ABSTRACT: Hepatic neutral cytosolic cholesteryl ester hydrolase
(hncCEH) is a key enzyme in the regulation of hepatic free choles-
terol (FC). In examining the effects of over-expression of this en-
zyme on cholesterol homeostasis, mice were infected with a re-
combinant adenovirus construct (AdCEH) of the rat hncCEH cDNA
driven by the human cytomegalovirus promoter. Cholesteryl es-
terase 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-galac-
tosidase (Ad
βGAL). The PNPC esterase activity of AdCEH mice
peaked threefold higher than controls on day 2, declining on sub-
sequent days. In contrast, cholesteryl esterase peaked eightfold
higher than controls on day 3, indicating a shift in substrate selec-
tivity of hncCEH. Hepatic FC peaked at 144% of controls, 7 d
postinfection. The mRNAs for cholesterol 7
α-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. Coinciding 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 re-
sistant to compensatory responses by other enzymes of cholesterol
homeostasis.
Paper no. L9627 in Lipids 40, 31–38 (January 2005).
Free cholesterol (FC) is an essential component of membranes
and an important determinant of their physicochemical proper-
ties. FC is also the precursor to bile acids, steroid hormones,
and oxysterols that regulate pathways of lipid metabolism, in-
cluding cholesterol biosynthesis (1–3). Indicative of the meta-
bolic and regulatory significance of these roles, intracellular
FC levels are maintained within relatively narrow ranges over
a broad range of dietary intakes and metabolic states (4–7).
Among the homeostatic processes contributing to this stability
is the cycle of esterification with long-chain FA and hydrolysis
of the resulting cholesteryl esters (CE) to regenerate metaboli-
cally active FC. The hydrolytic component of this CE cycle is
catalyzed by the hepatic neutral cytosolic CE hydrolase (hnc-
CEH), which has been cloned and characterized in this labora-
tory (8–11). The hncCEH is a broad-specificity carboxylesterase
that hydrolyzes TAG (12) and water-soluble esters of
p-nitro-
phenol, as well as CE (8,9,12). Similar to other enzymes in cho-
lesterol metabolism, the hncCEH promoter has active sterol re-
sponsive elements that regulate hncCEH expression (10,13,14).
Moreover, hncCEH mRNA, protein, and enzymatic activity are
altered in response to perturbations of sterol flux, consistent
with the hypothesis that hncCEH plays a significant role in cho-
lesterol homeostasis (8,10,15).
Cholesterol homeostasis is maintained in the liver by regu-
lation of the various input and output pathways for metabolism
and transport. In addition to hncCEH, these include (i) ACAT,
which catalyzes the esterification component of the CE cycle;
(ii) HMG-CoA reductase (HMGCoAR), the rate-determining
enzyme in the
de novo synthesis of cholesterol; (iii) cholesterol
7α-hydroxylase (CYP7A1), and sterol 27-hydroxylase
(CYP27), which catalyze the initial steps in the classic and al-
ternative pathways, respectively, for bile acid synthesis; and
(iv) lipoprotein synthesis, uptake, and degradation (16,17). The
response of each of these to perturbations in the metabolically
active cholesterol pools is the means by which the liver main-
tains cholesterol homeostasis.
Although a number of studies have examined the ef
fects of
perturbations of cholesterol homeostasis on hncCEH, the cur-
rent studies were designed to determine the effects of over-ex-
pression of hncCEH on hepatic sterol metabolism and other
key enzymes of cholesterol homeostasis. To this end, an adeno-
viral vector was constructed with a cytomegalovirus promoter
to constitutively express hncCEH. W
e describe the validation
of this construct in cell culture and mice and the effects of over-
expression of hncCEH on hepatic lipid levels and mRNAs of
several enzymes known to exhibit compensatory responses to
altered sterol flux through the liver.
MATERIALS AND METHODS
Chemicals and supplies. Radionuclides were purchased from
New England Nuclear (Boston, MA); p-nitrophenylcaprylate
Copyright © 2005 by AOCS Press 31 Lipids, Vol. 40, no. 1 (2005)
*To whom correspondence should be addressed at Department of Biochem
-
istry, Medical College of Virginia Campus, Virginia Commonwealth Uni
-
versity, Richmond, VA 23298. E-mail: grogan@hsc.vcu.edu
Abbreviations: Ad
βGAL, adenovirus over-expressing β-galactosidase; AdCEH,
adenovirus over-expressing cholesteryl ester hydrolase; CE, cholesteryl ester;
CYP7A1, cholesterol 7α-hydroxylase; CYP27, sterol 27-hydroxylase; FC, free
cholesterol; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; HMG-CoAR,
HMG-CoA reductase; hncCEH, hepatic neutral cytosolic cholesteryl ester hydro-
lase; HSL, hormone-sensitive lipase; pfu, plaque-forming units; PNPC, p-nitro-
phenylcaprylate.
Over-expression of Hepatic Neutral Cytosolic Cholesteryl
Ester Hydrolase in Mice Increases Free Cholesterol and Reduces
Expression of HMG-CoAR, CYP27, and CYP7A1
Timothy B. Langston
a
, Phillip B. Hylemon
b
, and W.M. Grogan
a,
*
Departments of
a
Biochemistry and Molecular Biophysics and
b
Microbiology and Immunology,
Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, Virginia 23298