Download Effect of ovarian hormones on mitochondrial enzyme activity in the

Am J Physiol Endocrinol Metab
281: E803–E808, 2001.
Effect of ovarian hormones on mitochondrial enzyme
activity in the fat oxidation pathway of skeletal muscle
S. E. CAMPBELL AND M. A. FEBBRAIO
Exercise Physiology and Metabolism Laboratory, Department of Physiology,
The University of Melbourne, Parkville, Victoria 3010, Australia
Received 18 December 2000; accepted in final form 8 May 2001
the ovarian hormones exert
significant metabolic effects, particularly in lipid metabolism (11, 18–20, 31). Accordingly, treatment of
male rats with estradiol (E2) significantly increased
lipid availability during submaximal exercise (19).
Furthermore, treatment of ovariectomized female rats
with E2 stimulated fatty acid oxidation during prolonged exercise, whereas treatment with E2 and progesterone (Prog) in combination inhibited this oxidative effect (11). The mechanisms underlying these
effects remain poorly understood; however, insight into
these functional differences might be gained by examining some of the key enzymes of the major pathways
for lipid metabolism in skeletal muscle. Enzymes representative of fatty acid flux in the mitochondria [carnitine palmitoyltransferase I (CPT I)], the ␤-oxidation
pathway [␤-3-hydroxyacyl-CoA dehydrogenase (␤-HAD)],
and the tricarboxylic acid cycle [citrate synthase (CS)]
were chosen for analysis, as these enzymes are important in the lipid oxidation pathways.
CPT I is considered to be the rate-limiting step in the
oxidation of long-chain fatty acids (24), and in the liver
its activity has previously been demonstrated to be
influenced by estrogens (39). Although skeletal muscle
and liver CPT I isozymes have exhibited many similarities, tissue-specific differences have been found in
molecular masses (40) and sensitivity to inhibition by
malonyl-CoA (26); therefore, further investigation of
the effect of the ovarian hormones on skeletal muscle
CPT I is warranted. Similarly, ␤-HAD is an important
enzyme in the ␤-oxidation pathway in skeletal muscle
(13), and its activity has been correlated with muscle
fatty acid oxidative capacity (27). ␤-HAD activity is
known to be altered in response to dietary manipulation and by exercise; the basis of this control is believed
to be, in part, through substrate availability (12).
Given that E2 seems to increase lipid availability (19,
31), it is possible that it may have some effect on
␤-HAD activity. Conversely, CS is thought to be a key
regulatory enzyme in the tricarboxylic acid cycle, and
thus of oxidative metabolism (23). Once through the
␤-oxidation pathway, fatty acid moieties enter the tricarboxylic acid cycle for further biochemical processing. Interestingly, Holt and Rhe (14) demonstrated
that both ␤-HAD and CS activities in epithelial cells of
the rat uterus increased with E2 treatment; however,
this response was cell specific and was not seen in
stromal or myometrial cells. This differential response
demonstrates the necessity for tissue-specific assessment of the effect of E2 on enzyme activities.
The present study aimed to examine the effect of
ovariectomy, as well as E2 and Prog replacement therapies, on the maximal activity of these key enzymes for
lipid oxidation. Because the ovarian hormones have
been demonstrated to have both individual (11, 18–20)
and concerted (11, 18) metabolic effects, with Prog
often inhibiting the effects of E2 (11, 18), replacement
therapies used physiological levels of E2 and Prog separately and in combination. It was hypothesized that
the maximal activities of fat oxidation enzymes would
Address for reprint requests and other correspondence: M. A.
Febbraio, Exercise Physiology and Metabolism Laboratory, Dept. of
Physiology, The Univ. of Melbourne, Parkville 3010, Australia
(E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
ovariectomy; estrogen; progesterone; lipid metabolism
IT IS WELL ESTABLISHED THAT
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Campbell, S. E., and M. A. Febbraio. Effect of ovarian
hormones on mitochondrial enzyme activity in the fat oxidation pathway of skeletal muscle. Am J Physiol Endocrinol
Metab 281: E803–E808, 2001.—To examine the roles of 17␤estradiol (E2) and progesterone (Prog) in lipid metabolism,
skeletal muscle enzyme activities were studied in female
Sprague-Dawley rats. Groups included sham-operated rats
(C) and ovariectomized rats treated with placebo (O), E2 (E),
Prog (P), both hormones at physiological doses (P ⫹ E), or
both hormones with a high dose of E2 (P ⫹ HiE). Hormone (or
vehicle only) delivery was via time-release pellets inserted at
the time of surgery, 15 days before metabolic testing. Results
demonstrated that carnitine palmitoyltransferase maximal
activity was 19, 21, and 19% lower (P ⬍ 0.01) in O, P, and
P ⫹ E rats, respectively, compared with C rats. Conversely,
activity in E and P ⫹ HiE rats was 14 and 19% higher (P ⬍
0.01) than in C. ␤-Hydroxyacyl-CoA dehydrogenase (␤-HAD)
maximal activity was 20% lower (P ⬍ 0.01) in O than in C
rats; similarly, P and P ⫹ E rats were 18 and 19% lower,
respectively (P ⬍ 0.01); however, treatment with E2 returned
␤-HAD activity to C levels. These results suggest that E2
plays a role in lipid metabolism by increasing the maximal
activity of key enzymes in the fat oxidative pathway of
skeletal muscle.
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EFFECT OF OVARIAN HORMONES ON OXIDATIVE ENZYMES
be negatively affected by the absence of the ovarian
hormones, whereas treatment with E2 would improve
their function. Prog was expected to inhibit the effect of
E2 but to have little effect on its own. This study will
help determine how the ovarian hormones affect lipid
oxidation in skeletal muscle.
MATERIALS AND METHODS
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Animals. Female Sprague-Dawley rats 12–15 wk old and
weighing 201.8 ⫾ 4.9 (SE) g were used in these experiments.
All animals were housed in a temperature-controlled room
(21 ⫾ 2°C) with a 12:12-h light-dark cycle. Water was available ad libitum, and rats were given 20 g of standard rat
chow/day to control food intake. The amount of rat chow
administered daily was selected on the basis of pilot work
from our laboratory that demonstrated that food intake of
10% of body weight was sufficient to maintain body weight
over the duration of the experiment. This experiment was
approved by the Animal Research Ethics Committee of the
University of Melbourne.
Experimental design. Rats were bilaterally ovariectomized
or sham operated under sodium brietal anesthesia (60 mg/kg
ip). Groups of ovariectomized rats were treated immediately
with time-release hormone pellets (Innovative Research of
America) inserted subcutaneously, with either E2 (2.5 ␮g/
day; E), Prog (1.5 mg/day; P), both hormones at the same
doses (P ⫹ E), or both hormones with pharmacological levels
of E2 (25 ␮g/day; P ⫹ HiE). Groups of intact (C) and ovariectomized (O) rats were treated with vehicle-only placebo pellets. Animals were treated for 14 full days postoperation
before metabolic testing. Efficacy of the ovariectomies and
sex steroid treatments was confirmed by plasma E2 and Prog
RIA kits (Diagnostic Products) at the end of the treatment
period.
Metabolic testing. Animals remained sedentary during the
14-day treatment period. Rats were fasted for 12 h before the
commencement of metabolic testing. On the morning of day
15, animals were effectively suffocated with CO2 (80:20,
CO2-O2), rendering them unconscious in ⬍20 s. A midline
incision was made, and the diaphragm was cut to ensure
death. A cardiac puncture was performed, the blood was spun
at 7,000 g for 2 min, and the plasma was removed and stored
at ⫺80°C for analysis of the sex steroids, insulin, free fatty
acid (FFA), and glycerol. The muscles of the hindlimb were
rapidly exposed, the gastrocnemius was removed and dissected into white and red portions, and all visible connective
tissue was removed. The red and white muscles were each
divided into the following two portions: the first was placed
on ice in precooled buffer for immediate isolation of mitochondria and determination of CPT I activity, and the second was
frozen in liquid nitrogen for later analysis of CS and ␤-HAD.
Isolation of intact mitochondria. The mitochondrial isolation method was as previously described by Jackman and
Willis (15), and the entire procedure was performed at 0–4°C.
The buffer solutions were as follows: solution I, 100 mM KCl,
40 mM Tris-methane (Tris 䡠 HCl), 10 mM Tris base, 5 mM
MgCl2, 1 mM EDTA, and 1 mM ATP, pH 7.4; solution II, 100
mM KCl, 40 mM Tris 䡠 HCl, 10 mM Tris base, 1 mM MgSO4,
0.1 mM EDTA, 0.2 mM ATP, and 1.5% fatty acid-free BSA,
pH 7.4; solution III, same as solution II without BSA; solution
IV, 220 mM sucrose, 70 mM mannitol, 10 mM Tris 䡠 HCl, and
1 mM EDTA, pH 7.4.
Muscle samples (⬃50–70 mg wet muscle) were weighed,
finely minced, gently homogenized with a hand-driven allglass homogenizer in 20 vol/wt of solution I, and centrifuged
at 700 g for 10 min. The supernatants were collected and
further spun at 14,000 g for 10 min. The mitochondrial
pellets were gently resuspended in 10 vol/wt of solution II
and centrifuged at 7,000 g for 10 min. This pellet was resuspended in 10 vol/wt of solution III and spun at 3,500 g for 10
min. Finally, the pellet was resuspended in 1 vol/wt of solution IV, gently rendered homogenous, and subsequently kept
on ice for enzymatic analysis. This procedure extracts mainly
subsarcolemmal mitochondria that contain very low levels of
contaminants, as previously described (2).
CPT I activity. CPT I (EC 2.3.1.21) activity measurements
were carried out with the sensitive forward radioisotope
assay, as previously described (25) and modified by Starritt
et al. (37). Briefly, this assay measured the amount of labeled
palmitoyl-L-carnitine formed when both palmitoyl-CoA and
labeled L-carnitine were added to the medium surrounding
the intact mitochondria. The standard incubation mixture
contained the following in a volume of 90 ␮l: 117 mM
Tris 䡠 HCl (pH 7.4), 0.28 mM reduced glutathione, 4.4 mM
ATP, 4.4 mM MgCl2, 16.7 mM KCl, 2.2 mM KCN, 40 mg/l
rotenone, 0.5% BSA, 100 ␮M palmitoyl-CoA, and 2 mM
3
L-carnitine with 1 ␮Ci of L-[ H]carnitine. The reaction was
initiated by the addition of 10 ␮l of mitochondrial suspension
(1:3 dilution) and was stopped 6 min later by the addition of
60 ␮l of ice-cold 1 M HCl. Palmitoyl-[3H]carnitine formed
during the reaction was extracted with 400 ␮l of watersaturated butanol in a process involving a series of washes
with distilled water and subsequent centrifugations (1,000 g
for 5 min) to separate the butanol phase. Radioactivity was
measured in 100 ␮l of the butanol layer and 5 ml of liquid
scintillation cocktail. All assays were performed in duplicate
at 37°C.
CPT I activities were expressed in terms of whole muscle
(␮mol 䡠 min⫺1 䡠 kg wet muscle⫺1). This was achieved by referencing the CPT I activity in the mitochondrial suspension to
the ratio of CS activity of the intact mitochondria in the
suspension to that of the whole muscle homogenate.
Whole muscle homogenate preparation. Muscle samples
(⬃20–30 mg wet muscle) were mechanically homogenized in
50 vol/wt of a 100 mM phosphate buffer solution containing
0.5 g/l BSA, pH 7.3. The homogenates were frozen in liquid
nitrogen and freeze-thawed three times to disrupt the mitochondrial membranes.
CS and ␤-HAD activities. CS activity was measured spectrophotometrically as previously described (36). CS activity
was assayed in the whole muscle homogenate samples, in the
intact mitochondrial suspension (1:20 dilution), and in the
mitochondrial suspension after disruption of the mitochondrial membranes by three freeze-thaw cycles and the addition of 1% Triton X-100 to the assay medium. ␤-HAD was
assayed spectrophotometrically as previously described (22).
Both assays were performed in duplicate at 25°C.
Blood hormone and metabolite assays. Plasma E2, Prog,
and insulin were measured by commercially available double-antibody RIA kits (Diagnostic Products and Pharmacia &
Upjohn, respectively). Plasma glycerol was measured by a
fluorometric assay linked to NADH. Samples were deproteinated and incubated for 1 h in a solution containing the
following: 1.0 M hydrazine, 0.2 M glycine, 1 mM EDTA, 2 mM
MgCl2, 0.2 mM NAD⫹, 0.5 mM ATP, pH 9.8, and the enzymes
glycerokinase and glycerol 3-phosphate. Production of NADH
is in a one-to-one ratio with glycerol.
Statistics. All statistical comparisons were made using
one- or two-way ANOVA tables, as appropriate, with significance set at the P ⬍ 0.05 level. Specific differences were
located with Scheffé’s F-test post hoc comparison. All data
statistics were compared using the Statistica software package, and data are reported as means ⫾ SE.
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Table 1. Animal characteristics
Final body wt, g
Plasma E2, pg/ml
Plasma Prog, ng/ml
Plasma insulin, pmol/l
C
(n ⫽ 10)
O
(n ⫽ 11)
E
(n ⫽ 9)
P
(n ⫽ 9)
P⫹E
(n ⫽ 9)
P ⫹ HiE
(n ⫽ 10)
216 ⫾ 5
36.3 ⫾ 3.7b–f
35.6 ⫾ 4.7b,c
45.4 ⫾ 3.2d*
209 ⫾ 5
16.9 ⫾ 0.9a,c,e,f
9.5 ⫾ 1.0a,d–f
54.7 ⫾ 3.2
212 ⫾ 5
46.0 ⫾ 3.4a,b,d–f
10.3 ⫾ 0.7a,d–f
45.2 ⫾ 3.4d
210 ⫾ 6
16.6 ⫾ 0.8a,c,e,f
45.8 ⫾ 1.5b,c
58.1 ⫾ 3.1a,c,e,f
212 ⫾ 7
50.4 ⫾ 4.6a–d,f
49.6 ⫾ 1.9b,c
45.9 ⫾ 1.9d
208 ⫾ 8
1,594 ⫾ 90a–e
52.3 ⫾ 3.5b,c
44.9 ⫾ 1.6d
Values are means ⫾ SE; n, no. of animals. C, intact placebo group; O, ovariectomized rats treated with placebo; E, ovariectomized estradiol
(E2)-treated group; P, ovariectomized progesterone (Prog)-treated group; P ⫹ E, ovariectomized E2 ⫹ Prog-treated group; P ⫹ HiE,
ovariectomized high E2 (pharmacological) ⫹ Prog. P ⬍ 0.05 compared with C (a), O (b), E (c), P (d), P ⫹ E (e), and P ⫹ HiE (f). * P ⬍ 0.05,
main effect of exercise compared with rest.
RESULTS
DISCUSSION
This study demonstrated that the ovarian sex steroids participate in the control of the maximal activity
of several key enzymes of lipid oxidation in both red
and white skeletal muscle. Ovariectomy decreased
CPT I and ␤-HAD enzyme activities to below control
levels. Maximal activities of these enzymes were restored by treatment with E2 (E) but not by treatment
with Prog (P). Interestingly, the combination of E2 and
Prog (P ⫹ E) produced similar maximal activities to
those of ovariectomized animals, demonstrating that
Prog inhibited E2 in physiological concentrations. However, this inhibitory effect was overridden in the pres-
Table 2. Maximal enzyme activities
C
O
P
P⫹E
390.2 ⫾ 4.6a,c,f
403.5 ⫾ 6.1a,c,f
E
P ⫹ HiE
Red gastrocnemius
CPT I, nmol 䡠 min⫺1 䡠 g
wet muscle⫺1
CS, ␮mol 䡠 min⫺1 䡠 g
wet muscle⫺1
␤-HAD, ␮mol 䡠 min⫺1 䡠 g
wet muscle⫺1
496.0 ⫾ 8.5b–f
410.2 ⫾ 7.0a,c,f
574.4 ⫾ 9.0a,b,d–f
614.2 ⫾ 11.3a–e
34.4 ⫾ 0.8
30.0 ⫾ 1.4f
34.0 ⫾ 1.3
34.2 ⫾ 1.3
33.3 ⫾ 0.9
36.8 ⫾ 1.4b
15.6 ⫾ 0.4b–e
12.5 ⫾ 0.8a,c,f
15.4 ⫾ 0.2b,d,e
13.6 ⫾ 0.6a,c,f
13.5 ⫾ 0.4a,c,f
15.6 ⫾ 0.4b–e
212.7 ⫾ 4.8a,c,f
204.6 ⫾ 5.8a,c,f
344.5 ⫾ 5.9a,b,d,e
White gastrocnemius
CPT I, nmol 䡠 min⫺1 䡠 g
wet muscle⫺1
CS, ␮mol 䡠 min⫺1 䡠 g
wet muscle⫺1
␤-HAD, ␮mol 䡠 min⫺1 䡠 g
wet muscle⫺1
255.1 ⫾ 6.9b–f
206.5 ⫾ 3.4a,c,f
320.4 ⫾ 7.2a,b,d,e
11.5 ⫾ 1.0
10.0 ⫾ 0.6
11.6 ⫾ 0.8
10.8 ⫾ 0.8
11.5 ⫾ 0.8
3.5 ⫾ 0.2
2.9 ⫾ 0.3f
3.7 ⫾ 0.2
3.3 ⫾ 0.2f
2.9 ⫾ 0.1f
12.9 ⫾ 0.8
4.4 ⫾ 0.2b–e
Values are means ⫾ SE. CPT I, carnitine palmitoyltransferase; CS, citrate synthase; ␤-HAD, ␤-hydroxyacyl-CoA dehydrogenase. P ⬍ 0.05
compared with C (a), O (b), E (c), P (d), P ⫹ E (e), and P ⫹ HiE (f).
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Characteristics of experimental animals. The animal
characteristics are presented in Table 1. Plasma E2
and Prog concentrations confirmed the efficacy of the
ovariectomies and sex steroid treatments. No significant differences were found among the mean values of
either initial or final body weight. Plasma insulin values were significantly elevated in the P rats (P ⬍ 0.05)
when compared with C, E, P ⫹ E, and P ⫹ HiE rats.
There were no differences in insulin levels among any
of the other treatment groups. Additionally, there were
no significant differences in plasma FFA or glycerol
values among any of the six groups of rats (Fig. 1).
Enzyme activities. Enzyme activities are presented
in Table 2. In red gastrocnemius, CPT I maximal activity was 19, 21, and 19% lower (P ⬍ 0.01) in O, P, and
P ⫹ E rats, respectively, compared with C rats. Activity in E and P ⫹ HiE rats was 14 and 19% higher (P ⬍
0.01), respectively, compared with C. Findings were
similar in white gastrocnemius, with CPT I maximal
activity 19, 17, and 19 lower (P ⬍ 0.01) in O, P, and
P ⫹ E rats, respectively, than C rats, and E and
P ⫹ HiE rats were 20 and 25% higher (P ⬍ 0.01),
respectively. Results indicated no between-group differences in CS maximal activity in either red or white
gastrocnemius at physiological concentrations; however, CS maximal activity in red gastrocnemius was
19% higher (P ⬍ 0.02) in P ⫹ HiE compared with O.
Maximal activity of ␤-HAD was 20% lower (P ⬍ 0.01)
in O than in C rats in red gastrocnemius. ␤-HAD
activity was also reduced in P and P ⫹ E rats (18 and
19% lower, respectively, P ⬍ 0.01) compared with C
rats; however, treatment with E2 (E) returned ␤-HAD
activity to C levels. Interestingly, pharmacological
doses of E2 restored levels to normal, despite the presence of Prog, as the P ⫹ HiE group was 19% (P ⬍ 0.01)
higher than the P ⫹ E group. No significant differences
were found in ␤-HAD maximal activity in the white
gastrocnemius in physiological concentrations of E2;
however, pharmacological levels of E2 increased the
maximal activity of ␤-HAD to at least 25% above (P ⬍
0.01) O, P, and P ⫹ E groups.
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ence of pharmacological concentrations of E2
(P ⫹ HiE). These data suggest that the ovarian hormones are capable of altering the capacity for skeletal
muscle to oxidize FFAs. Although this study did not
directly measure fatty acid oxidation, E2 has previously been demonstrated to increase fat oxidation during exercise in ovariectomized rats, whereas E2 and
Prog together had no effect (11). Hence the alteration
in CPT I and ␤-HAD maximal activity observed in the
present study provides a possible mechanism for these
changes in oxidative capacity. Of note, the ␤-HAD
enzyme measured in this study catalyzes the oxidation
of short-chain fatty acids, whereas CPT I is involved in
long-chain fatty acid oxidation. Because both enzymes
were affected by ovariectomy and hormone replacement therapy, this suggests that the ovarian hormones
can influence fat oxidation regardless of chain length.
Accordingly, the absence of E2 has recently been demonstrated to negatively affect the activity of very long-,
long-, and medium-chain acyl-CoA dehydrogenases in
the liver (28), indicating the E2 has a similar effect on
all of the acyl-CoA dehydrogenase enzymes, regardless
of the fatty acyl chain length they bind.
By controlling the food intake, and hence weight
gain, of the rats in this study, we maintained similar
plasma FFA and glycerol levels across all treatment
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Fig. 1. Plasma free fatty acid (FFA; A) and glycerol (B) levels in
intact and ovariectomized animals treated with sex steroids after 30
min of rest or exercise. C, sham-operated rats; O, ovariectomized rats
treated with placebo; E, rats given estrogen; P, rats given progesterone; P ⫹ E, rats given both hormones at physiological doses; P ⫹ HiE,
rats given progesterone and a high dose of estrogen. *Main effect
(P ⬍ 0.05) of exercise compared with rest.
groups. Additionally, with the exception of P rats,
plasma insulin was also similar between treatment
groups. Although previous studies have found differences in these parameters (5, 30), these groups did not
control food intake and therefore had significant differences in weight gain, which is likely to affect insulin
homeostasis. Insulin is a known inhibitor of lipolysis
(41); thus, differences in plasma insulin levels would
result in differences in plasma FFA and glycerol values. Of note, short-term administration (5 days) of E2
to male rats also resulted in elevated plasma FFA
concentrations, with no change in body weight (8).
Unfortunately, this study did not measure insulin levels, which can also be affected by short-term administration of the ovarian hormones (9), so it is unknown if
this effect was independent of changes in plasma insulin. Furthermore, because of differences in receptor
populations, males can respond differently to E2 administration (10), which could also account for the
discrepancy in these results. Interestingly, despite elevated insulin levels in Prog-treated rats, plasma FFA
and glycerol levels were not different from those of
other groups, indicating that the insulin sensitivity
had not yet become severe enough to alter lipid availability. This suggests that controlling for diet may help
attenuate the negative effects of ovariectomy, or treatment with Prog, on insulin homeostasis and lipolysis,
at least in the short term. This is not too surprising,
given that the first intervention in the treatment of
type II diabetes includes diet regulation.
Results from this study demonstrate that the ovarian hormones can alter the activity of CPT I; however,
it is interesting to note that a recent study has reported
no gender differences in this parameter (1). Although
these results may at first seem paradoxical, several
factors must be considered. First, there is no mention
of the menstrual cycle by these authors, so it is unknown whether this variable was controlled for in their
female subjects. In the present study, we observed that
the ovarian hormones had divergent effects on the
activity of CPT I and that these effects varied depending on their relative concentrations. Both the absolute
and relative concentrations of estrogen and Prog
change throughout the menstrual cycle and could
therefore alter the activity of CPT I. Thus, if the menstrual cycle was not controlled for, this could result in
a high variability that could mask the subtle effects of
the ovarian hormones. Additionally, testosterone is
also a steroid hormone that is capable of altering muscle metabolism and hence should be considered in
gender comparisons as well. Therefore, although no
gender differences were observed (1), this does not
exclude the possibility of the ovarian hormones altering the activity of CPT I. Finally, differences in the
regulation of lipid metabolism in the rat compared
with human have previously been observed (35); thus,
rat CPT I may respond differently to the ovarian hormones. Hence, interspecies differences remain a limitation in this study.
A possible mechanism by which the ovarian hormones alter the activities of CPT I and ␤-HAD is by
EFFECT OF OVARIAN HORMONES ON OXIDATIVE ENZYMES
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of skeletal muscle to oxidize lipids, particularly in
times of metabolic stress.
The importance of matching cellular fatty acid utilization rates to energy demand is emphasized by the
dramatic consequence of genetic defects that result in a
limited capacity for fatty acid oxidation. Clinical symptoms of mitochondrial enzymatic defects can include
hypoglycemia, liver dysfunction, and cardiomyopathies
(32). Hence, alteration in lipid homeostasis by the
ovarian hormones may result in mild manifestations of
these symptoms, particularly in cases with additional
metabolic or cardiovascular complications, as are often
prevalent in aging postmenopausal women. Interestingly, women treated with tamoxifen, a potent antiestrogen, are at increased risk of developing a fatty
liver and nonalcoholic steatohepatitis (33). Furthermore, Djouadi et al. (6) have recently demonstrated
that male peroxisome proliferator-activated receptor-␣
null mice treated with etomoxir, a CPT I inhibitor,
have a 75% mortality rate compared with only 25% in
female mice. Additionally, pretreatment of the males
with E2 could reverse this effect, thereby implicating
E2 in the maintenance of energy homeostasis. Correspondingly, pathologies such as obesity or diabetes
could also be affected by altered metabolic capacity. In
fact, decreased lipid oxidation in both of these conditions has been linked to an impaired capacity of lipolytic and oxidative enzymes in skeletal muscle (4, 17,
34) and therefore may also be influenced by the ovarian
hormones. Consequently, consideration of hormonal
status (amenorrhea, pregnancy, or menopause) could
be helpful in the control of these and other metabolic
diseases.
Of particular interest in these results is the inhibition of the lipolytic effects of E2 by Prog at physiological
concentrations seen in both CPT I and ␤-HAD. This
inhibition was abolished with pharmacological concentrations of E2, demonstrating that, in high enough
concentrations, E2 can override the inhibitory effect of
Prog. This could have significant clinical effects when
considering long-term treatment of amenorrheic or
postmenopausal women with oral contraceptive or hormone replacement therapy. In these situations, it may
be prudent to treat with an estrogen-based therapy vs.
either Prog or combination therapies. Conversely, if
considering a combination hormone replacement therapy, our data suggest the utilization of elevated levels
of E2 to offset any inhibition by Prog. It is interesting to
note that similar results have been found with carbohydrate metabolism. Women using combination therapy
demonstrated poorer glucose homeostasis and insulin
sensitivity than those using estrogen alone (7, 21).
In conclusion, the absence of the ovarian hormones
in female rats resulted in a decrease in the maximal
capacity of skeletal muscle to oxidize lipids. Treatment
with E2 restores this ability to at least control levels,
whereas treatment with Prog alone has no effect. If
ovariectomized animals are treated with both E2 and
Prog in physiological concentrations, Prog inhibits the
lipolytic effect of E2, and this is only restored with
pharmacological concentrations of E2.
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upregulating their gene transcription, which would
result in changes in the protein levels of these enzymes. This could have a potent effect on the overall
activity of these enzymes, particularly when expressed
per gram of tissue wet weight. Indeed, increases in
hepatic CPT I activity as a result of dehydroepiandrosterone were found to be closely correlated to increases
in transcription rates and mRNA concentration (3).
Dehydroepiandrosterone is a precursor for steroid hormone synthesis and can result in elevated estrogen or
testosterone levels. Additionally, the estrogen-signaling pathway has recently been recognized to play a
pivotal role in constitutive hepatic expression of genes
involved in ␤-oxidation (6, 28). Both the estrogen and
Prog receptors are present in skeletal muscle (16);
hence, this is a viable pathway for the ovarian hormones to exert their metabolic effects.
Of note was the difference in the enzyme activities
between the P ⫹ E group, which received physiological
concentrations of both E2 and Prog, and the intact C
rats. This may best be explained by the fact that the
intact rats were cycling normally and were therefore
exposed to elevated Prog levels (40–50 ng/ml) for only
⬃12 h during the proestrous phase of their 4- to 5-day
cycle (38). Furthermore, control rats were killed at
various times throughout their estrous cycle, so that a
more representative collection of data points could be
obtained; therefore, these pooled results incorporate
the changing levels of the ovarian hormones. Conversely, the P ⫹ E rats were exposed continuously to
high levels of Prog; therefore, the P ⫹ E rats would
have a greater tendency to exhibit any inhibitory characteristics of Prog.
It is tempting to associate the enhanced enzymatic
activities seen in the presence of E2 with increased
fatty acid oxidation in skeletal muscle. There is, however, some concern as to whether the maximal flux rate
in a metabolic pathway depends on the absolute activities of the enzymes that catalyze the near-equilibrium
reactions (29). Because fat oxidation was not measured
in this study, this brings into question the physiological significance of the difference in the metabolic patterns observed with different hormone therapy treatment. The lower maximal activities of the fat oxidative
enzymes would imply a lower maximal flux rate in the
O, P, and P ⫹ E animals; however, such speculations
must be treated with caution. Nonetheless, the data
help offer a possible explanation for the increase in
lipid oxidation seen with hormone replacement therapies in ovariectomized rats (11). It is interesting to note
that the effect of E2 and that of E2 ⫹ Prog on enzyme
activities seen in this study are both of a comparable
magnitude to the altered lipid oxidation data from
Hatta et al. (11). Furthermore, the activity of ␤-HAD
has recently been correlated with whole body fat oxidation during exercise in humans, suggesting that
changes in ␤-HAD activity result in changes in the
capacity for fat oxidation capacity (27). Hence, it is
likely that, by altering the maximal activity of CPT I
and ␤-HAD, the ovarian hormones influence the ability
E807
E808
EFFECT OF OVARIAN HORMONES ON OXIDATIVE ENZYMES
We acknowledge the advice of Lawrence Spriet and Emma Starritt.
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AJP-Endocrinol Metab • VOL
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