Age-Related Hypercholesterolemia and HMG-CoA Reductase Dysregulation: Sex Does Matter (A Gender Perspective)

Abstract

Although cardiovascular diseases are less prevalent in premenopausal women than in men, their occurrence in women increases at the onset of menopause, and the loss of female sex hormones contributes to the striking increase in cardiovascular morbidity and mortality in postmenopausal women. We present here a description of age-related disruption of lipid homeostasis, which particularly affects 3-hydroxy 3-methylglutaryl Coenzyme A reductase, the key rate-limiting enzyme in the cholesterol biosynthetic pathway. We further discuss the age- and gender-related dysregulation of this enzyme, providing new evidence for the different mechanisms driving dyslipidemia in elderly men and women. In addition, we introduce pharmacological methods of regulating HMGR and maintaining cholesterol homeostasis.

Full text

Hindawi Publishing Corporation
Current Gerontology and Geriatrics Research
Volume 2010, Article ID 420139, 7pages
doi:10.1155/2010/420139
Review Article
Age-Related Hypercholesterolemia and HMG-CoA Reductase
Dysregulation: Sex Does Matter (A Gender Perspective)
Laura Trapani and Valentina Pallottini
Department of Biology, University of Roma Tre, Viale Marconi, 446, 00146 Rome, Italy
Correspondence should be addressed to Valentina Pallottini, vpallott@uniroma3.it
Received 29 October 2009; Revised 26 January 2010; Accepted 18 February 2010
Academic Editor: Jacek Witkowski
Copyright © 2010 L. Trapani and V. Pallottini. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Although cardiovascular diseases are less prevalent in premenopausal women than in men, their occurrence in women increases
at the onset of menopause, and the loss of female sex hormones contributes to the striking increase in cardiovascular morbidity
and mortality in postmenopausal women. We present here a description of age-related disruption of lipid homeostasis, which
particularly affects 3-hydroxy 3-methylglutaryl Coenzyme A reductase, the key rate-limiting enzyme in the cholesterol biosynthetic
pathway. We further discuss the age- and gender-related dysregulation of this enzyme, providing new evidence for the different
mechanisms driving dyslipidemia in elderly men and women. In addition, we introduce pharmacological methods of regulating
HMGR and maintaining cholesterol homeostasis.
1. Introduction
Aging has been defined as the series of the deteriorative
changes occurring during the adult period of life that
underlie increased vulnerability to challenges and decreased
survival [1]. This deterioration is responsible for both the
commonly recognized sequential changes that accompany
advancing age and the progressive increase in the chance of
disease and death and is usually manifested as a progressive
decrease in physiological functions.
Aging is characterized by the loss of homeostasis [2]
that leads to changes in the biochemical composition
of tissues [3–5], reduced ability to respond adaptively
to environmental stimuli [6], and increased susceptibility
and vulnerability to diseases [7] including coronary artery
diseases (CAD). The term CAD refers to pathologic changes
within the coronary artery walls that result in diminished
blood flow through these vessels. CAD can cause myocardial
ischemia and possibly lead to acute myocardial infarction
through three mechanisms—profound vascular spasm of the
coronary arteries, formation of atherosclerotic plaques, and
thromboembolism.
Although it is widely accepted that abnormal levels of
lipids and/or lipoproteins in blood are modifiable risk factors
for CAD [8,9], the importance of lipid levels as prognostic
factors in older adults is controversial. Several studies have
suggested that the association between cholesterol concen-
tration and atherosclerotic CAD weakens with age, and that
screening and treating older adults for dyslipidemia provides
little potential benefit [10,11]. In contrast, other reports
suggest that lipoprotein levels remain a significant risk factor
for CAD in the elderly and that treatment of dyslipidemia in
the elderly may have a greater impact on CAD mortality than
in younger people because the total attributable risk from
dyslipidemia is greater in the older age group [12,13].
The mechanisms behind this age-related dyslipidemia
are incompletely characterized. Some evidence demonstrates
that the causes of age-related disruption of lipid homeostasis
include the gradual decline in fractional clearance of LDL
with increasing age, the progressively reduced ability to
remove cholesterol through conversion to bile acids, and the
decreased activity of the rate-limiting enzyme in bile acid
biosynthesis, cholesterol 7α-hydroxylase (C7αOH). More-
over, an interesting hypothesis states that critical changes
in cholesterol and lipoprotein metabolism depend on the
progressive decrease in growth hormone (GH) secretion, a
characteristic feature of aging. GH plays an important role in
cholesterol homeostasis by either modulating the expression
of hepatic LDLr [14] or controlling the activity of cholesterol
7α-hydroxylase [15].

2 Current Gerontology and Geriatrics Research
Cholesterol
Heme A Isoprenylated
proteins Dolichol
2,3 -Oxiydosqualene
Squalene Geranylgeranyl-PP
Farnesyl-PP
Geranyl-PP
Isopentenyl-PP
Mevalonate
HMG CoA
Acetyl CoA
HMGR
Figure 1: Schematic illustration of the biosynthetic pathway of HMGR end-products.
CAD is less prevalent in premenopausal women than in
men, but its occurrence in women increases at the onset of
menopause, and the loss of female sex hormones contributes
to the striking increase in the incidence of cardiovascular
morbidity and mortality in postmenopausal women. Estro-
gen replacement therapy results in improved lipoprotein
profiles in postmenopausal women, but these improved
profiles account for less than half of the cardioprotective
effects of estrogen replacement therapy. The cardioprotective
effects of estrogens include beneficial changes in plasma lipid
levels (increased high-density lipoprotein (HDL), decreased
total cholesterol and low-density lipoprotein (LDL), and
decreased LDL oxidation) [16,17], antiplatelet and antiox-
idant effects, and preservation of endothelium-mediated
vasodilation [18,19]. Providing further evidence for the car-
dioprotective role of estrogen, studies performed in estrogen-
deficient animal models have demonstrated a disruption of
lipid homeostasis [20].
Cholesterol plays an essential role in the synthesis of
new membranes, the turnover of lipids in existing mem-
branes, and the biosynthesis of products such as steroid
hormones and bile acids [21]. Cholesterol homeostasis is
maintained by a feedback regulatory system that senses the
level of cholesterol in cell membranes and modulates both
the transcription of genes encoding proteins involved in
cholesterol biosynthesis and posttranscriptional events along
with the uptake of cholesterol from plasma lipoproteins
[22]. Maintenance of cholesterol homeostasis is regulated
by both the receptor-mediated endocytosis of LDL by LDL
receptors (LDLr) and de novo cholesterol synthesis via the
rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme
A reductase (HMGR) [23]. Because of the pivotal role of
HMGR in cholesterol and nonsterol isoprenoid compound
biosynthesis, most of the mechanisms controlling cholesterol
homeostasis are related to short- and long-term regulation of
HMGR.
To provide new evidence for the different mechanisms
driving dyslipidemia in elderly men and women, this review
will focus on age-related disruption of lipid homeostasis, and
in particular on the age- and gender-related dysregulation
of HMGR, the key rate-limiting enzyme in the cholesterol
biosynthetic pathway.
2. HMGR Regulation in Adults
Cholesterol biosynthesis occurs through a tightly regulated
pathway that employs multiple feedback mechanisms to
maintain homeostasis [24].Over the past several decades,
much work has focused on the regulation of HMGR, which
catalyzes the conversion of HMG-CoA to mevalonate (MVA)
through a four-electron oxidoreduction. This reaction is
the rate-limiting step in the synthesis of cholesterol and
other isoprenoids such as dolichol, isopentenyladenine,
which is present in some tRNAs, heme A, ubiquinone, and
prenylated proteins such as Ras and Rab proteins (Figure 1)
[24].
Encoded by the HMGR gene located on chromosome
5 of human genome, HMGR consists of a single 888
amino acid polypeptide chain. The N-terminal membrane
domain contains 339 hydrophobic residues that span the
endoplasmic reticulum (ER) membrane and contains the
sterol-sensing domain (SSD), which is responsible for the
binding of sterols and other MVA derivatives that accelerate
enzyme degradation [25], while the catalytic site is located in
the hydro-soluble C-terminal cytoplasmic domain. A linker
region (residues 340–459) connects these two portions of the
protein [2].

Current Gerontology and Geriatrics Research 3
Short-term regulation of HMGR is achieved through its
phosphorylation and dephosphorylation, both of which can
affect enzyme activity. Phosphorylation of residue S872 of
HMGR decreases its catalytic activity, and removal of this
phosphate results in reactivation [26,27]. AMP-activated
kinase (AMPK) appears to be the major HMGR kinase
in the liver, where cholesterologenesis takes place. AMPK
is a heterotrimeric serine/threonine kinase consisting of a
catalytic αsubunit and regulatory βand γsubunits [28].
AMPK is activated by phosphorylation of the αsubunit at a
specific threonine residue (Thr172) [29]. HMGR activation
is mediated by its dephosphorylation by protein phosphatase
2A (PP2A), which regulates a significant network of cellular
events [30].
In addition to this short-term regulation, HMGR is sub-
ject to transcriptional, translational, and posttranslational
control [31].Theselevelsofcontrol,whicharemediatedby
changes in intracellular sterol levels and cholesterol uptake by
LDLr, can result in changes of over 200-fold in HMGR levels
[32]. Both LDLr and HMGR are produced in response to
activation of the Sterol Regulatory Element Binding Proteins
(SREBPs), and particularly SREBP-2, in the liver [33,34].
Long-term regulation of HMGR is mediated by a pair
of membrane-bound proteins, SREBP cleavage activating
protein (Scap) and Insulin-induced gene (Insig), which
directly bind sterols and thereby sense sterol concentration in
the membranes of the ER. As a result of these binding events,
both Scap and Insig undergo conformational changes that
initiate a series of molecular events blocking Scap’s ability
to transport SREBPs to the Golgi, terminating cholesterol
synthesis and uptake [32]. Furthermore, the intracellular
accumulation of sterols induces HMGR to bind Insig,
promoting ubiquitination and proteasomal degradation of
HMGR [35].
Several hormones, including insulin, glucagon, gluco-
corticoids, thyroid hormone, and estrogen, regulate the
expression of hepatic HMGR in animals. Insulin likely
stimulates HMGR expression by increasing its rate of
transcription, while glucagon opposes this effect. Hepatic
HMGR activity undergoes significant diurnal variations due
to changes in the levels of immunoreactive proteins, which
are primarily mediated by changes in insulin and glucagon
levels. Thyroid hormone increases hepatic HMGR levels by
acting to increase both transcription and mRNA stability,
while glucocorticoids decrease hepatic HMGR expression by
destabilizing HMGR mRNA [36]. The effects of estrogen
on HMGR expression are still debated. Some studies sug-
gest that estrogens act to increase hepatic HMGR activity
primarily by stabilizing HMGR mRNA and that deficiencies
in those hormones that act to increase hepatic HMGR gene
expression lead to elevated serum cholesterol levels [36]. On
the other hand, studies using the DLD1 cell line suggest that
estrogens induce an early increase in LDLr at both the mRNA
and the protein level and later cause decreases in HMGR
activity and protein expression [37].
Although the mechanisms that regulate cholesterol
homeostasis are well known, [22] the literature describ-
ing putative physiological sex differences in cholesterol
homeostasis-related proteins is limited [38–42]. Further-
more, most of these papers are fragmented and very old,
and none of them focus on the mechanisms underlying
these sex-related differences. De Marinis and coworkers [43]
provided evidence of sex-related physiological differences in
proteins involved in cholesterol homeostasis. In particular,
activity and expression levels of HMGR are lower in 3-
month-old female rats and in 17-β-estradiol-treated 3-
month-old male rats than in 3-month-old untreated male
rats. Moreover, 3-month-old female rats express lower levels
of SREBP-2 and higher levels of Insig than their male
counterparts. Sex-related variations in expression of these
regulatory proteins are functionally consistent with the well-
accepted classical model of HMGR behavior [22,32], and
no sex-related differences have been observed in either LDLr
expression or cholesterol levels, excluding the involvement of
end-product feedback in presence of physiological content
of estradiol. The difference in the expression pattern of
regulatory proteins in males and females seems to be related
to the presence of estrogen, and altered expression of these
regulatory proteins drives the sex-related differences in
HMGR expression.
2.1. Sex-Related Differences in HMGR Dysregulation during
Aging. Due to the serious health-related consequences of
aging, significant efforts have been made to provide a
more complete understanding of this particular stage of
life. Current research aiming to delineate the biological
mechanisms of aging has yielded valuable information about
the molecular basis of age-related physiological deteriora-
tion. One of the critical problems associated with aging
is the increased incidence of CAD and, more generally,
cardiovascular diseases (CVD). Many risk factors predispose
elderly people to develop pathologies related to failure of
the heart vasculature, including hypercholesterolemia. Thus,
understanding the mechanisms driving increased cellular
and plasma cholesterol content during aging is essential in
defining specific intervention points.
During aging, hepatic lipid modifications occur. In par-
ticular, studies of 24-month-old male rats showed increased
plasma cholesterol levels and increased hepatic cholesterol
synthesis accompanied by full activation of HMGR [44,
45], which was dependent on the well-known age-related
increaseinreactiveoxygenspecies(ROS)[46,47]. The age-
related increase in activation of HMGR has been associated
with an increase in ROS [48,49]. The current model
proposes that increased ROS levels result in activation
of both p38 and AMPKα. In turn, p38 activation may
result in an increase in association of PP2A with HMGR,
leading to dephosphorylation and increased activation of
HMGR. AMPKαkinase activity is impaired by the enhanced
association of PP2A with HMGR [50]. Moreover, findings in
H2O2-stimulated HepG2 cells demonstrate that the effect of
ROS on HMGR dephosphorylation is mediated by activation
of the p38/MAPK pathway [45].
In addition to the short-term regulation mediated by
phosphorylation and dephosphorylation, long-term regu-
lation of HMGR also appears to be affected by aging.
Age-related variations in hormone levels and hormone

4 Current Gerontology and Geriatrics Research
sensitivity induce a decreased ability to maintain homeostatic
potential, and these hormonal changes are always associated
with changes in the expression or functionality of some
molecules. In particular, it has been clearly demonstrated
that the age-related decrease in insulin sensitivity induces
changes in some factors involved in cholesterol metabolism,
such as Insig-1 protein. This age-related reduction in Insig
expression results in slower degradation of HMGR [51,52].
While many studies have established that susceptibility
to CAD increases with age, little is known about the
mechanisms underlying the increased incidence of CAD in
postmenopausal women as compared to men of the same
age.
Previous studies have shown that 12-month-old
estropausal rats, in which estrogen levels are decreased, have
higher levels of plasma cholesterol, increased activation
of HMGR, and decreased LDLr membrane exposure than
3-month-old female rats. These changes result in decreased
cholesterol uptake and increased cholesterol synthesis,
supporting the correlation between hypercholesterolemia,
aging, and estropause. Increased activation of HMGR does
not depend on an increase in ROS as seen in aged-matched
male rats [53]. Instead, HMGR activation seems to be
due to decreased activation of AMPK during the period
of 17-βestradiol deficiency that occurs at the beginning
of estropause; this decrease in AMPK activation results in
decreased phosphorylation of HMGR.
Treatment of older female rats with 17-βestradiol results
in restoration of normal cholesterol levels, decreased activa-
tion of HMGR, and increased LDLr exposure on the plasma
membrane. Furthermore, while 17-βestradiol treatment
does not fully restore AMPK activation, AMPK is sufficiently
activated in older 17-βestradiol-treated female rats to
phosphorylate HMGR, reestablishing HMGR activity [54].
This estradiol-induced enhancement in AMPK activation
is supported by studies by Schulz and coworkers, who
demonstrated that estradiol-mediated AMPK activation was
independent of estrogen receptor ligand engagement and
involved catechol metabolism of estradiol [55].
The decrease in estradiol levels that occurs at the onset of
estropause does not affect long-term regulation of HMGR,
but mediates short-term HMGR regulation by controlling
activation of AMPK. Thus, a relationship exists between
changes in estrogen levels and HMGR-related modulation
of cholesterolemia in older female rats. The protective
role played by estrogens in modulating the lipid profile
is mediated not only through increases in plasma HDL,
decreases in plasma LDL, and decreased oxidation, but
also through regulation of AMPK activation, which inhibits
HMGR and cholesterol synthesis.
3. Conclusion and Future Perspectives
In elderly men and women, HMGR is highly activated;
however, the mechanisms driving dysregulation of HMGR
appear to be gender-dependent. Studies of aged male rats
suggest that in males, HMGR dysregulation is due to
increased association between PP2A and HMGR, which
results in increased activation of HMGR. On the other hand,
studies of estropausal female rats, in which estrogen levels
are decreased, suggest that the menopause-related increase
in HMGR activity is caused by the decreased activation of
AMPK observed during estrogen deficiency.
The regulation of HMGR activity has been an attrac-
tive target for pharmacological treatment of hypercholes-
terolemia, the main risk factor for CAD. Consequently,
better understanding of the molecular mechanisms that drive
dysregulation of HMGR activity and hypercholesterolemia in
aged men and women could provide gender-specific targets
for treatments to lower plasma cholesterol content, resulting
in both prevention and reduction of one of the main risk
factors for cardiovascular diseases.
Decreased cellular cholesterol synthesis leads to a home-
ostatic response involving up-regulation of cell-surface
receptors that bind atherogenic lipoproteins such as LDL
and VLDL. These lipoproteins are taken up by the cell
anddegraded[56], resulting in a reduction in circulating
atherogenic lipoproteins. This process helps to explain the
clinical usefulness of HMGR inhibitors (statins).
HMGR inhibition results in not only reductions in
cellular and plasma cholesterol levels, but also reductions in
other products synthesized through the cholesterol biosyn-
thetic pathway, such as ubiquinone, prenylated proteins, and
dolichol. The restoration of HMGR to its normal activation
state regulates the physiological synthesis of cholesterol
within cells, and restored cellular cholesterol levels are in turn
reflected in proper membrane LDLr presence [22].
Statins are effective means of primary and secondary
prevention of ischemic heart disease (IHD) in middle-aged
men; however, proof of the efficacy of statins in preventing
development and progression of IHD in women and elderly
people is less convincing. In the PROSPER (Prospective
Study of Pravastatin in the Elderly at Risk of vascular disease)
trial, pravastatin not only had no effectinmenandwomen
aged 70–82 years, but also significantly increased the rate
of breast cancer in these patients. In the ALLHAT-LLT
(Antihypertensive and Lipid-Lowering Treatment to Prevent
Heart Attack Trial) trial, pravastatin lowered neither the total
number of nonfatal myocardial infarctions and IHD deaths
nor total mortality in patients aged 65 years and older and in
women [57].
Considering the undesirable side effects of statins and
the effects of these drugs on other important compounds
in addition to cholesterol, development of new pharma-
cologically active compounds capable of regulating plasma
cholesterol content is critical to effectively control this
important CVD risk factor.
Many recent studies have described new compounds able
to decrease plasma cholesterol content. Although the exact
mechanisms by which these compounds act are unknown,
some of these compounds affect expression levels and activity
of HMGR. These novel compounds could thus represent
the future of hypercholesterolemia therapy and should be
studied further.
Hypercholesterolemia can also be approached using
other therapeutic targets; for example, proprotein convertase
subtilisin/kexin type 9 (PCSK9) has been implicated as

Current Gerontology and Geriatrics Research 5
an important regulator of LDL metabolism. PCSK9 belongs
to the subtilisin family of serine proteases and is highly
expressed in the liver [58]. Secreted PCSK9 modulates LDL
levels through posttranslational down-regulation of hepatic
LDLr protein [34]. Down-regulation of PCSK9 could thus
be effective in decreasing plasma cholesterol content by
increasing LDLr levels without affecting activity of HMGR
and its end-products.
In addition, squalene synthase, an enzyme that is
downstream of HMGR in the cholesterol synthesis pathway,
modulates the first committed step of hepatic cholesterol
biosynthesis at the final branching point of the cholesterol
biosynthetic pathway. Pharmacologic inhibitors of squalene
epoxidase and oxidosqualene cyclase, two enzymes that act
downstream of squalene synthase, may thus be useful in
reducing plasma LDL content [59].
Moreover, the identification of Scap and Insig as sterol-
binding proteins in mammalian cells has added a new level
of molecular detail to the understanding of regulation of
the SREBP pathway and subsequent regulation of HMGR
levels, providing new potential targets for pharmacological
intervention.
Much work remains to define the relationship between
hormonal changes and their effects on transcription factors
and cholesterol metabolism in different physiological and
pathological conditions. For example, some studies suggest
that estrogens are able to regulate cholesterol homeosta-
sis without directly affecting HMGR [43]. Additionally,
more detailed studies are required to define the specific
roles of Insig proteins and to determine the metabolic
consequences of their reciprocal regulation. In fact, since
they are required for feedback regulation of SREBP pro-
cessing and HMGR degradation, Insigs may represent a
new target for pharmacological intervention to maintain
blood cholesterol levels within the optimal range. None of
the papers we have cited focus on the sex-related differ-
ences.
Thus, although HMGR plays a pivotal role in regulating
cholesterol metabolism, future studies should address sex-
related differences in the cholesterol biosynthetic pathway
to identify new targets for customized pharmacological
treatment of hypercholesterolemia.
Acknowledgments
The authors wish to thank past and present members of their
laboratories who contributed with data and discussions to
the ideas presented here. In particular, the invaluable and
dedicated work of our mentor Professor Anna Trentalance
is warmly acknowledged.
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