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© 1999 American Heart Association, Inc.
Original Contributions |
Cholesterol and Lipoprotein Metabolism in Aging
Reversal of Hypercholesterolemia by Growth Hormone Treatment in Old Rats
From the Metabolism Unit, Center for Metabolism and Endocrinology, Department of Medicine, and the Molecular Nutrition Unit, Center for Nutrition and Toxicology, Novum, Karolinska Institute at Huddinge University Hospital, Huddinge, Sweden.
Correspondence to Mats Rudling, MD, PhD, CME, M63, Huddinge University Hospital, S-141 86 Huddinge, Sweden. E-mail mats.rudling{at}cnt.ki.se
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Abstract |
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Abstract—Plasma cholesterol levels increase with age, as does the incidence of coronary heart disease. The mechanisms responsible for the age-related hypercholesterolemia are not well understood. An interesting hypothesis suggests that the relative deficiency in growth hormone (GH), which occurs with aging, contributes to the development of the age-related hypercholesterolemia, because GH has beneficial effects on cholesterol metabolism. In the present work, we tested this hypothesis by the administration of GH to normal rats of varying ages. Plasma lipids and hepatic cholesterol metabolism were characterized in 2-, 12-, and 18-month-old male Sprague-Dawley rats. In 2-month-old rats, GH specifically stimulated the hepatic low density lipoprotein (LDL) receptor expression in a dose-dependent way, both at the protein level and at the mRNA level. Concomitantly, plasma cholesterol increased by
30% within the large high
density lipoprotein and LDL fractions. In 12-month-old animals,
cholesterol 7
-hydroxylase (C7OH) activity was
reduced, whereas hepatic LDL receptors and plasma total
cholesterol were unchanged. GH treatment (1 mg ·
kg-1 · d-1) normalized the activity of
C7OH and had effects on plasma cholesterol and LDL
receptors similar to those seen in 2-month-old animals. In 18-month-old
rats, plasma cholesterol was increased 2-fold, whereas
hepatic LDL receptor expression and C7OH activity were similar to
those of the 12-month-old animals. Infusion of GH to 18-month-old rats
had similar effects on hepatic C7OH and LDL receptors as seen in
12-month-old rats. However, GH treatment strongly reduced the
hypercholesterolemia in 18-month-old animals.
We conclude that the age-dependent increase of plasma
cholesterol in rats can be reversed by the administration
of GH, presumably through the pleiotropic effects of this hormone on
lipoprotein metabolism.
Key Words: aging • cholesterol • cholesterol 7-hydroxylase • growth hormone • LDL receptor
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Elevated levels of plasma cholesterol, particularly LDL cholesterol, are known to be associated with an enhanced risk for atherosclerosis and coronary heart disease.1 2 Total and LDL cholesterol levels increase with age,3 4 as does the incidence of cardiovascular disease.5 The mechanisms behind this age-related increase in plasma cholesterol are still incompletely characterized. Of particular interest is the finding of a gradual decline in the fractional clearance of LDL from the circulation with age6 7 8 9 and evidence of the reduced expression of hepatic LDL receptors (LDLRs) with increasing age in some species.10 11 The capacity for body cholesterol removal through the conversion of cholesterol to bile acids is also progressively reduced with age,12 and a decrease in the activity of the rate-limiting enzyme in bile acid biosynthesis, cholesterol 7
-hydroxylase (C7OH), has been demonstrated in the aging
rat.13 14 In addition, there is some evidence that the
synthesis of apolipoprotein (apo) B-100 in VLDL may be increased with
age.9
A number of explanations to these findings have been discussed,
including both dietary and hormonal factors. An interesting
hypothesis15 states that the critical changes in
cholesterol and lipoprotein metabolism depend
on the progressive decrease in growth hormone (GH) secretion, which
occurs with normal aging.16 17 In previous studies, mainly
based on experiments in hypophysectomized rats, we established that GH
has an important role in cholesterol
homeostasis,15 both by modulating the expression of
hepatic LDLRs18 19 20 21 and by controlling the activity of
C7OH.22 In addition, GH stimulates hepatic
VLDL23 and apoE24 secretion and may influence
the ratio between apoB-100 and apoB-48 through stimulation of apoB mRNA
editing in the rat.20 25
To further characterize the role of GH in the physiological regulation of cholesterol and lipoprotein metabolism and particularly to evaluate the possible relevance of reduced GH secretion for the development of hypercholesterolemia with age, we performed studies in normal rats of varying ages, both with and without infusion of GH. We were able to show that when plasma cholesterol was significantly increased in old rats, the hyperlipidemic pattern could be reversed by infusion with GH. These effects may be partially mediated via stimulation of LDLR activity and bile acid synthesis but probably also reflect the pleiotropic effects of GH on lipoprotein metabolism.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Materials
Osmotic minipumps (model 2001, delivering 1 µL solvent per hour) were from Alza Corp. Recombinant human GH (Genotropin) and recombinant human insulin-like growth factor I (IGF-I) were kindly provided by AB Pharmacia & Upjohn, Stockholm, Sweden. All other materials were obtained from previously described sources.19 20 26 27 28 29
Animals and Experimental Procedures
Altogether, 75 male Sprague-Dawley rats (ALAB, Sollentuna,
Sweden) were used in 4 separate experiments. The animals were adapted
to the environment at least 1 week before the experiments. Young rats
were purchased at 7 weeks of age, whereas old animals were obtained at
6 months of age and kept at the animal facilities until 12 or 18 months
of age. Rats were housed under standardized conditions, with free
access to water and chow; the light cycle hours were between 6
AM and 6 PM The studies were approved by the
Institutional Animal Care and Use Committee. Osmotic minipumps were
implanted subcutaneously in the dorsal region. A small (5- to 10-mm)
incision was made, and a subcutaneous tunnel was created with a pair of
forceps. Minipumps were put in place and the skin was sutured. Controls
were sham-operated with the same surgical procedure. The presence of
minipumps only did not alter plasma lipoproteins; this was verified in
a separate control experiment (not shown). When the experiments were
terminated, the rats were anesthetized with ether between 10
and 11 AM, blood was drawn by cardiac puncture, and the
animals were killed by cervical dislocation. The livers were
immediately removed, and 1 piece of fresh liver (1 g) was taken for
preparation of microsomes for subsequent assay of enzyme activities as
described below. The remaining liver was immediately frozen in
LN2.
Plasma IGF-I was determined in serum by radioimmunoassay after separation of IGFs from IGF-binding proteins by acid-ethanol extraction and cryoprecipitation. To minimize interference from the remaining IGF-binding proteins, des1-3–IGF-I was used as the radioligand.30
Size Fractionation of Lipoproteins by Fast Protein Liquid
Chromatography (FPLC)
Equal volumes of plasma from every rat were pooled (5 mL) for
each group, and the density was adjusted to 1.21 g/mL with KBr. After
ultracentrifugation at
100x103g for 48 hours, the supernatant was
removed and adjusted to 2 mL with 0.15 mol/L NaCl, 0.01% EDTA, and
0.02% NaN3, pH 7.3. One milliliter of this
solution (corresponding to 2.5 mL of plasma) was injected onto a
540x18-mm Superose 6B column after filtration through a Millipore
0.45-µm HA filter; 2-mL fractions were collected at a flow rate of 1
mL/min.29 In the experiment illustrated in Figure 1
, the column was 470 mm
long.
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, Control group;
, 1 mg · kg-1 · d-1; 
, 2
mg · kg-1 · d-1; and 
, 3.5
mg · kg-1 · d-1 of GH. C, LDLR
expression assayed by ligand blotting, using rabbit
125I–ß-VLDL. Hepatic membranes were prepared from pooled
liver samples from each group and separated by SDS-PAGE (6%
polyacrylamide). D, Hepatic LDLR mRNA levels assayed by
solution hybridization in total RNA extracts. Data represent
mean±SEM. *P<0.05, **P<0.005 vs
untreated animals.
SDS–Polyacrylamide Gel Electrophoresis (PAGE) Separation
of Apolipoproteins
Pooled plasma from each group was separated in 2-mL fractions by
FPLC. Equal volumes from fractions 22 to 24 (VLDL), 25 to 27 (IDL), 30
to 32 (LDL), and 35 to 37 (HDL) were pooled, and 1.1 mL was
precipitated with trichloroacetic acid (15% final concentration),
delipidated by washing twice with acetone, and solubilized in 200 µL
of loading buffer as described elsewhere.29 After boiling
the samples for 5 minutes in the presence of 5% (vol/vol)
2-mercaptoethanol, 100 µL was loaded onto 4% to 20% gradient
SDS-polyacrylamide gels and separated for 4 hours at 45 mA.
Gels were stained with Coomassie blue. For reference, wide-range
molecular-mass standards (Bio-Rad Laboratories), as well as human LDL
and HDL, were used.
Lipid Extracts
Liver homogenates and hepatic microsomes were
prepared as previously described.27 28 The lipid fraction
was obtained by extraction with chloroform/methanol (2:1, vol/vol),
dried under a stream of N2, and dissolved in 200
µL methanol.
Cholesterol Assay
Cholesterol in plasma, FPLC fractions, liver
extracts, and hepatic microsome extracts was assayed with
Boehringer Mannheim free or total cholesterol assay
kits, with the use of a 5.2 mmol/L cholesterol
standard (Merck, catalog No. 141 64).
Ligand Blot Assay of LDLR
Hepatic membranes were prepared as described
previously.18 Membranes from pooled liver samples were
separated by 6% SDS-PAGE under nonreduced conditions. Size markers
were reduced with 2-mercaptoethanol and boiled. Filters were incubated
with 125I-labeled rabbit ß-migrating VLDL
(ß-VLDL), as described.18 Filters were exposed on Dupont
Cronex film. The LDLR expression was quantified from the 120-kDa bands
by using a Fujix Bio-imaging analyzer (BAS 2000, Fuji Photo
Film Co). Background was obtained by counting irrelevant filter parts
of the same area. The data are presented as photostimulated
luminescence per microgram of protein loaded.
Activities of C7OH and HMG-CoA Reductase
Microsomes were prepared by differential
ultracentrifugation of individual liver
homogenates in the absence of fluoride as described
previously.12 26 27 The activity of C7OH was determined
as the formation of 7-hydroxycholesterol (pmol · mg
protein-1 · min-1) from
endogenous microsomal cholesterol by using
isotope dilution mass spectrometry.27 Microsomal
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was
assayed by determining the conversion of HMG-CoA to mevalonate and
expressed as picomoles formed per milligram protein per
minute.26 The enzyme assays were carried out in
duplicate.
Total Nucleic Acid Preparation
Total nucleic acid (TNA) extracts were prepared as
described.31 The concentration of TNA in the samples was
measured by absorbance at 260 nm and assuming that 1 optical
density unit was equivalent to 40 µg TNA per mL.
Quantification of mRNA
The mRNA levels for the LDLR and C7OH were quantified by a
solution hybridization titration assay with mouse
-35S–UTP-cRNA-probes corresponding to
nucleotides 1247 to 1308 in the human LDLR cDNA and to
nucleotides 646 to 813 in the rat C7OH genomic
DNA.32 Editing of apoB mRNA was determined by primer
extension analysis with the use of total
RNA.20 33
Statistics
Data are presented as mean±SEM. The significance of
differences between groups was tested by 1-way ANOVA followed by a
planned comparison or by post hoc comparisons of group means according
to least significant difference or honestly significantly difference
methods (Statistica software, Stat Soft). To stabilize the variances,
data were logarithmically transformed when the assumption for no
correlation between means and variances was
violated.34
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Previous information on the role of GH in lipoprotein metabolism has mainly been gained from studies in hypophysectomized rats,18 19 20 23 24 25 and thus, we first wanted to assess the effects of GH administration in normal young rats. Eight-week-old male rats were given a subcutaneous infusion of recombinant human GH at increasing doses 1, 2, and 3.5 mg GH · kg-1 · d-1. After 6 days of infusion, the animals were killed and plasma and livers were collected. GH infusions increased plasma total cholesterol slightly, although this rise was not statistically significant (Figure 1A
), and FPLC
separation of plasma lipoproteins showed a slight increase,
predominantly within the large HDL particles (Figure 1B).
Determination of the hepatic LDLR expression showed a dose-dependent
stimulatory effect after the infusion of GH (Figure 1C), and
this effect was evident also at the mRNA level (Figure 1D).
Many of the actions of GH are mediated by the GH-dependent
production of IGF-I.16 To determine whether the
effects on lipoprotein metabolism observed in normal young
rats given GH could be reproduced by the administration of IGF-I to
normal rats, we repeated the experiment by using recombinant human
IGF-I. After 6 days of infusion of 2 and 4 mg ·
kg-1 · d-1 of
IGF-I, respectively, hepatic LDLR expression was not altered
(Figure 2A). Plasma total
cholesterol was unchanged (Figure 2B), but
separation of plasma lipoproteins by FPLC indicated a slight increase
within LDL particles (Figure 2C). Thus, in consonance with our
previous findings in hypophysectomized rats,20 IGF-I could
not reproduce the effects of GH on lipoprotein metabolism
in normal rats.
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, Control group;
To explore the possible relationship between GH secretion and hepatic
lipoprotein metabolism, we then investigated young (8
weeks) and 12-month-old rats that were given a GH infusion of 1 mg
· kg-1 · d-1 for
6 days in comparison with untreated animals. The plasma levels of IGF-I
were 38% lower in the old untreated controls (Table 1), indicative of a reduced
secretion of GH in these rats.17 The infusion of GH did
not alter this difference between young and old animals.
Analysis of plasma total cholesterol revealed a
modest increase of cholesterol in old untreated rats (Table 1), which was due to an increase within LDL and large HDL
particles (Figure 3A). GH infusion
significantly increased plasma total cholesterol in both
young and old animals (Table 1). This treatment did not reverse
the increases in LDL and HDL cholesterol in the old rats,
and both groups of rats responded with an elevation of
cholesterol within large HDL particles (Figure 3A).
Determination of the triglyceride content in FPLC fractions
showed no difference between untreated young and old rats (Figure 3B), whereas the infusion of GH increased
triglycerides slightly (30%) in the VLDL of old
animals.
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Quantification of the hepatic LDLR expression showed a similar level of
expression in 8-week- and 12-month-old rats, and the infusion of GH
increased hepatic LDLR expression slightly (by 25% to 50%) in both
groups (Figure 3C). The hepatic LDLR mRNA levels were similar in
young and old rats (Table 1), and there was no alteration by GH
treatment. Analysis of the C7OH enzymatic activity in
hepatic microsomes revealed a 60% lower level (P<0.001) in
the 12-month-old rats compared with the young rats (Figure 3D).
In the old animals, GH infusion increased the activity of C7OH (to
130%, P<0.001) to a level similar to that observed in
untreated young controls. A response to GH treatment was also seen in
the young animals, the C7OH activity being increased by 40%
(P<0.05, Figure 3D). Despite the differences found
in C7OH activity, no differences were found between young and old
rats when the hepatic C7OH mRNA abundance was determined by solution
hybridization (Table 1). GH infusion doubled the C7OH mRNA
abundance in the young rats (P<0.05), whereas only a slight
increase was observed in the old animals. There were no significant
differences in hepatic total cholesterol between the
groups, whereas GH treatment significantly increased hepatic microsomal
free cholesterol in the young rats (P<0.05,
Table 1).
Thus, there was a clear reduction of C7OH activity in the
12-month-old rats, and this situation could be normalized by GH
treatment. However, the modest increase of plasma total
cholesterol in these animals was not statistically
significant compared with the corresponding values in young controls.
In the rat, a doubling of plasma cholesterol has been
reported between 12 and 18 months of age.35 To further
explore whether treatment with GH could improve age-induced changes in
lipid metabolism, we therefore repeated the experiment in
18-month-old animals. GH was infused at a rate of 1 mg ·
kg-1 · d-1 for 6 days,
and 8-week-old rats served as the reference. Determination of plasma
IGF-I showed a 34% reduction in the untreated 18-month-old rats (Table 2). Again, GH treatment did not
alter the difference in plasma IGF-I levels between young and old
rats.
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Analysis of plasma total cholesterol in this
experiment revealed a 2-fold increase in the 18-month-old rats compared
with the young ones (Table 2). In agreement with our previous
results, GH treatment tended to increase plasma total
cholesterol in young rats. In contrast, GH infusion
dramatically reduced plasma total cholesterol in the
18-month-old animals (P<0.001), to a level that was similar
to that of young GH-treated rats (Table 2). Lipoprotein profile
analysis by FPLC showed that the age-induced plasma
cholesterol in 18-month-old rats was within all lipoprotein
classes (Figure 4A). The infusion
of GH to these animals clearly reduced cholesterol in all
fractions, so that the lipoprotein pattern of GH-treated, 18-month-old
rats resembled that of GH-treated young rats. In young GH-infused rats,
plasma cholesterol again increased mainly within large HDL
and LDL. Analysis of triglycerides in FPLC
fractions showed higher concentrations in VLDL particles of untreated
18-month-old rats (Figure 4B). GH infusion reduced VLDL
triglycerides in old animals, whereas VLDL
triglycerides increased in 8-week-old rats. SDS-PAGE
separation of the apolipoproteins in FPLC fractions revealed changes in
consonance with the alterations of the lipid profiles (Figure 4C). Particularly, there was an increase in LDL apoB-100 in
18-month-old rats; this abnormality was reversed by the infusion of GH.
Thus, it was evident that GH had contrasting effects on the lipoprotein
pattern in young and 18-month-old animals.
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Despite the dramatic differences in plasma cholesterol and
lipoprotein profiles, the hepatic LDLR expression in the 18-month-old
rats was also similar to that of young animals (Figure 5A). GH infusion did not alter
hepatic LDLR expression in either old or young animals. It was thus
clear that there was no major change in hepatic LDLR expression with
aging.
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P<0.05 vs 18-month-old controls. W indicates weeks;
M, months.
Microsomal activity of C7OH in the 18-month-old animals was 35%
lower (P<0.05) than that in the young ones (Figure 5B). In young rats, GH treatment stimulated C7OH activity,
inducing a 70% increase. Also in 18-month-rats, a stimulation of
C7OH activity was seen, though of lesser magnitude (Figure 5B). Similar results were obtained when the mRNA abundance for
C7OH was measured in hepatic TNA extracts (Table 2). As in
the previous experiment, no differences were detected in hepatic
microsomal free cholesterol content between young and old
rats. GH infusion again increased hepatic microsomal free
cholesterol in young rats. Hepatic total
cholesterol was similar in young and old animals, and GH
infusion significantly reduced its concentration in young animals
only.
The drastic effect of GH treatment on plasma lipoproteins in the
18-month-old animals occurred in contrast to more moderate changes in
LDLR and C7OH activities. This prompted us to search for other
changes induced by GH. Assay of the activity of liver microsomal
HMG-CoA reductase, the rate-limiting enzymatic step in
cholesterol biosynthesis, did not show any difference
between young and 18-month-old animals, however (Figure 5C).
This finding suggests that the
hypercholesterolemia is not explained by an
elevated hepatic synthesis of cholesterol. GH treatment
stimulated HMG-CoA reductase activity in both groups of animals.
Finally, we determined the apoB mRNA editing by primer extension
analysis of total hepatic RNA. No differences were found in the
degree of mRNA editing between the 18-month-old rats compared with
young animals.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The results of the present study strongly support the concept that GH has an important role in the physiological regulation of cholesterol and lipoprotein metabolism. The reversal of the disturbed lipoprotein pattern in 18-month-old male rats by a moderate dose of GH implies that, although other mechanisms may be involved, a relative GH deficiency contributes to the development of age-related hypercholesterolemia in this species. However, the present data do not permit any definite identification of a single mechanism by which GH exerts its effect in this situation. Instead, the data rather indicate that the pleiotropic effects of GH on lipoprotein metabolism result in compensatory adjustments of age-related changes at a number of regulatory steps.
The present data confirm and extend previous studies of cholesterol metabolism in the aging rat.13 14 35 36 37 38 39 Thus, a significant hypercholesterolemia was present in 18-month-old rats, whereas only a minor increase was detected in 12-month-old animals. The age-induced increase in plasma cholesterol was within all lipoprotein classes and was associated with an increased apoB-100 protein in LDL. Triglycerides were also elevated, particularly in VLDL. The hepatic content of total and microsomal cholesterol was unaltered even in the 18-month-old rats. Ståhlberg et al13 reported unchanged levels of free cholesterol in hepatic microsomes of 1-, 6-, and 24-month-old rats and observed an increased total cholesterol only in livers from 24-month-old animals, indicating that hepatic cholesterol may accumulate in rats of very old age. Our findings of an essentially unaltered hepatic LDLR expression are in agreement with previous reports by Stange and Dietschy39 and Nanjee et al.37 The activity of HMG-CoA reductase was unchanged in old rats despite their hypercholesterolemia, again in agreement with Ståhlberg et al,13 who found no change in HMG-CoA reductase activity when comparing 6- and 24-month-old animals. From the literature, it appears that HMG-CoA reductase activity is higher in rats up to 4 to 5 weeks of age,13 14 which may indicate that a decrease occurs during sexual maturation and not later in life.
Of particular interest is the finding of a clear reduction of C7OH
activity already in 12-month-old rats. This observation supports the
data of Ståhlberg et al,13 who found a progressive
reduction of C7OH activity when comparing animals at the ages of 1
month, 6 months, and 24 months. Thus, a reduced C7OH activity,
and—since HMG-CoA reductase activity is maintained—a diminished
relative conversion of cholesterol to bile acids occurs
relatively early in the aging process. It is important to note that no
change in microsomal free cholesterol, the substrate pool
for the C7OH enzyme, was observed, and that the changes in enzyme
activity were not paralleled by any change in the C7OH mRNA
levels. The latter observation would indicate that nontranscriptional
mechanisms of regulation are involved in the age-induced reduction of
enzyme activity. In most situations, hepatic C7OH activity is
strictly regulated at the transcriptional level in the
rat.40 41 42 However, evidence of posttranscriptional
regulation has actually been observed in hypophysectomized
(GH-deficient) rats, wherein enzyme activity is considerably reduced
despite increased mRNA levels.22
Despite a pronounced reduction of C7OH activity, there was only a
minor increase in plasma cholesterol in 12-month-old rats.
C7OH has previously been regarded as an important determinant for
the level of cholesterol in plasma because of its key role
in the excretion of cholesterol from the body. Stimulation
of bile acid production by interruption of the enterohepatic
circulation of bile acids is well known to lower plasma total and LDL
cholesterol, presumably through induction of the hepatic
LDLR.43 44 45 46 Although it has been shown that plasma
cholesterol may drop after the transient overexpression of
hepatic C7OH,47 the elimination of this enzyme by gene
knockout does not result in altered plasma lipids.48
Previous studies on hypophysectomized rats have also indicated that the
link between plasma cholesterol and hepatic C7OH may at
times be absent.22 Although it is still reasonable to
assume that a decreased C7OH activity may amplify the effects of
other age-related changes in lipoprotein metabolism, our
data would indicate that the
hypercholesterolemia appearing in 18-month-old
rats is not solely dependent on a reduced activity of C7OH. In
preliminary experiments, we did not find any change in the editing of
apoB mRNA in old rats, an observation that would speak against the
possibility that a relative predominance of apoB-100 in VLDL secreted
from the liver explains a slower lipoprotein turnover in the older
animals.49 In conclusion, the clear age-induced
hypercholesterolemia in 18-month-old rats
cannot simply be explained by a single alteration of hepatic
cholesterol synthesis, bile acid production, LDLR
expression, or apoB mRNA editing. However, combined discrete effects on
all of these processes may very well be of great importance. Such
effects are indeed difficult to demonstrate reliably with available
assays.
The administration of GH to young rats was associated with a dose-dependent increase of hepatic LDLR expression, both at the protein and the mRNA level. Plasma cholesterol increased slightly, predominantly within large HDL and LDL particles. The mechanisms responsible for the latter change were not explored further. Thus, GH also exerts clear effects on lipoprotein metabolism in normal rats when the doses are adequately high. Furthermore, these effects are specific for GH, in that they could not be reproduced by the infusion of IGF-I. In consonance with our present results, we have not been able to obtain similar effects on LDLR or apoB mRNA editing in hypophysectomized rats when GH was substituted with IGF-I.20 Also, in normal middle-aged humans, GH and IGF-I exert different effects on plasma LDL cholesterol levels.50
To evaluate the possible contribution of a relative GH deficiency to
the age-related hypercholesterolemia in rats,
we chose to substitute old animals (12-month- and 18-month-old) with a
dose of GH (1 mg · kg-1 · d-1) that did
not influence cholesterol metabolism to a major
degree in young rats. This treatment did not significantly change
plasma IGF-I levels, and the dose may thus be considered relatively
low. Nevertheless, the infusion of GH to
hypercholesterolemic 18-month-old rats was associated
with a very clear reversal of the changes in plasma lipoproteins. It
was more difficult to exactly define a specific point of action
responsible for this overall effect. GH affects a number of regulatory
steps in lipid and lipoprotein
metabolism.15 16 51 The fact that the
lipoprotein pattern was reversed despite an increased HMG-CoA reductase
activity would indicate that the turnover of lipoproteins was
stimulated. The increase in hepatic LDLR expression was relatively
modest, as was the change in C7OH activity. The fact that more
pronounced effects were observed in response to GH treatment in the
12-month-old animals may suggest that the oldest rats were relatively
resistant to infusion with GH. The 12-month-old rats displayed
a response that was similar to that obtained in the young animals, both
in C7OH and lipoprotein pattern. It is of interest to note that
plasma IGF-I levels were not changed in these groups of rats, either.
The possible contribution of a GH-induced stimulation of the hepatic
secretion of apoE or of an increased peripheral clearance
of lipoproteins cannot be evaluated from the present study. It
seems reasonable to assume that the pleiotropic effects of GH on
lipoprotein metabolism are responsible for the favorable
changes in plasma lipoprotein patterns in the 18-month-old rats.
In conclusion, our studies have demonstrated the reversal of age-related hypercholesterolemia in the rat by infusion of GH. Our findings further support the concept15 that GH has an important role in modulating lipoprotein metabolism under physiological conditions.
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Acknowledgments |
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This study was supported by grants from the Swedish Medical Research Council (03X-7137); the Swedish Society for Medical Research; the Swedish Foundation for Strategic Research; the Ax:son Johnson, Jeansson, Widengren, and Ruth and Richard Julin Foundations; the Swedish Heart-Lung Foundation; "Förenade Liv" Mutual Group Life Insurance Co; the foundation of Female Old Servants; and the Karolinska Institute. We thank Ingela Arvidsson, Ewa Ellis, and Lilian Larsson for technical assistance.
Received March 24, 1998; accepted July 28, 1998.
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References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Steinberg D. Metabolism of lipoproteins and their role in the pathogenesis of atherosclerosis. Atheroscler Rev. 1988;18:1–23.
2.
Levine GN, Keaney JF Jr, Vita JA.
Cholesterol reduction in cardiovascular
disease: clinical benefits and possible mechanisms. N Engl
J Med. 1995;332:512–521.
3.
Heiss G, Tamir I, Davis CE, Tyroler HA.
Lipoprotein-cholesterol distributions in selected North
American populations: the Lipid Research Clinics Program prevalence
study. Circulation. 1980;61:302–315.
4.
Abbott RD, Garrison RJ, Wilson PW, Epstein FH,
Castelli WP, Feinleib M, LaRue C. Joint distribution of lipoprotein
cholesterol classes: the Framingham Study.
Arteriosclerosis. 1983;3:260–272.
5. Castelli WP, Wilson PW, Levy D, Anderson K. Cardiovascular risk factors in the elderly. Am J Cardiol. 1989;63:12H–19H.[Medline] [Order article via Infotrieve]
6. Miller NE. Why does plasma low density lipoprotein concentration in adults increase with age? Lancet. 1984;1:263–267.[Medline] [Order article via Infotrieve]
7.
Grundy SM, Vega GL, Bilheimer DW. Kinetic mechanisms
determining variability in low density lipoprotein levels and rise with
age. Arteriosclerosis. 1985;5:623–630.
8. Ericsson S, Eriksson M, Vitols S, Einarsson K, Berglund L, Angelin B. Influence of age on the metabolism of plasma low density lipoproteins in healthy males. J Clin Invest. 1991;87:591–596.
9. Millar JS, Lichtenstein AH, Cuchel M, Dolnikowski GG, Hachey DL, Cohn JS, Schaefer EJ. Impact of age on the metabolism of VLDL, IDL, and LDL apolipoprotein B-100 in men. J Lipid Res. 1995;36:1155–1167.[Abstract]
10. Mahley RW, Hui DY, Innerarity TL, Weisgraber KH. Two independent lipoprotein receptors on hepatic membranes of dog, swine, and man: apo-B, E and apo-E receptors. J Clin Invest. 1981;68:1197–1206.
11. Mahley R, Innerarity TL. Lipoprotein receptors and cholesterol homeostasis. Biochim Biophys Acta. 1983;737:197–222.[Medline] [Order article via Infotrieve]
12. Einarsson K, Nilsell K, Leijd B, Angelin B. Influence of age on secretion of cholesterol and synthesis of bile acids by the liver. N Engl J Med. 1985;313:277–282.[Abstract]
13. Ståhlberg D, Angelin B, Einarsson K. Age-related changes in the metabolism of cholesterol in rat liver microsomes. Lipids. 1991;26:349–352.[Medline] [Order article via Infotrieve]
14. Choi YS, Ide T, Sugano M. Age-related changes in the regulation of cholesterol metabolism in rats. Exp Gerontol. 1987;22:339–349.[Medline] [Order article via Infotrieve]
15. Angelin B, Rudling M. Growth hormone and hepatic lipoprotein metabolism. Curr Opin Lipidol. 1994;5:160–165.[Medline] [Order article via Infotrieve]
16.
Corpas E, Harman SM, Blackman MR. Human growth hormone
and human aging. Endocr Rev. 1993;14:20–39.
17.
Sonntag WE, Steger RW, Forman LJ, Meites J. Decreased
pulsatile release of growth hormone in old male rats.
Endocrinology. 1980;107:1875–1879.
18.
Rudling M, Norstedt G, Olivecrona H, Reihnér E,
Gustafsson JÅ, Angelin B. Importance of growth hormone for the
induction of hepatic low density lipoprotein receptors. Proc Natl
Acad Sci U S A. 1992;89:6983–6987.
19.
Rudling M, Angelin B. Loss of resistance to dietary
cholesterol in the rat following hypophysectomy: importance
of growth hormone for the expression of hepatic low density lipoprotein
receptors. Proc Natl Acad Sci U S A. 1993;90:8851–8855.
20. Rudling M, Olivecrona H, Eggertsen G, Angelin B. Regulation of rat liver low density lipoprotein receptors: in vivo stimulation by growth hormone is not mediated by insulin-like growth factor I. J Clin Invest. 1996,97:292–299.
21. Parini P, Angelin B, Lobie P, Norstedt G, Rudling M. Growth hormone specifically stimulates the expression of low density lipoprotein receptors in human hepatoma cells. Endocrinology. 1995;136:3767–3773.[Abstract]
22.
Rudling M, Parini P, Angelin B. Growth hormone and bile
acid synthesis: key role for the activity of hepatic microsomal
cholesterol 7-hydroxylase in the rat. J
Clin Invest. 1997;99:2239–2245.[Medline]
[Order article via Infotrieve]
23.
Elam MB, Wilcox HG, Solomon SS, Heimberg M. In vivo
growth hormone treatment stimulates secretion of very low density
lipoprotein by the isolated perfused rat liver.
Endocrinology. 1992;131:2717–2722.
24.
Sjöberg A, Oscarsson J, Edén S, Olofsson
SO. Continuous but not intermittent administration of growth hormone to
hypophysectomized rats increases apolipoprotein-E secretion from
cultured hepatocytes. Endocrinology. 1994;134:790–798.
25.
Sjöberg A, Oscarsson J, Boström K,
Innerarity TL, Edén S, Olofsson SO. Effects of growth hormone on
apolipoprotein-B (apoB) messenger ribonucleic acid editing, and apoB 48
and apoB 100 synthesis and secretion in the rat liver.
Endocrinology. 1992;130:3356–3364.
26. Angelin B, Einarsson K, Liljeqvist L, Nilsell K, Heller RA. 3-Hydroxy-3-methylglutaryl coenzyme A reductase in human liver microsomes: active and inactive forms and cross-reactivity with antibody against rat liver enzyme. J Lipid Res. 1984;25:1159–1166.[Abstract]
27.
Einarsson K, Angelin B, Ewerth S, Nilsell K,
Björkhem I. Bile acid synthesis in man: assay of hepatic
microsomal cholesterol 7 -hydroxylase activity by
isotope dilution-mass spectrometry. J Lipid Res. 1986;27:82–88.[Abstract]
28. Einarsson K, Benthin L, Ewerth S, Hellers G, Ståhlberg D, Angelin B. Studies on acyl-coenzyme A:cholesterol acyltransferase activity in human liver microsomes. J Lipid Res. 1989;30:739–746.[Abstract]
29. Rudling M, Angelin B. Stimulation of rat hepatic low density lipoprotein receptors by glucagon: evidence of a novel regulatory mechanism in vivo. J Clin Invest. 1993;91:2796–2805.
30.
Bang P, Wivall IL, Eriksson U, Sara V, Hall K.
Comparison of acid ethanol extraction and acid gel filtration before
IGF-I and IGF-II radioimmunoassays: improvement of determinations in
acid ethanol extracts by the use of truncated IGF-I as
radioligand. Acta Endocrinol (Copenh). 1991;124:620–629.
31.
Parini P, Angelin B, Rudling M. Importance of estrogen
receptors in hepatic LDL receptor regulation. Arterioscler Thromb
Vasc Biol. 1997;17:1800–1805.
32. Rudling M. Hepatic mRNA levels for the LDL receptor and HMG-CoA reductase show coordinate regulation in vivo. J Lipid Res. 1992;33:493–501.[Abstract]
33.
Baum CL, Teng BB, Davidson NO. Apolipoprotein B
messenger RNA editing in the rat liver: modulation by fasting and
refeeding a high carbohydrate diet. J Biol Chem. 1990;265:19263–19270.
34. Snedecor GW, Cochran WG. Statistical Methods. Ames, Iowa: Iowa State University Press; 1980.
35. Story JA, Gomolinski E, Czarnecki SK, Tepper SA, Kritchevsky D. Age-strain interrelations in lipid metabolism of rats. Lipids. 1981;16:87–92.[Medline] [Order article via Infotrieve]
36. Carlson LA, Fröberg SO, Nye ER. Effect of age on blood and tissue lipid levels in the male rat. Gerontologia. 1968;14:65–79.
37. Nanjee MN, Miller NE. Sex difference in the saturable binding of low-density lipoprotein by liver membranes in ageing rats. Biochim Biophys Acta. 1988;959:378–385.[Medline] [Order article via Infotrieve]
38. Schneeman BO, Richter D. Changes in plasma and hepatic lipids, small intestinal histology and pancreatic enzyme activity due to aging and dietary fiber in rats. J Nutr. 1993;123:28–37.
39. Stange EF, Dietschy JM. Age-related decreases in tissue sterol acquisition are mediated by changes in cholesterol synthesis and not low density lipoprotein uptake in the rat. J Lipid Res. 1984;25:703–713.[Abstract]
40. Vlahcevic ZR, Heuman DM, Hylemon PB. Regulation of bile acid synthesis. Hepatology. 1991;13:590–600.[Medline] [Order article via Infotrieve]
41.
Sundseth SS, Waxman DJ. Hepatic P-450
cholesterol 7-hydroxylase: regulation in vivo at
the protein and mRNA level in response to mevalonate, diurnal rhythm,
and bile acid feedback. J Biol Chem. 1990;265:15090–15095.
42.
Noshiro M, Nishimoto MM, Okuda K. Rat liver
cholesterol 7- -hydroxylase: pretranslational
regulation for circadian rhythm. J Clin Invest. 1990;265:10036–10041.
43.
Brown MS, Kovanen PT, Goldstein JL. Regulation of
plasma cholesterol by lipoprotein receptors.
Science. 1981;212:628–635.
44.
Kovanen PT, Bilheimer DW, Goldstein JL, Jaramillo JJ,
Brown MS. Regulatory role for hepatic low density lipoprotein receptors
in vivo in the dog. Proc Natl Acad Sci U S A. 1981;78:1194–1198.
45.
Rudling MJ, Reihnér E, Einarsson K, Ewerth
S, Angelin B. Low density lipoprotein receptor-binding activity in
human tissues: quantitative importance of hepatic receptors and
evidence for regulation of their expression in vivo. Proc Natl
Acad Sci U S A. 1990;87:3469–3473.
46. Åkerlund JE, Reihnér E, Angelin B, Rudling M, Ewerth S, Björkhem I, Einarsson K. Hepatic metabolism of cholesterol in Crohn's disease: effect of partial resection of ileum. Gastroenterology. 1991;100:1046–1053.[Medline] [Order article via Infotrieve]
47.
Spady DK, Cuthbert JA. Adenovirus-mediated transfer of
a gene encoding cholesterol 7-hydroxylase into
hamsters increases hepatic enzyme activity and reduces plasma total and
low density lipoprotein cholesterol. J Clin
Invest. 1995;96:700–709.
48.
Schwarz M, Lund EG, Setchell KDR, Kayden HJ, Zerwekh
JE, Björkhem I, Herz J, Russell DW. Disruption of
cholesterol 7- -hydroxylase gene in mice, II: bile
acid deficiency is overcome by induction of oxysterol
7--hydroxylase. J Biol Chem. 1996;271:18024–18031.
49. Sparks CE, Marsh JB. Metabolic heterogeneity of apolipoprotein B in the rat. J Lipid Res. 1981;22:519–527.[Abstract]
50.
Olivecrona H, Johansson AG, Lindh E., Ljunghall S,
Berglund L, Angelin B. Hormonal regulation of serum lipoprotein (a)
levels: contrasting effects of growth hormone and insulin-like growth
factor-I. Arterioscler Thromb Vasc Biol. 1995;19:847–849.
51.
Davidsson MB. Effect of growth hormone on carbohydrate
and lipid metabolism. Endocr Rev. 1987;8:115–131.
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