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1.
Neutral cholesteryl ester hydrolase activity (EC 3.1.1.13) present in microsomes isolated from lactating rat mammary glands
was found to be inhibited by a factor (or factors) occurring in the cytosolic fraction of male rat liver. The inhibitor was
heat-labile, non-dialyzable, destroyed by proteolysis, and was stable following preparation of an acetone/diethyl ether powder
of the cytosolic fraction. The protein also inhibited the activity of hormone-sensitive lipase (HSL) (from bovine adipose
tissue) and esterase fromCandida cylindracea, but seemed to be more active against the neutral hydrolase found in rat liver microsomes. For the mammary gland microsomal
cholesteryl ester hydrolase, the extent of the inhibitory effect was dependent on the concentration of the cytosolic protein,
50% inhibition being achieved by about 100 μg of cytosolic protein, and on the method of initiating the enzyme assay. Kinetic
analysis indicated that, under circumstances where the reaction was initiated by the addition of substrate, the inhibition
was characterized as “uncompetitive”. When an inhibitor/substrate complex was allowed to form in the absence of enzyme, an
element of “competitive” inhibition was introduced into the reaction. Food withdrawal reducted the activity of the inhibitor
in live by 56%, but activity was fully restored by short-term re-feeding. In contrast, feeding a diet high in fat led to a
34% increase in activity. The present findings suggest that the inhibitory factor(s) may be involved in the regulation of
the hydrolysis of cholesteryl esters in the liver and also in other cell types. 相似文献
2.
The role of lipoproteins and serum in the formation and accumulation of cholesteryl esters in human monocyte-derived macrophages
(HMD macrophages) was investigated; studies were also carried out with IC21 cells (a cell line derived from mouse peritoneal
macrophages). Following preincubation of HMD macrophages with lipoprotein-depleted serum (LPDS), both native and acetylated
low density lipoprotein (LDL and AcLDL, respectively) stimulated the formation of cholesteryl esters with a resultant increase
in cellular cholesteryl ester content. Cholesteryl ester formation and accumulation was also stimulated in macrophages exposed
continuously to 25-hydroxycholesterol. However, the stimulation of cholesterol esterification by either lipoproteins or 25-hydroxycholesterol
was not inhibited by progesterone in HMD macrophages, but was in the IC21 cells. Cholesterol efflux and the hydrolysis of
cellular cholesterol ester, promoted by serum components, were studied in HMD macrophages preloaded with cholesteryl ester
by incubation with 25-hydroxy cholesterol. Replacement of the medium with one devoid of 25-hydroxycholesterol resulted within
24 hr in at least a 30% decrease in the cholesteryl ester content of the HMD macrophages; replacement with a medium high in
cholesterol acceptor content (LPDS or high density lipoprotein) and incubation for three days led to the most marked decreases
in cellular cholesterol content. Thus, hydrolysis of the cholesteryl esters by HMD macrophages was not dependent on the presence
of cholesterol acceptors in the medium, but cellular cholesterol content was. 相似文献
3.
Kathleen M. Botham 《Lipids》1991,26(11):901-906
An acid cholesteryl ester hydrolase activity associated with a fraction containing mitochondria and lysosomes from rat lactating
mammary glands was found to have a pH optimum of 5.0. Its sedimentation pattern was closely related to that of the lysosomal
enzyme markers acid phosphatase and β-glucuronidase, suggesting that the activity is associated with the lysosomes. The enzyme
was strongly inhibited by Cu2+, but was inhibited little by other divalent metal ions. Acid cholesteryl ester hydrolase activity was almost completely abolished
byp-hydroxymercuribenzoate, but this effect was reversed in the presence of an equimolar concentration of reduced glutathione
(GSH), indicating that the enzyme requires free sulfhydryl groups for activity. These properties are similar to those of acid,
lysosomal cholesteryl ester hydrolases found in other tissues. Acid cholesteryl ester hydrolase activity was 8–14 fold higher
in mammary tissue from lactating as compared to virgin rats. Neutral cholesteryl ester hydrolase activities associated with
the microsomal and cytosolic subcellular fractions were also increased in lactating glands, but to a lesser extent. In addition,
a 2-fold increase in the activities of both the acid and microsomal neutral enzymes was seen during the first few days of
lactation, while the cytosolic neutral activity remained constant. These results suggest that mammary gland cholesteryl ester
hydrolases have a role in the regulation of cholesterol metabolism in mammary cells, and in the provision of cholesterol for
secretion into milk. 相似文献
4.
A cytosolic protein, that is inhibitory to neutral cholesteryl ester hydrolase, has been investigated in the livers of female
rats using microsomes isolated from the mammary gland of lactating rats as an enzyme source. To facilitate comparisons, inhibitory
activity is expressed in terms of the amount (μg) of cytosolic protein required to reduce esterase activity by 50% and is
compared to the hepatic content of both cholesterol and cholesteryl esters. The experiments revealed a sexual difference in
the level of inhibitory activity, with the livers of both suckling and mature male animals containing less of the material
than the corresponding females. Alterations in the physiological status of the females, such as pregnancy and lactation, led
to a decrease in the activity of the protein. This was reversed by blocking lactation with a combination of an antiserum to
rat growth hormone and the anti-prolactin drug, bromcoriptine, but not by premature weaning of the animals. Food withdrawal
for 24 hr also had the effect of increasing inhibitory activity. In general the cholesteryl ester content of the livers correlated
with the level of inhibitory activity. Thus the activity of the cytosolic inhibitor of neutral cholesteryl ester hydrolase
responded to changes in both the hormonal and the nutritional status of the female animal. It is suggested that the presence
of the greater cholesteryl ester hydrolase inhibitory activity in the female liver may help to explain the lower risk of coronary
heart disease in premenopausal females by facilitating increased hepatic storage of the sterol in the form of the ester. 相似文献
5.
Short term regulation of hepatic cholesterol ester hydrolase by reversible phosphorylation is described. Two different kinase
systems seem to be involved in this regulation. The addition of ATP, cyclic AMP and Mg2+ to rat liver 104,000× g supernatant (S104) produced a 100–140% increase in cholesterol ester hydrolase activity. This stimulation
was abolished when protein kinase inhibitor was added prior to the addition of ATP, cyclic AMP and Mg2+. Cholesterol ester hydrolase activity was also stimulated when calcium ions, phosphatidylserine, and diolein were added to
S104 along with ATP and Mg2+. Diolein in this reaction could be substituted by phorbol 12-myristate 13-acetate. Preincubation of S104 with alkaline phosphatase
resulted in a deactivation of cholesterol ester hydrolase. The addition of increasing concentrations of Mg2+ to S104 produced increasing inhibition of cholesterol ester hydrolase activity, and this effect was blocked by NaF.
It is suggested that rat liver cholesterol ester hydrolase is activated by cyclic AMP dependent protein kinase and protein
kinase C. Deactivation is accomplished by dephosphorylation catalyzed by a phosphoprotein phosphatase, dependent on Mg2+.
This work was presented at the Twenty-Third Southeastern Regional Lipid Conference, held October 26–28, in Cashiers, North
Carolina. 相似文献
6.
7.
The effects of 5 μg/ml of 25-hydroxycholesterol; cholestane-3β, 5α,6β-triol; and cholesterol on acyl CoA cholesterol acyltransferase,
acid cholesteryl ester hydrolase and neutral cholesteryl ester hydrolase was studied in cultured rabbit aortic smooth muscle
cells. After 1 hour incubation, 25-hydroxycholesterol resulted in a fourfold stimulation of acyl CoA cholesterol acyltrans-ferase
activity. No stimulation by 25-hydroxycholesterol was noted before 15 minutes or after 5 hours of incubation. Neither cholestane-3β,5α,6β-triol
nor cholesterol influenced acyl CoA cholesterol acyltransferase activity at any time interval. No significant effects of any
of the sterols were noted on acid cholesteryl ester hydrolase or neutral cholesteryl ester hydrolase activity. The imbalance
between acyl CoA cholesterol acyl trans-ferase and hydrolase activities induced by 25-hydroxycholesterol could result in cholesteryl
ester accumulation by arterial smooth muscle cells, which may be associated with atherosclerosis. 相似文献
8.
Repeated oral administrations of ethanol to rats induced accumulation of cholesteryl ester, as well as triglyceride in the livers. The contents of free cholesterol and phospholipid in the livers were not changed significantly in the present experiment in which ethanol ingestions were repeated four times. Although in vitro esterification of cholesterol by particle fractions of the alcoholic fatty liver was not affected, hydrolysis of cholesteryl palmitate by the supernatant fraction of the liver homogenate was reduced when compared with those of the control group which was given water or isocaloric glucose. The results of in vitro esterification of cholesterol and hydrolysis of cholesteryl palmitate in the liver of the rats which were ingested with glucose were larger than those of the control rats which were given water. 相似文献
9.
10.
The suppression of plasma very low density lipoprotein (VLDL) triglyceride levels by dietary fish oils rich in polyunsaturated
n−3 fatty acids has been attributed to decreased hepatic VLDL secretion. To investigate the effect of n−3 fatty acids on lipid
metabolism and VLDL secretion in a tissue culture system, we incubated rabbit hepatocytes with oleic acid and eicosapentaenoic
acid (EPA) and examined [3H]glycerol and [14C]fatty acid incorporation into hepatocyte triglyceride and phospholipid and into media VLDL. Glycerol incorporation studies
showed that EPA failed to stimulate VLDL triglyceride secretion from hepatocytes as occurred with oleic acid (P<0.05). Oleic
acid preferentially enhanced hepatocyte triglyceride synthesis while EPA stimulated significantly phospholipid synthesis (P<0.01).
Varying the relative concentrations of oleic acid and EPA at a constant total fatty acid concentration corroborated preferential
triglyceride synthesis from oleic acid. Synthesis shifted predominantly to phospholipids with increasing concentrations of
EPA and lower levels of oleic acid. Incorporation of the [14C]fatty acids (800 μM) followed similar patterns: 87% of [14C]oleic acid was incorporated into hepatocyte triglyceride and 44% of [14C]EPA was assimilated in hepatocyte phospholipid (p<0.001). Fatty acids at trace concentrations (53 nM) showed a more divergent
pattern of lipid incorporation: 60% of [14C]oleic acid was incorporated into triglyceride while 91% of [14CEPA was incorporated into phospholipid (p<0.001). We conclude that in primary rabbit hepatocyte culture, which appears to
be a useful model to study lipid metabolism and VLDL secretion, EPA is avidly incorporated into phospholipid while oleic acid
predominantly becomes esterified in triglyceride. In addition, EPA, unlike oleic acid, fails to stimulate hepatocyte VLDL
secretion. These divergent effects on hepatocyte lipid metabolism are, at least in part, likely to be responsible for fish
oil induced suppression of plasma triglycerides. 相似文献
11.
Short-term activation of microsomal cholesterol ester hydrolase by glucagon, cAMP analogues, and vasopressin in isolated rat
hepatocytes is described. Glucagon led to a dose-and time-dependent activation of cholesteryl oleate hydrolysis, but values
returned to basal levels within 120 min. Exposure of isolated hepatocytes to 0.5 mM concentrations of dibutyryl-cAMP or 8-[4-chlorophenylthio]-cAMP,
or 25 μM forskolin caused persistent activation of cholesterol ester hydrolase activity after a lag period of 30 min. The
three agents resulted in early marked intracellular accumulation of cAMP that declined progressively, and moderate and sustained
reductions in the diacylglycerol content. The actions of glucagon on hepatocytes were inhibited by pretreatment of cells with
10 nM [8-arginine] vasopressin. Vasopressin elicited a consistent and sustained increase in cholesterol ester hydrolase activity
and diacylglycerol without affecting cAMP while reducing the effect of glucagon on cAMP. Furthermore, the effects of glucagon
and vasopressin on the activation of cholesterol ester hydrolase were not additive despite the similarity of their stimulation
of diacylglycerol formation. Blockade of vasopressin-mediated activation of cholesterol ester hydrolase and diacylglycerol
content were induced by excess prazosin. These data suggest that stimulation of microsomal cholesterol ester hydrolase in
isolated liver cells may involve at least two signal transduction systems. 相似文献
12.
The utility of 2-hydroxypropyl-β-cyclodextrin for increasing the sensitivity of assays for the microsomal acyl-CoA:cholesterol
acyltransferase, and the acid lysosomal and the neutral microsomal and cytosolic cholesterol ester hydrolase activity was
studied in rat hepatocytes. Enzyme assays, at optimal concentrations of cyclodextrin, were validated by assessing: (i) linearity
of product formation with incubation time and protein amount, and saturation with substrate, and (ii) the effect of treatments
of cells or of subcellular fractions on enzyme activities. Delivery of cholesterol dissolved in 2-hydroxypropyl-β-cyclodextrin
to the acyl-CoA:cholesterol acyltransferase assay mixture raised the enzyme activity more than 8-fold and was twice that measured
when cholesterol was added in Triton WR-1339. 2-Hydroxypropyl-β-cyclodextrin itself was partially effective, apparently by
making endogenous cholesterol more accesible to the enzyme. Inclusion of 2-hydroxypropyl-β-cyclodextrin in cholesterol ester
hydrolase assays using standard micellar substrates doubled the activity estimated in lysosome and microsome preparations
and enhanced the cytosolic cholesterol esterase activity by about 50%. Differences in the catalytic activity of acyl-CoA:cholesterol
acyltransferase and cholesterol ester hydrolases caused by treatment of hepatocytes with compound 58-035 or 25-hydroxycholesterol,
or of subcellular fractions with NaF, were maintained when enzymes were assayed with cyclodextrin. The results indicate that
2-hydroxypropyl-β-cyclodextrin is a suitable vehicle for delivering cholesterol to acyl-CoA:cholesterol acyltransferase and
enhances the sensitivity of standard assays of the enzymes governing the intrahepatic hydrolysis of cholesteryl esters. 相似文献
13.
Hepatic neutral cytosolic cholesteryl ester hydrolase (hncCEH) is a key enzyme in the regulation of hepatic free cholesterol
(FC). In examining the effects of over-expression of this enzyme on cholesterol homeostasis, mice were infected with a recombinant
adenovirus construct (AdCEH) of the rat hncCEH cDNA driven by the human cytomegalovirus promoter. Cholesteryl esterase and
p-nitrophenylcaprylate (PNPC) esterase activities were measured in liver postmitochondrial supernatants at 1, 3, 7, and 11
d after infection with AdCEH or a control virus expressing β-galactosidase (AdβGAL). The PNPC esterase activity of AdCEH mice
peaked threefold higher than controls on day 2, declining on subsequent days. In contrast, cholesteryl esterase peaked eightfold
higher than controls on day 3, indicating a shift in substrate selectivity of hncCEH. Hepatic FC peaked at 144% of controls,
7 d postinfection. The mRNAs for cholesterol 7α-hydroxylase, sterol 27-hydroxylase, and HMG-CoA reductase decreased to 47,
46, and 58% of controls, respectively, on day 7, coinciding with peak FC concentrations. Coiniding with increased cholesteryl
esterase activity, hepatic esterified cholesterol dropped precipitously from day 3 onward, to 11% of controls by day 11. Hepatic
TAG levels also declined, consistent with the reported TAG lipase activity of hncCEH. These results demonstrate elevation
of FC and depletion of cholesteryl esters by over-expression of hncCEH, which were resistant to compensatory responses by
other enzymes of cholesterol homeostasis. 相似文献
14.
Glycodihydrofusidate, which has the same detergent properties as bile salts, is excreted almost exclusively by the bile duct
after intravenous injection in the rat. As with bile salts, it leads to a significant (P≤0.05) increase in excretion of lecithins
and cholesterol (0.15 μmol lecithin and 0.026 μmol cholesterol per 1 μmol of glycodihydrofusidate excreted). In addition,
this drug simultaneously inhibits excretion of both endogenous bile salts and bile pigments. 相似文献
15.
M. G. Horning R. M. Hebert R. J. Roth D. L. Davis E. C. Horning E. P. Fischer G. L. Jordan Jr. 《Lipids》1972,7(2):114-120
The effect of chronic administration of ethylp-chlorophenoxyisobutyrate (CPIB) on the secretion of bile lipids was studied in four dogs with surgically implanted Thomas
cannulae for periods of 2–7 months. The concentration of cholesterol, triglycerides andp-chlorophenoxyisobutyric acid in serum and of bile acids, cholesterol and phospholipids (phosphatidyl cholines) in bile were
measured. Chronic administration of CPIB resulted in a marked increase in the concentration of cholesterol, bile acids and
phosphatidyl cholines in the bile of all dogs, and a decrease in serum cholesterol and triglyceride concentration in serum
in three of the four dogs. Serum concentrations ofp-chlorophenoxyisobutyric acid were monitored to insure the presence of the drug in the dogs; however, no correlation between
serum levels ofp-chlorophenoxyisobutyric acid and the concentration of biliary lipids was noted. The bile acids andp-chlorophenoxyisobutyric acid were determined by gas chromatographic procedures and the structures were confirmed by gas chromatography-mass
spectrometry.
One of eight papers presented at the symposium “Recent Advances in Drugs Affecting Lipid Metabolism,” AOCS Meeting, Houston,
May 1971. 相似文献
16.
R. N. Redinger 《Lipids》1979,14(3):277-284
The effect of 1.7–2.2 mg/day oral phenobarbital over short (1 MO) and long term (6–24 MO) treatment on primary bile acid (BA)
secretion, composition, synthesis, pool size, and enterohepatic cycling rates as well as phospholipid (PL) and cholesterol
(C) secretion rates and biliary composition was determined in 12 asymptomatic cholesterol gallstone subjects while 5 normals
had only short term studies. Phenobarbital enhanced BA and C secretion (BA-636±166 to 2110±382 mg/hr, p<0.001 and C-42±5 to
224±48 mg/hr, p<0.001) and BA cycling rate in all subjects studied during stimulated enterohepatic circulation but, during
fasting, it only enhanced BA secretion (451±129 vs. 759±159 mg/hr, p<0.05) in gallstone subjects. Cholic acid (CA) production
rate (171±28 to 395±9 mg/hr, p<0.05) and pool size (727±80 to 1209±132 mg/hr, p<0.05) were increased during long term treatment
of gallstone subjects, while the proportion of CA in bile and deoxycholic acid (DCA) in feces increased. Treatment decreased
biliary cholesterol from supersaturated to saturated levels (9.5±0.6 vs. 6.1±0.9 moles%, p<0.02) in all fasting gallstone
subjects and decreased cholesterol crystal loads during long term treatment; but, while prohibiting gallstone growth, it did
not affect stone dissolution over 24 month's treatment. Phenobarbital also failed to affect biliary lipid composition or bile
acid pool size in short term treatment of normals. Thus, phenobarbital affected hepatic metabolism of CA by enhancing production
rate, secretion, and pool size; and in testinal metabolism of both CA and chenodeoxycholic (CDC) acids by increasing their
cycling rates. Phenobarbital may have failed to produce stone dissolution by enhancing CA production and pool size more than
that of CDC.
Portions of this work were presented at the National Meeting of the American Federation of Clinical Research, Atlantic City,
April 29, 1973 and at the Annual Meeting of the Canadian Society of Clinical Investigation, Winnepeg, Manitoba, January 21,
1975 as well as that of the Royal College of Physicians and Surgeons of Canada, January 27, 1977 at Toronto, Canada. 相似文献
17.
Effects of passive smoking on the regulation of rat aortic cholesteryl ester hydrolases by signal transduction 总被引:2,自引:0,他引:2
The effects of exogenous oxidative stress due to passive smoking on cholesteryl ester (CE)-metabolizing enzymes and their
regulatory kinases were examined by exposing rats to cigarette smoke (CS) for a 1-h period twice a day for 8, 12, or 20 wk.
An oxidatively modified low density lipoprotein (Ox-LDL) with a high lipid peroxide was identified in three CS groups after
all three exposure periods. The rat aortic acid and neutral CE hydrolases (ACEH and NCEH) were activated to similar extents
by both cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) in the presence of their respective cofactors. The
aortic PKC activity in the three CS groups exhibited significant reductions of 72, 84, and 75% as compared with the respective
controls, which coincided with the reductions in the ACEH activities (86, 71, and 80%, respectively), whereas the PKA activities
increased to 121, 197, and 252% in the three CS groups, respectively. Reflecting the increase of the PKA activity, the NCEH
activity exhibited increases of 112% at 8 wk and 140% until 12 wk of exposure and decreased by 50% of the control value at
20 wk of exposure, suggesting inactivation of NCEH itself. The activation of acyl-CoA:cholesterol O-acyl-transferase activity was associated with an increase of free cholesterol in aorta. The vitamin E diet prevented the
formation of Ox-LDL and the oxidative inactivation of most enzymes, especially PKC, until 12 wk, but was less effective by
20 wk. The oxidative inactivation of PKC, particularly its activated form that translocated to the membrane fraction, was
confirmed in the in vitro exposure to active oxygen generators at an optimal concentration; this inactivation was prevented by catalase and superoxide
dismutase. These results suggested that the formation of Ox-LDL and alterations in CE-metabolizing enzymes caused by passive
smoking could contribute to a twofold increase in the aortic CE content, thereby contributing to one of the mechanisms for
atherosclerosis associated with smoking. 相似文献
18.
Effects of ethanol diets on cholesterol content and phospholipid acyl composition of rat hepatocytes
Chronic treatment of adult male rats with ethanol liquid diets resulted in alterations in phospholipid and cholesterol contents
as well as the acyl composition of phosphatidylethanolamine (PE), phosphatidylinositol (PI)-phosphatidylserine (PS) mixture,
and phosphatidylcholine (PC) of isolated hepatocytes. The influence of ethanol on these lipids was largely dependent on the
proportion of dietary fat. Phospholipid and total cholesterol contents were elevated 23 and 27%, respectively, by ethanol
when offered in a low-fat diet (5% corn oil). Only the percentage of arachidonic acid from PI-PS was significantly reduced
in the low-fat ethanol group. Exposure to a high-fat (34% corn oil) diet in the presence of ethanol for 4–5 weeks resulted
in a significant decrease in arachidonate/linoleate ratios of hepatic PE, PS-PI and PC, while total phospholipid content remained
constant. In the high-fat, ethanol-treated group, hepatic cholesterol content was increased 2-fold. These results suggest
that the level of dietary fat plays an important role in determining the effects of chronic ethanol consumption on hepatic
cholesterol content and phospholipid acyl composition. 相似文献
19.
The isolated perfused rat liver technique was utilized to study the effect of biliary obstruction on the rates of uptake,
synthesis and secretion of14C-labeled cholesterol in an effort to elucidate the mechanism of hypercholesterolemia in this condition. By perfusing normal
and biliary obstructed livers with a medium containing14C-labeled acetate or14C-labeled lipoprotein cholesterol and by measuring cholesterol biosynthesis in vitro, it was possible to clarify some of the
questions concerning these aspects of lipid metabolism. The results of these studies revealed that the uptake of cholesterol
was not altered in biliary obstruction. Instead the hypercholesterolemia of biliary obstruction arises from an increased rate
of secretion of cholesterol from liver, which is accompanied by an increased rate of hepatic cholesterolgenesis. Evidence
is also presented to suggest that a substance is present in biliary obstructed blood which is capable of initiating increased
cholesterolgenesis in normal livers.
Deceased. 相似文献
20.
A 28 kDa inhibitory protein was purified from ratestis cytosol by sequential 40–65% ammonium sulfate precipitation, cation
exchange chromatography, anion exchange chromatography, and preparative SDS-polyacrylamide gel electrophoresis. The heat-stable,
trypsin-labile protein exhibited nonenzymatic, concentration-dependent inhibition of testicular and pancreatic cholesteryl
ester hydrolases at all stages of putification. Copurifying at each stage was a 26.5 kDa protein which comprised 25% of the
mass of the two proteins. Polyclonal antibodies raised to either or both 28 kDa and 26.5 kDa proteins by direct injection
of excised electrophoretic bands cross-reacted with both proteins on western blots, immunoprecipitated both proteins, and
neutralized inhibitory activity. Amino acid compositions of the individual proteins electroeluted from SDS-polyacrylamide
gels were different from those of other surface-active proteins of similar molecular weights. Both proteins exhibited identical
pl of 4.8 on chromatofocusing columns and two-dimensional gel electrophoresis. Although the subcellular distribution of the
28 kDa protein is unknown, its testicular cytosolic concentration, calculated from the purified protein mass, was 8×10−9 mols/L, which probably underestimates the actual concentration by an order of magnitude. This is greater than the minimum
concentration required forin vitro inhibition (10−9 mols/L), consistent with a physiological role for this protein. 相似文献