor is on delayed gastric emptying.

Inhibition of lymphatic absorption of cholesterol by cholestane-3 beta, 5 alpha, 6 beta-triol.

The effect of cholestane-3,5alpha,6-triol (CT) on the intestinal absorption of cholesterol and oleic acid, as well as the absorption of labeled CT, was studied in lymph ductcannulated rats. Intragastric administration of 50 mg of CT in an emulsion with cholesterol-7alpha-(3)H and oleic acid-1-(14)C resulted in 50% inhibition of sterol transfer into lymph but only 8% depression of fatty acid absorption over an 8 hr period. The absorption of labeled CT into lymph was only 2-3% compared with 50% absorption of cholesterol when each was fed alone. 10% of the fed CT was recovered in the intestinal mucosa, and of this, one-half was associated with the brush border fraction. In rats fed CT 6 days prior to cholesterol and fatty acid administration, there was no effect on fatty acid absorption, while cholesterol absorption was reduced by almost 30%. When the intestinal mucosa from these animals were investigated by electron microscopy, it appeared that CT feeding resulted in numerous enlarged mitochondria and a marked increase in length of the microvilli. If animals were allowed to recover for 6 days from the CT prefeeding regime, the intestinal mucosa appeared normal, and the absorption of cholesterol approached that in controls. A possible mechanism for CT inhibition of cholesterol absorption was shown to be competition for the enzyme cholesterol esterase which esterifies cholesterol prior to entrance into the lymphatic system. CT itself is poorly esterified and poorly absorbed, but it is effective in inhibiting esterification of cholesterol in vitro.

Differential transport of cholesterol and oleic acid in lymph lipoproteins: sex differences in puromycin sensitivity.

Adult rats of both sexes were prepared with indwelling drainage catheters in the left thoracic lymphatic duct, and with duodenal infusion catheters. Control and puromycin-treated animals were administered an aqueous test emulsion containing [7alpha-(3)H]cholesterol and [1-(14)C]oleic acid, followed two hours later, by a tracer dose of [1-(14)C]-leucine. Successive 2-hr lymph samples were subjected to ultracentrifugal separations of the major lipoprotein classes. These were specifically extracted for lipids, and for DNA- and lipid-free protein. In both sexes, oleic acid absorption was largely associated with the d < 1.006 g/ml chylomicron fraction throughout the 6-hr experimental period. Small but consistent levels of labeled fatty acid appeared in the 1.006 < d < 1.019 g/ml VLDL fraction. However, with both sexes 25-35% of the absorbed cholesterol appearing in lymph was recovered in the VLDL fraction. Furthermore, there were statistically greater levels of cholesterol in this lymph fraction in females than in males. Cumulative protein levels and leucine incorporation into chylomicron proteins was comparable in both sexes. However, VLDL protein in the female was significantly greater than in the male and this difference was mimicked by the greater incorporation of leucine into VLDL proteins in the female. In males, there were no significant effects of puromycin on cholesterol or oleic acid absorption, despite a marked inhibition in chylomicron protein levels and leucine incorporation into this fraction. There was also no effect of the inhibitor on VLDL protein levels or on leucine incorporation into VLDL peptides. Cholesterol but not oleic acid absorption in females was significantly depressed by administration of puromycin, and this was largely attributed to a decrease in VLDL transport of the sterol. Also, unlike males, leucine incorporation into VLDL peptides was inhibited by 75% by puromycin administration. These results emphasize the importance of non-chylomicron transport of cholesterol during absorption and suggest a hormonal influence on intestinal VLDL synthesis in female rats.-Vahouny, G. V., E. M. Blendermann, L. L. Gallo, and C. R. Treadwell. Differential transport of cholesterol and oleic acid in lymph lipoproteins: sex differences in puromycin sensitivity.

Enzymatic assay for cholesterol ester hydrolase activity.

A rapid and accurate method is described for the assay of cholesterol ester hydrolase (CEH) activity. Aliquots of the enzyme-substrate incubation mixture are extracted into isopropanol. The free cholesterol concentration in each extract is determined enzymatically using a single aqueous reagent containing cholesterol oxidase and peroxidase. The free cholesterol remaining after the cholesterol ester hydrolase-catalyzed esterification is converted to delta 4-cholestenone and hydrogen peroxide (H2O2); peroxidase couples H2O2 with phenol and 4-amino-antipyrine to yield a stable rose-colored product absorbing at 500 nm. The method is highly reproducible and the values correlate well with those obtained with the chromatographic radioassay of CEH activity.

Cholesterol arachidonate as a prostaglandin precursor in adrenocortical cells.

Rat adrenocortical cells were incubated with labeled arachidonate, and the radioactivity in unesterified fatty acids was reduced by washing with 2% albumin solutions. These cells were then incubated for two hours in the absence and presence of 7.1 x 10(-10)M ACTH. During subsequent incubation of prelabeled cells with ACTH, both the mass and radioactivity of arachidonate in adrenocortical cholesteryl esters was depleted to the same extent (30--32%). The released arachidonate was in part incorporated into phospholipids, and there was also a significant increase in unesterified arachidonic acid. During this period, there was also increased incorporation of arachidonate into labeled prostaglandins. Of this increase, 92% by isotope analysis, and 88% by radioimmunoassay techniques was attributable to prostaglandins of the E pathway. These data demonstrate that prostaglandin E synthesis is specifically increased during ACTH stimulation of rat adrenocortical cells and suggest that a major source of the arachidonate substrate for this synthesis is derived from hormone-dependent hydrolysis of cortical cholesteryl esters.