© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Human Growth Hormone Increases Apo(a) Expression in Transgenic Mice
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Abstract |
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Abstract—Levels of Lp(a), an atherogenic lipoprotein that circulates in human plasma, are increased by the administration of growth hormone (GH). Many of the physiological effects of GH are mediated through insulin-like growth factor-1 (IGF-1), but ironically, IGF-1 treatment of humans is associated with a fall in plasma Lp(a) levels. To glean insight into the mechanism responsible for the GH-associated increase in plasma levels of Lp(a), we administered recombinant human GH (rhGH) to mice expressing a 370-kb human genomic fragment containing the apo(a) gene, 40 kb of 5'-, and 200 kb of 3'-flanking sequence [YAC-apo(a) transgenic mice]. The plasma levels of apo(a) and hepatic levels of apo(a) mRNA rose dramatically in the post-pubertal male mice in response to rhGH treatment. To determine whether the increase in plasma apo(a) was mediated by IGF-1, we treated castrated and noncastrated YAC-apo(a) transgenic mice with a continuous infusion of IGF-1 (100 µg/d) for 2 weeks, and plasma levels of apo(a) fell by
50%. Thus the effects of rhGH and IGF-1 administration on plasma
levels of apo(a) in the YAC-apo(a) transgenic mice simulate those seen
in humans. The coordinate changes in apo(a) mRNA and plasma levels of
apo(a) in response to rhGH and IGF-1 strongly suggest that these 2
hormones have independent effects on the transcription of the
apo(a) gene.
Key Words: apo(a) • growth hormone • insulin-like growth factor-1
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Introduction |
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Lp(a) is an atherogenic plasma lipoprotein composed of a particle of LDL attached to a large glycoprotein, apo(a).1 Plasma levels of Lp(a) vary over a 1000-fold range (from <0.1 mg/dL to >100 mg/dL), and most of the interindividual variation is due to inherited differences in the size and sequence of the apo(a) gene.2 The rate of synthesis of Lp(a), rather than its degradation, correlates best with its plasma levels.3
Our knowledge regarding the mechanism by which sequence variations in apo(a) affect plasma levels of Lp(a) is limited due to the relative paucity of appropriate model systems in which to study the synthesis and metabolism of apo(a). In humans, the apo(a) that circulates in plasma is synthesized in the liver, but cultured human hepatocytes synthesize only a trace amount of apo(a).4 Thus no readily accessible human tissue is available for the analysis of hepatic apo(a) synthesis, and apo(a) is not present in the blood of any nonhuman species other than great apes or hedgehogs.5 6
Apo(a) transgenic mice have been available for 6 years but cannot be
used to examine the regulation of apo(a) synthesis because the cDNA is
under the control of the mouse metallothionein
promoter.7 In 1995 Frazer and colleagues developed
an apo(a) transgenic mouse that expresses the human apo(a) gene within
its own genomic context.8 The mice contain a 270 kb human
genomic fragment that includes the entire coding sequence of the apo(a)
gene and at least 60 kb of 5'- and 3'-flanking sequences. The male and
female transgenic mice have similar plasma levels of apo(a) (20
mg/dL) until puberty.9 At puberty the concentration of
plasma apo(a) falls to almost undetectable levels in the male mice, an
effect that can be completely reversed by castration.8 The
plasma apo(a) and hepatic apo(a) mRNA levels return to adult levels if
the castrated male mice are given
5
-dihydrotestosterone.8 From these experiments, it was
concluded that testosterone is responsible for the postpubertal
decrease in plasma apo(a) levels in the transgenic mice. Treatment of
female mice with estrogen also results in a decrease in plasma apo(a),
although the effect is less pronounced.9 Thus
pharmacological levels of sex steroids profoundly affect the plasma
levels of apo(a) in these mice.
We propose an alternative explanation for the low plasma apo(a) levels
in postpubertal male mice expressing the human apo(a) gene. The fall in
plasma levels of apo(a) may in part be an indirect rather than direct
effect of testosterone on hepatic apo(a) expression. In rodents, sex
steroids determine the secretion pattern of growth hormone (GH).
Postpubertal male and female rats have similar mean plasma levels of
GH; however in males, the pattern of secretion is pulsatile, whereas in
females, it is more continuous.10 11 In mice, the pulse
interval of GH secretion is longer in males (2.5 hours) than in
females (1.4 hours), so the plasma level of GH remains more constant
in the female mice.12 The specific pattern of GH
secretion, in turn, affects the expression levels of numerous hepatic
genes by stimulating the expression of some genes and suppressing the
expression of others.13 14 The male-specific pattern of GH
secretion may contribute to the fall in peripubertal plasma apo(a)
levels in postpubertal male YAC-apo(a) transgenic mice.
Another stimulus for these studies is the finding that high plasma levels of GH are associated with elevations in plasma concentrations of Lp(a) in humans. Acromegalics have increased plasma levels of Lp(a), and successful treatment of this condition results in a fall in the plasma level of Lp(a) that is proportional to the decrease in GH levels.15 16 Administration of GH to normal or GH-deficient humans results in a significant increase in plasma levels of apo(a) (for review see reference 17). The reason why GH therapy increases plasma levels of apo(a) in humans is not known. Many of the physiological effects of GH are mediated by insulin-like growth factor-1 (IGF-1),18 but ironically, IGF-I administration to humans results in a decrease rather than increase in plasma levels of Lp(a).19 20 21 22
In this study, we have used a recently developed transgenic mouse line that expresses a 370 kb fragment containing the entire apo(a) gene as well as 40 kb of 5' and 200 kb of 3' sequence to examine the effect of GH and IGF-1 treatment on the expression of apo(a).23
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Methods |
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Animals and Experimental Design
Mice (University of Texas Southwestern Medical Center, Dallas) expressing a 370-kb human DNA fragment containing the entire apo(a) gene, 40 kb of 5'-flanking and 200 kb of 3'-flanking sequence in a mixed C57Bl/6 and SJL background [YAC-apo(a) mice] were used in these experiments.23 The mice were back-crossed into the C57Bl/6 strain, and at 1 month of age, venous blood was collected by retro-orbital puncture from the N2 and N3 generations. Plasma was subjected to immunoblot analysis,7 and mice with immunodetectable apo(a) were entered into the study. All the mice in the study were heterozygous for the YAC-apo(a) transgene.
The transgenic mice were housed in a conventional, non–germ-free animal facility with 12-hour dark/light cycles. The mice were weaned at 21 days and fed a cholate-free, standard mouse chow diet containing 6% animal fat and <0.04% cholesterol (Teklad, mouse/rat diet 7002: Harlan Teklad) with free access to water. Surgical castration was performed in male and female mice at 10 weeks of age, and these mice were entered into the study 4 weeks after surgery.
Recombinant human growth hormone (rhGH) was a gift from Eli Lilly (Indianapolis, IN). The rhGH was diluted in 150 mmol/L NaCl and 10 mmol/L Tris, pH 7.4, and administered to 10-week-old mice by continuous infusion using an osmotic mini-pump, type 2001(Alza Corporation) implanted subcutaneously through a small incision below the scapula of the mouse. Control mice received vehicle alone. The mice receiving intermittent rhGH therapy received the same daily dose of GH administered directly into the peritoneal cavity in divided doses every 12 hours. The mice were weighed daily at 10 AM.
A total of 200 µL of venous blood was obtained by retro-orbital
puncture using heparinized capillary tubes (Microvette CB1000,
Sarstedt) before initiation of rhGH treatment. At the indicated time
intervals, the mice were fasted for 5 hours before administering sodium
phenobarbital (Abbott Laboratories). The animals were exsanguinated by
cannulating the left ventricle of the heart with an 18 gauge needle and
slitting open the right atrium. The mice were perfused with 50 mL of
phosphate buffered saline (pH 7.4), and the livers were collected and
stored in liquid nitrogen.
Recombinant human insulin-like growth factor-I (rhIGF-1) was kindly provided by Genentech (South San Francisco, CA). The IGF-1 was diluted with 0.9% NaCl at a concentration of 8.4 µg/µL and administered to mice (100 µg/d) by a continuous infusion (0.5 µL/h) using a mini-osmotic pump (Alzert 2002).
Immunoblot Analysis of Apo(a)
Venous blood was collected in heparinized capillary tubes from
the retro-orbital plexus before treatment and at the end of each
experiment. Plasma was isolated by centrifugation at
5000g for 10 minutes, aliquoted, and stored at -80°C. A
total of 0.3 µL of plasma was subjected to immunoblot
analysis using an anti-apo(a) monoclonal antibody (IgG-a5) or
rabbit anti-rat apoAI antibody,24 exactly as
previously described.25
Measurement of Plasma Apo(a), Growth Hormone, and IGF-I
Plasma samples were stored at -80°C, and the apo(a) levels
were quantified within 3 months of collection using a sandwich
monoclonal antibody-based enzyme-linked immunosorbent
assay.26 Plasma concentrations of human GH were
quantitated in duplicate using an hGH ELISA Kit (Boehringer
Mannheim). The plasma levels of IGF-1 were measured at Genentech
Laboratories using a radioimmunoassay as described.27 The
plasma was subjected to acid/methanol precipitation to remove the IGF-1
from its binding proteins before measuring the total plasma IGF-1
level. The plasma levels of cholesterol and
triglycerides were measured using
Cholesterol/HP (Boehringer Mannheim) and
Triglyceride (GPO-Trinder, Sigma) kits, respectively.
Northern Blot Analysis
Total cellular RNA was prepared from mouse tissues using RNA
STAT-60 (TelTest B Inc) according to the instructions of manufacturer.
Fifteen micrograms of RNA from each tissue was size-fractionated on a
1%(wt/vol) agarose, 0.6 mol/L formaldehyde gel and then transferred to
nylon membranes (Hybond N+, Amersham). A cDNA probe was
generated from the apo(a)8 K4-type 2 repeat using reverse
transcriptase-PCR (Promega RT-PCR kit, Promega) and 2
oppositely-oriented oligonucleotides
(5'-TGACAC- CACACTCGCATAGTCGGAC-3' and
5'-GATGACCAAGCT-TGGCAGGTTCTTCC-3')8 to
amplify a 338 bp fragment from hepatic poly (A)+
mRNA isolated from our YAC-mice apo(a). The fragment was gel-purified
and radiolabeled with [32P]-dCTP (3000 Ci/mmol)
using Megaprime DNA Labeling System (Amersham). The filters were
allowed to hybridize with the 32P-labeled probes
(1x106 cpm/mL) for 2 hours at 65°C using
Rapid-hyb buffer (Amersham).
A mouse IGF-I fragment containing the sequences of exon 3 was amplified using PCR, 2 oppositely-oriented oligonucleotides(5'-AGCCCACAGGCTATGGCTCC-3' and 5'-CTTCTGAGTCT-TGGGCATGTC-3'), and mouse genomic DNA.28 A radiolabeled 1141-bp fragment from the rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragment29 was included in the hybridization buffer. The filter was washed with 0.1% (wt/vol) SDS, 0.1xSSC (1xSSC: 150 mmol/L NaCl and 15 mmol/L NaCitrate) at 70°C for 30 minutes before being exposed to Reflection NEF 496 film (Dupont-NEN) at -80°C with an intensifying screen. A cDNA probe for mouse apoAI was obtained from Drs Jay Horton and Hitoshi Shimano (University of Texas Southwestern Medical Center, Dallas).30
Statistical Methods
The values are expressed as means±SD. Comparisons were made
using a 2-tailed Student's t test.
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Results |
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To examine the effect of GH on plasma levels of apo(a), we administered rhGH to mice expressing a human apo(a) gene in its own genomic context. We used mice containing a human genomic fragment that includes the entire apo(a) gene, 40 kb of 5'- and 200 kb of 3'-flanking sequence, respectively. In this article these mice are referred to as YAC-apo(a) transgenic mice.23 Before puberty, the male and female mice have similar plasma levels of apo(a).23 After puberty, the male YAC-apo(a) transgenic mice have a significantly lower mean plasma level of apo(a) than their female counterparts [1.2±0.91 (n=20) versus 8.14±2.6 mg/dL (n=20)].
To determine whether GH treatment increases plasma apo(a) levels in the
YAC-apo(a) transgenic mice, postpubertal male mice (10 weeks old) were
treated with a continuous infusion of either rhGH or vehicle alone for
7 days. The dosage of rhGH administered ranged from 0.625 mg ·
kg-1 · d-1 (daily
dose per animal:15 to 19 µg) to 10 mg ·
kg-1 · d-1 (daily
dose: 250 to 300 µg). The plasma rhGH levels were measured using an
ELISA assay that detects human but not mouse GH (Figure 1
). A direct relationship was seen
between the daily dosage of rhGH administered to the mice and the
plasma level of hGH (Panel A). No hGH was detected in the plasma of the
mice given vehicle alone. The levels of hGH ranged from 7.6 ng/mL in
the mice treated with 0.625 mg ·
kg-1 · d-1 to 40.2
ng/mL in the mice given 10 mg · kg-1
· d-1. To confirm that the infused growth
hormone was physiologically active, we
monitored the body weights of the mice. A dose-dependent increase in
weight was seen in association with rhGH treatment (Panel B). Animals
receiving the highest daily dose of rhGH (10 mg ·
kg-1 · d-1) had a
mean increase in body weight of 10%.
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A significant dose-dependent increase in the plasma levels of apo(a)
was seen in the rhGH treated mice (Figure 1C
). The mean plasma
levels of apo(a) increased 3.5-fold at the lowest dose (0.625 mg
· kg-1 · d-1)
and 15-fold at the highest dose (10 mg ·
kg-1 · d-1). No
significant change in plasma level of apo(a) was seen in the animals
treated with buffer alone. Thus, administration of rhGH is associated
with a significant increase in plasma apo(a) in post-pubertal male
YAC-apo(a) transgenic mice.
The serum glucose, alanine transaminase, aspartate aminotransferase,
bilirubin, and albumin, as well as the plasma
cholesterol and plasma triglycerides, were
measured on pooled samples from each group. No consistent
changes or trends were observed in the serum levels of glucose, alanine
transaminase, bilirubin, or albumin. The plasma levels of
aspartate aminotransferase increased with rhGH treatment, although the
effect was not dose-dependent. No significant change was seen in plasma
cholesterol levels. The plasma triglyceride
level increased by 28% at the highest dose of rhGH (Table 1). To determine the distribution of
cholesterol between the lipoprotein classes, the plasma
samples were pooled and subjected to size-fractionation on a Superose 6
column. In rhGH treated animals, there tended to be a reduction in the
cholesterol content of the HDL fraction associated with a
slight shift to larger sized HDL particles. No significant changes were
seen in the VLDL or LDL fraction (data not shown).
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Immunoblot analysis of plasma apo(a) from
representative postpubertal adult male mice is shown in
Figure 2. The animals that received
vehicle alone had only a trace amount of immunodetectable apo(a) in
their plasma. A dramatic increase in the amount of apo(a) was seen in
the mice treated with rhGH. The effect of rhGH was not a generalized
effect on all plasma proteins synthesized by the liver. No change was
seen in the plasma levels of immunodetectable apoAI.
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To determine whether rhGH treatment was associated with an increase in
the amount of apo(a) mRNA, Northern blot analysis was performed
using total RNA from multiple tissues of mice treated with the highest
dose of rhGH. No apo(a) mRNA transcript was detected in the brain,
heart, kidney, muscle, adipose tissue, spleen, testes, and ovary of the
YAC-apo(a) transgenic mice (data not shown). Northern blot
analysis of total hepatic mRNA using an apo(a)-specific probe
revealed a single band of 7.5 kb in all the mice (including the mice
treated with vehicle alone when the film was subjected to a longer
exposure) (Figure 3A). The relative
amount of apo(a) mRNA was 12-fold higher in the mice receiving the
highest dose of rhGH (10 mg · kg-1
· d-1) than in the mice receiving vehicle
alone. Although there were interindividual variations in the amount of
apo(a) mRNA, the level correlated with the plasma levels of apo(a) in
the 18 animals studied (R2=0.93)
(Figure 3B). No significant changes in the level of hepatic
apoB-100 mRNA were observed with rhGH treatment (data not shown). Taken
together, these data are consistent with the increase in plasma
apo(a) levels associated with rhGH treatment being due to an effect of
rhGH on the rate of apo(a) transcription or on the stability of the
apo(a) mRNA in the liver.
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To determine the time course of the increase in hepatic apo(a) mRNA and
the plasma levels of apo(a), adult male mice were treated with 2.5 mg
rhGH · kg-1 ·
d-1 for 1 to 7 days. A 1.7-fold increase in
hepatic apo(a) mRNA levels was seen within the first 24 hours, and a
further increase was observed over the ensuing 2 days (Figure 4, top panel); no change in apo(a) mRNA
level was seen between days 3 and 7. Parallel changes were seen in the
plasma levels of apo(a) (Figure 4, bottom panel), which also
reached a plateau by day 3. These results are consistent with
the rhGH-associated increase in plasma apo(a) resulting from the effect
of the hormone on the levels of hepatic apo(a) mRNA.
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To determine whether rhGH has a similar apo(a)-elevating effect in
female mice, we treated 3 females for 7 days with rhGH by continuous
infusion at a dosage of 10 mg · kg-1
· d-1 (Table 2). The mean percent increase in
body weight was -1.5% in the control group and 4.6% in rhGH-treated
group. The percent change in plasma apo(a) level was determined by
comparing the mean of the pretreatment values to the posttreatment
levels. No significant change was seen in the mice treated with vehicle
alone (n=3). In the female mice treated with high-dose rhGH, a 2-fold
increase in plasma apo(a) was seen. Northern blot analysis of
the hepatic mRNA from these mice showed a 1.5-fold increase in the mRNA
level after rhGH treatment (data not shown). Thus rhGH had a more
modest elevating effect on hepatic apo(a) mRNA and plasma levels of
apo(a) in female YAC-apo(a) transgenic mice than their male
counterparts.
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Adult male (n=6) and female mice (n=6) were castrated to examine the
effect of endogenous sex steroids on the apo(a)-elevating
effect of rhGH. Recombinant hGH was given by continuous infusion at 2.5
mg · kg-1 ·
d-1 for 4 days starting 1 month after
castration. Castration has no significant effect on plasma levels of
apo(a) in the female mice (Table 3),
which is similar to what was previously reported in the other
YAC-apo(a) transgenic mouse line.9 In noncastrated female
mice, a 33% increase in plasma apo(a) was seen in the rhGH-treated
mice. In the castrated females, treatment with rhGH resulted in a
larger increase in plasma apo(a) levels (6.8±1.9 to 14.0±5.8 mg/dL)
than was seen in the noncastrated mice. This suggests that estrogen may
antagonize somewhat the apo(a)-elevating effect of rhGH in the mice,
although more animals need to be studied to confirm this finding.
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Castration was associated with a much more dramatic increase in plasma
apo(a) levels in the male than female YAC-apo(a) transgenic mice. The
plasma level of apo(a) increased 6-fold in the noncastrated male mice
that were treated with 2.5 mg · kg-1
· d-1 of rhGH by continuous infusion, which
was similar to the results of our prior experiment (see Figure 1). In the castrated male mice, no significant increase in
plasma levels of apo(a) was seen in association with rhGH
treatment.
To determine whether the method of delivery of rhGH to the animals
influenced the effect of the hormone on plasma levels of apo(a), we
administered 2.5 mg · kg-1 ·
d-1 of rhGH or vehicle alone via
intraperitoneal injection every 12 hours for 4 days
to adult male mice (n=3 in each group). Measurement of human GH at 1,
3, and 6 hours revealed that at 1 hour the level was between 250 to 400
ng/mL, and the level decreases to below the detectable range after 6
hours. No difference in plasma levels of apo(a) were seen between the
control (1.36±1.04 mg/dL) and rhGH-treated groups (1.26±0.96 mg/dL)
(Table 4). When the treatment was
extended for 6 days, a modest increase in the mean plasma apo(a) level
(from 1.56±1.04 to 2.43±0.75 mg/dL) was seen (data not shown). In the
same experiment, mice were treated with identical daily dose of rhGH
but administered as a continuous infusion. These mice had plasma levels
of human GH range from 12 to 26 ng/mL. As expected, there was a
significant increase in plasma levels of apo(a). Thus the same daily
dose of rhGH that caused an increase in plasma levels of apo(a) when
given as a continuous infusion did not result in a significant
elevation in the plasma level of apo(a) when administered as an
intermittent injection despite observing an increase in body
weight.
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One of the effects of GH in the mouse is to increase the synthesis and
secretion of IGF-1 by the liver. To determine whether the
administration of rhGH was associated with an increase in IGF-1
expression, we examined the level of hepatic IGF-1 mRNA and plasma
levels of IGF-1 in the rhGH-treated adult male YAC-apo(a) transgenic
mice (Figure 5). Two mRNA transcripts of
0.8 and 7.5 kb are generated from the murine IGF-1 gene,28
and both increased significantly (P<0.05) in response to
administration of recombinant hGH treatment. A 2.2-fold increase in the
level of the 7.5 kb transcript and a 2.7-fold elevation in the amount
of the 0.8 kb transcript was seen at the highest dose. A
radioimmunoassay assay that measures total IGF-1 was used to
measure the plasma IGF-1 levels in these animals (Lyn Powell-Braxton,
Genetech). The mean level of plasma IGF-1 in normal mice is 164.8±11.6
ng/mL.31 The highest dose of GH was associated with
only a 22% increase in the plasma level of IGF-1, which was not
statistically significant.
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To determine whether the apo(a)-elevating effect of rhGH was mediated
by IGF-1, 3-month-old castrated and noncastrated male and female
YAC-apo(a) transgenic mice were treated with 100 µg of IGF-1 by
continuous infusion for 14 days. Plasma was obtained 7 and 14 days
after initiation of treatment, and the apo(a) levels were measured
(Figure 6). In the noncastrated male
YAC-apo(a) transgenic mice, no change was seen in the plasma levels of
apo(a). In contrast, the mean plasma levels of apo(a) in the
noncastrated female mice fell 2.5 fold (from 9.4±2.0 to 3.7±14 mg/dL)
after IGF-1 treatment. No change in plasma apo(a) levels was seen in
the animals administered vehicle alone. A decrease of similar magnitude
was seen in the plasma levels of apo(a) after IGF-1 treatment (60%
decrease) in both the male and female castrated mice (Figure 6A, panels C and D). Thus, the apo(a) elevating effect of rhGH in these
mice was not mediated by IGF-1.
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Northern blot analysis was performed using total hepatic RNA,
and the relative amounts of apo(a) mRNA were determined using a
phosphoimager. The relative amounts of apo(a) mRNA tended to correlate
with the levels of plasma apo(a) (Figure 6B). These results are
consistent with the effects of IGF-1 on the plasma levels of
apo(a) being due to changes in the level of transcription of the apo(a)
gene or stabilization of the apo(a) mRNA.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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We have shown that administration of rhGH to mice expressing a human apo(a) transgene under the control of its own regulatory sequences is associated with a significant increase in the hepatic content of apo(a) mRNA and levels of plasma apo(a). The apo(a)-elevating effect of rhGH was most pronounced in the postpubertal male mice; a significant increase in the plasma levels of apo(a) occurred within 24 hours of initiating rhGH treatment, and the levels reached a maximum at 3 days. To determine whether the effect of rhGH on plasma apo(a) levels was mediated by IGF-1, IGF-1 was administered to the YAC-apo(a) mice. IGF-1 treatment resulted in an
50% fall in
plasma apo(a) levels in the transgenic mice. Therefore, the increase in
plasma apo(a) associated with GH treatment is not due to its
stimulatory effect on IGF-1. The directional effects of rhGH and IGF-I
on the plasma levels of apo(a) in these animals simulate those seen in
humans, and thus the YAC-apo(a) transgenic mice provide an excellent
animal model in which to study the regulation of apo(a) expression by
these agents.
Two YAC-apo(a) transgenic mice lines have been developed, and both
demonstrate a sexually dimorphic pattern of apo(a)
expression.8 23 Prepubertal male and female YAC apo(a)
transgenic mice have similar plasma levels of apo(a) until puberty and
then the levels fall dramatically in the males.8 23 The
peripubertal fall in plasma apo(a) in the male YAC-apo(a) transgenic
mice has been presumed to be due solely to the effect of testosterone
because castration results in an increase in plasma apo(a) levels and
administration of 5-dihydrotestosterone to the castrate male lowers
plasma levels to the post-pubertal range.8 An alterative
explanation for these observations is that some of the effect of
testosterone is indirect and due to the male-specific pattern of
secretion of GH.
Adult male and female rodents have similar mean plasma levels of GH, but the sex steroid profile determines whether the GH is secreted intermittently in bursts (testosterone) or more continuously (estrogen).11 32 Interpulse intervals of at least 2 hours duration are required to achieve a male pattern of hepatic gene expression.14 In male rodents, the male pulsatile pattern of GH secretions activates a number of members of the STAT transcription factor family (including STAT5), which have been implicated as mediating the GH-associated male-specific pattern expression of hepatic genes.33
The male pattern of GH secretion may inhibit expression of the apo(a)
gene in the postpubertal YAC-apo(a) transgenic mice. Consistent
with this scenario is our observation that administration of a GH as a
continuous infusion to postpubertal male mice resulted in an increase
in plasma apo(a) to levels that are similar to those seen in females.
Males that received intermittent administration of the same total daily
dose of rhGH had no significant increase in their plasma levels of
apo(a) (Table 4). Thus it is not just the amount of rhGH
administered but also the pattern of its delivery that affects plasma
levels of apo(a).
The elevating response of plasma apo(a) levels to a continuous infusion of rhGH in the male transgenic mice is similar to what has previously been observed for another murine hepatic gene, the prolactin receptor. Hepatic prolactin receptor mRNA levels are much lower in male than female mice, but administration of GH by continuous infusion to male mice increases the level of the hepatic prolactin receptor into the "female" range. The same dose of GH, if administered as an intermittent infusion, has no effect on prolactin hormone receptor mRNA levels.34
Humans differ from mice in that the pattern of GH secretion is not as dramatically different between the sexes, and there is no sexually dimorphic pattern of hepatic gene expression. Nor is there a significant difference between men and women in plasma levels of Lp(a). However, plasma levels of Lp(a) in humans are significantly affected by the circulating levels of GH (for review, see reference 35). Acromegalics have elevated plasma Lp(a) levels, and successful treatment of this disorder is associated with a decrease in plasma apo(a) levels proportional to the reduction in GH.16 15 36 GH administration to adult individuals with GH deficiency result in a 30% to 200% increase in plasma Lp(a) levels.32 37 A significant increase in plasma apo(a) level also occurs when GH is given to normal adults.38
Many of the biological effects of GH are mediated via IGF-1, but that
is not true for the apo(a)-elevating effect of this hormone in mice (or
men). Although recombinant hGH treatment was associated with detectable
increase in hepatic IGF-1 mRNA levels, only a modest increase in the
plasma levels of IGF-1 was seen in the YAC-apo(a) transgenic mice; it
may be that mice develop tolerance to effects of the rhGH as has been
proposed previously.28 IGF-1 treatment of YAC-apo(a)
transgenic mice resulted in a significant fall in plasma apo(a) levels,
which mirrors the direction of effect of IGF-1 on plasma levels of
Lp(a) in humans. Recombinant IGF-1 (80 µg ·
kg-1 · d-1)
administration to adult men for 1 week results in a 10% reduction
in plasma apo(a) levels.19 More prolonged treatment of
Laron dwarfs (who are resistant to the effects of GH so have
high plasma levels of GH and low levels of IGF-1 in their plasma)
results in a much more pronounced (66%) reduction in plasma levels of
Lp(a),21 which may be due to the lower initial plasma
levels of IGF-1 in these individuals. IGF-1 is also effective at
lowering plasma Lp(a) levels in GH-deficient subjects, who also have
low IGF-1 levels.20 Thus the fall in plasma Lp(a) levels
with IGF-1 treatment must be independent of its it effect on reducing
the plasma levels of GH.
Our finding that oophorectomy was not associated with any increase in the plasma level of Lp(a) is similar to what was previously reported for the other strain of YAC-apo(a) transgenic mice.9 In the mouse, plasma GH levels are not significantly affected by oophorectomy, which is in contrast to the situation in humans.39 Postmenopausal women tend to have higher plasma levels of Lp(a),40 and administration of oral estrogen to humans is associated with a fall in IGF-1, increase in GH,41 and reduction in plasma apo(a) levels.42 These observations taken together make it unlikely that the reduction in plasma Lp(a) associated with estrogen therapy in humans is due to the effects of the hormone on the GH/IGF-1 axis.
Based on the results of our studies, we would predict that if the
YAC-apo(a) transgenic mice were crossed with mice expressing a GH
transgene,43 the resultant mice that express both
transgenes would have high plasma levels of apo(a). It would be
interesting to cross the YAC-apo(a) transgenic mice with mice in which
the STAT5b gene has been inactivated
(Stat5b-/-) because STAT5b has been
implicated as being the transcription factor that mediates the effect
of a pulsatile GH pattern on hepatic gene expression in
rodents.33 44 The male
Stat5b-/- mice have higher hepatic
levels of many of the mRNA species that are low in wild-type males,
including the CYP15/2A4, prolactin receptor, and CYP6B/3A mRNAs. The
levels of these mRNA transcripts in the male
Stat5b-/- mice more closely resemble
the levels seen in wild-type females.44 It would be
expected that the expression levels of apo(a) would be higher in
the male mice
YAC-apo(a)+/-;Stat5b-/-.
Recently a YAC-apo(a) transgenic rabbit was developed using the same construct used to generate one of the mice lines.45 Interestingly, the plasma levels of apo(a) do not fall at puberty in the male YAC-apo(a) transgenic rabbits. Unlike rats and mice, rabbits do not have sexually differentiated livers, which again is consistent with the puberty-associated reduction in plasma apo(a) levels in the YAC-apo(a) transgenic mice being mediated by the rodent- and male-specific pattern of GH secretion rather than solely as a direct effect of testosterone.
Finally, the results of these studies demonstrate that changes in apo(a) gene transcription [or apo(a) mRNA stability] have significant effects on the plasma level of apo(a). These mice provide a much needed animal model that can be used to define the sequences that regulate the expression of the apo(a) gene. The mice can also be used to examine the effects of potential therapeutic agents on apo(a) gene transcription, mRNA translation, and the plasma level of apo(a).
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Acknowledgments |
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We wish to thank Melissa and Tommy Hyatt for excellent technical assistance. We thank Bo Angelin for helpful conversations. This work was supported by NIH HL47619 and HL20948 and the Perot Family Fund.
Received December 16, 1998; accepted February 19, 1999.
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