Keywords
mitochondria - non-alcoholic fatty liver disease - obesity - ovariectomy - reactive
oxygen species
Descritores
mitocôndrias - doença hepática gordurosa não alcoólica - obesidade - ovariectomia
- espécies reativas de oxigênio
Introduction
Menopause is associated with a higher incidence of metabolic syndrome (MS) and related
comorbidities, including obesity, insulin resistance, type 2 diabetes, dyslipidemia
and nonalcoholic fatty liver disease (NAFLD).[1]
The mechanisms involved in the pathogenesis of NAFLD and its progression to nonalcoholic
steatohepatitis (NASH) have not been fully elucidated to date, but the most accepted
is the “two-hit” hypothesis.[2] According to this paradigm, the primary abnormality (“first hit”) is most likely
insulin resistance, which leads to the accumulation of triacylglycerols (TG) within
the hepatocytes. Then, the oxidative stress established as a result of fat accumulation
represents the “second hit,” which induces hepatocyte injury and inflammation, both
factors contributing to the progression of the disease.
Therefore, in postmenopausal women, the insulin resistance can arise as an important
contributing factor to the development of NAFLD. Furthermore, through genomic actions,
estrogen exerts important effects on the liver lipid metabolism, in such a way that
in a condition of estrogen deficiency, the hepatic metabolism is diverted from lipid
oxidation to fatty acid biosynthesis, thus favoring its esterification and accumulation
as triacylglycerols (TGs) in the cytosol of hepatocytes.[3] In fact, NAFLD has been demonstrated in animal models of estrogen deficiency[3] and in postmenopausal women.[4]
A question that arises is whether livers from OVX mice preserve the capacity of buffering
fatty acids without metabolic disturbances even after a longer period of ovarian hormone
production cessation, a condition corresponding to a postmenopausal period in women.
It has long been known that estrogen functions as an in vitro and in vivo antioxidant,
and not only because of its phenolic chemical structure. In the liver, estrogen can
bind to estrogen receptors[5] and reduce the mitochondrial reactive oxygen species (ROS) generation.[6]
[7] In addition, estrogen can up-regulate the nuclear expression and/or the activity[6]
[7]
[8] of several antioxidant enzymes. Thus, in a condition of estrogen deficiency, an
oxidative status could arise as result of higher ROS generation by mitochondria and/or
peroxisomes, or decrease in the effectiveness of antioxidant systems, or both. This
could play a crucial role in the pathogenesis of a wide range of chronic and degenerative
disorders (aging, atherosclerosis, neurodegeneration, cancer, cataract) as well as
in acute clinical conditions.[9]
In this way, this work was planned to investigate the changes of the liver mitochondrial
oxidation and liver redox status in an animal model of estrogen deficiency. The OVX
mice constitute a very suitable model of estrogen deficiency, which is why they were
used in this work. This could be helpful to a better understanding of the pathogenesis
of diseases as well as to open new perspectives for therapeutic approaches targeting
these antioxidant enzymes.
Methods
Material
Substrates adenosine triphosphate (ATP), adenosine diphosphate (ADP), phenylmethylsulfonyl
fluoride (PMSF), o-phthalaldehyde (OPA), horseradish peroxidase, 2',7'-dichlorofluorescein diacetate
(DCFH-DA), 2',7'-dichlorofluorescein (DCF), reduced glutathione (GSH) and oxidized
glutathione (GSSG) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Animals
Female Swiss CD1 mice weighing 25–30 g (7 weeks of age) were randomly assigned to
sham-operation (SHAM/control) or bilateral ovariectomy. For surgery, the mice were
anesthetized with a mixture of ketamine-xylazine (50–10 mg/kg, intraperitoneal [IP].),
and their body weight was monitored each week. The mice were fed ad libitum with a standard laboratory diet (Nuvilab, São Paulo, SP, Brazil) for 10 weeks. All
procedures performed in this study were conducted in strict adherence to the guidelines
of the Ethics Committee for Animal Experimentation of the University of Maringá (079/2008).
Analysis of Serum Lipid Profile and Glycemia
The mice were fasted for 12 h and the blood was collected by cardiac puncture. Plasma
glucose concentrations were determined using a glucose analyzer (Optium). Total cholesterol
and triacylglycerols (TGs) were analyzed by standard methods (Gold Analisa Diagnóstica
Ltda., Belo Horizonte, MG, Brazil). Very low-density lipoprotein (VLDL) cholesterol
was calculated from TG levels (Friedewald equation) and low-density lipoprotein (LDL)
cholesterol was determined by subtracting high-density lipoprotein (HDL) and VLDL
from the total cholesterol.
Liver Determination of the Total Lipid Content
The total lipid content of the liver was measured by gravimetry and expressed in grams
per 100 g of wet liver weight following the method described by Folch et al.[10]
Isolation of Mitochondria and Peroxisomes
Mitochondria and peroxisomes from female mice livers were isolated by differential
centrifugation according to Bracht et al[11] and Natarajan et al,[12] respectively.
Determination of Protein Contents
Protein contents of the mitochondrial, peroxisomes and homogenate suspensions were
measured using the method of Lowry et al[13] and using bovine serum albumin as a standard.
Determination of Oxygen Uptake by Isolated Mitochondria Oxidizing Fatty Acids
Oxygen uptake by intact mitochondria was determined polarographically. The reaction
was initiated by the addition of (a) 20 μM palmitoyl-L-carnitine, (b) 20 μM palmitoyl-CoA + 2.0 mM
L-carnitine, or (c) 20 μM octanoyl-CoA + 2.0 mM L-carnitine to mitochondrial suspensions
(0.6–1.2 mg/ml).[14]
Membrane-bound Enzymatic Activities
Freeze-thawing disrupted mitochondria were used as enzymes sources to evaluate the
membrane-bound enzymatic activities. Nicotinamide adenine dinucleotide plus hydrogen
(NADH)-oxidase and succinate-oxidase activities were assayed polarographically.[11]
Liver Perfusion Experiments
The non-recirculating perfusion technique with Krebs-Henseleit-bicarbonate buffer
described by Scholz and Bucher[15] was used. For the surgical procedure, the animals were anesthetized by IP injection
of ketamine-xylazine (50–10 mg/kg). The perfusion flow was constant in each individual
experiment, and it was adjusted to 10 ml/min depending on the liver weight. The livers
of 24-hour fasted mice were used for the measurement of fatty acid oxidation. A mixture
of octanoate (0.3 mM) plus [1-14C] octanoate (0.01 μCi/ml) and 50 μM fatty acid-free bovine serum albumin was dissolved
in the perfused fluid.
Analytical
The oxygen concentration in the venous perfusate was continuously monitored with a
Teflon-shielded platinum electrode. β-hydroxybutyrate and acetoacetate were measured
by standard enzymatic procedures.[16] The carbon dioxide production was measured by trapping 14CO2 in phenylethylamine. The radioactivity was measured by liquid scintillation spectroscopy.
The following liquid scintillation solution was used: toluene/ethanol (2/1) containing
5 g/L of 2.5-diphenyloxazole (PPO) and 0.15 g/L of 2.2-p-phenylene-bis (5-phenyloxazole)
(POPOP). Metabolic rates were calculated from input–output differences and the total
flow rates were referred to the weight of the liver.
Determination of Reduced Glutathione (GSH) and Malondialdehyde (MDA) Contents
The GSH and MDA levels were measured in liver homogenates and isolated mitochondria.
To prepare the homogenates, samples of the livers were freeze-clamped with liquid
nitrogen and homogenized in a cold medium containing 250 mM of sucrose, 1 mM of ethylene
glycol tetraacetic acid (EGTA) and hydroxyethyl piperazine ethanesulfonic (HEPES)
10 mM pH 7.2 using a Van-Potter homogenizer (1:4, w:v).
The GSH levels were determined fluorimetrically, according the method described by
Hissin and Hilf.[17] The results were expressed as µg GSH/mg protein present in the supernatant. The
MDA concentrations were measured by direct spectrophotometry, using the thiobarbituric
acid reactive substances(TBARS) method, considered a biomarker of lipid peroxidation
and oxidative stress.[18] The results were expressed as nmol MDA/mg protein using a molar extinction coefficient
for MDA of 1.56 × 105 M−1 × cm−1.
Determination of Mitochondrial Hydrogen Peroxide (H2O2) Generation
The mitochondrial H2O2 formation was monitored with 2',7'-dichlorofluorescein diacetate (DCFH-DA). The results
were expressed as pmol dichlorofluorescein (DCF) produced/min/mg protein.[19]
Determination of Peroxisomal Hydrogen Peroxide (H2O2) Generation
The H2O2 generated by the peroxisomal oxidation of palmitoyl-CoA was measured fluorimetrically.[20] The results were expressed as pmol of DCF produced/min/mg protein.
Determination of Antioxidant Enzymes Activities
The chymotrypsin (CT) activity in the peroxisomal factions was assayed spectrophotometrically,
according to Aebi.[21] The enzymatic activity was expressed as μmol H2O2 reduced/min/mg of peroxisomal protein using the coefficient of molar extinction of
H2O2 of 0.036 mM−1 × cm−1.
The activities of the other antioxidant enzymes (cytosolic superoxide dismutase [SOD1],
glutathione peroxidase [GSH-Px], Glutathione reductase [GSSG-Red] and glucose-6-phosphate dehydrogenase activity [G6PD]) were measured in homogenates, using the Dounce homogenizer, prepared from
livers of overnight fasted mice. The activity of the antioxidant enzymes was determined
in the supernatant after a 30,000 × g centrifugation, for 10 minutes. In the case of superoxide dismutase (SOD), in addition
to the SOD1, the mitochondrial SOD (SOD2) activity was assessed in the supernatant
(6,000 × g) of sonicated mitochondrial fractions. The procedure described below was adopted
for both.
Superoxide dismutase:
The SOD activities were determined,[22] and the enzymatic activity was expressed as U of superoxide dismutase/mg protein
Glutathione peroxidase:
The glutathione peroxidase activity was measured according to Paglia and Valentine.[23] The enzymatic activity was expressed as nmol of NADPH oxidized/min × mg protein
using the molar extinction coefficient, ε=6.22 mM−1 × cm−1.
Glutathione reductase:
Glutathione reductase activity was determined according to Mize and Langdon.[24] The enzymatic activity was calculated as nmol of NADPH oxidized/min × mg protein.
Glucose-6-phosphate dehydrogenase activity:
The G6PD activity was determined spectrophotometrically.[25] To obtain the accurate enzyme activities for G6PD and PGD, both PGD activity alone
and total dehydrogenase activity (G6PD + PGD) were measured. The G6PD activity was
expressed as nmol of NADPH produced/min × mg protein using the molar extinction coefficient,
ε = 6.22 mM−1 × cm−1.
Treatment of Data
The data in the figures and tables are expressed as means ± standard error (SEM).
The significant differences among the means were identified by the Student t-test
and two-way analysis of variance (ANOVA) followed by a Newman-Keuls posttest. The
results are given in the text as probability values (P). A p-value < 0.05 was adopted as a criterion of significance. The statistical analysis
was performed by means of GraphPAD Software programs (GraphPAD Software, La Jolla,
CA, USA).
Results
Body Weight and Fasting Plasma and Serum Profiles
As confirmation of successful ovariectomy-induced suppression of endogenous estrogen
production, at 10 weeks after operation, the OVX mice exhibited an evident uterine
atrophy, with uterine/BW ratios 4 times minor than SHAM operated animals (placebo
surgery) ([Table 1]). In the OVX mice, the body weight was also 42.5% higher, and this was accompanied
by increased adiposity, as can be seen by the differences in the periuterine adipose
tissue (PUAT) to body weight ratios of these animals (+ 109%). The liver to body weight
ratio was also 8.7% higher in OVX mice. This result was corroborated by the quantification
of the total liver lipid content by gravimetry. [Table 2] depicts the plasma dosages of these animals. Although no differences have been noted
in the food intake between these two animal groups ([Table 2]), the fasting plasma glucose levels were significantly elevated in the OVX mice,
as well as the total cholesterol and LDL cholesterol levels ([Table 2]). Average fasting TGs, HDL and VLDL cholesterol levels were not significantly different
between the groups. Total lipids contents were also evaluated ([Table 2]). Although livers from control groups exhibited normal total lipid content of ∼
5.8%,[26] the livers from OVX mice presented significantly higher lipid contents (+61%), characterizing
extensive hepatic steatosis.
Table 1
Body weight and tissue weight changes of female SHAM and ovariectomized mice 10 weeks
after operation
|
Variable
|
SHAM (n = 6)
|
OVX (n = 6)
|
|
Glucose (mmol/L)
|
4.79 ± 0.27
|
6.30 ± 0.71*
|
|
Total cholesterol (mmol/L)
|
2.93 ± 0.30
|
5.83 ± 0.65*
|
|
HDL cholesterol (mmol/L)
|
1.53 ± 0.33
|
1.83 ± 0.23
|
|
LDL cholesterol (mmol/L)
|
1.29 ± 0.21
|
3.88 ± 0.63*
|
|
VLDL cholesterol (mmol/L)
|
0.11 ± 0.04
|
0.12 ± 0.03
|
|
Triacylglycerols (mmol/L)
|
0.56 ± 0.19
|
0.63 ± 0.16
|
|
Liver total lipid content (%)
|
5.59 ± 0.78
|
8.06 ± 0.48*
|
Abbreviations: HDL, high-density lipoprotein; LDLD, low-density lipoprotein; VLDL,
very low-density lipoprotein; OVX, ovariectomized
Values are means ± standard error of the mean (SEM).
*p < 0.05 compared with SHAM values as identified by Student t-test.
Table 2
Physiological measurements of female SHAM and ovariectomized mice 10 weeks after operation
|
Variable
|
SHAM (n = 15)
|
OVX (n = 15)
|
|
Body weight (g)
|
34.66 ± 3.73
|
49.40 ± 3.39*
|
|
Food intake (g/day/kg BW)
|
186.485 ± 21.77
|
160.369 ± 15.39
|
|
Uterus/BW (%)
|
0.39 ± 0.07
|
0.094 ± 0.06*
|
|
Liver/BW (%)
|
3.30 ± 0.23
|
3.59 ± 0.15
|
|
PUAT/BW (%)
|
2.61 ± 0.38
|
5.46 ± 0.90*
|
Plasma measures of triglycerides, total cholesterol, HDL-cholesterol, LDL-cholesterol,
VLDL-cholesterol and glucose from SHAM and OVX mice, fasted overnight, were analyzed
by standard methods as described in the section of Materials and Methods. Liver fragments
from the SHAM and OVX mice were homogenized in a 2:1 chloroform–methanol mixture to
determine the liver total lipid contents by gravimetric analyses. Values are means ± standard
error of the mean (SEM).
*p < 0.05 compared with SHAM values as identified by the Student t-test
Effects of Estrogen Deficiency in Isolated Mitochondria
To evaluate the effects of estrogen deficiency on mitochondrial β-oxidation of fatty
acids, the medium chain fatty acid octanoate and long-chain fatty acid palmitate were
used. In this series, these fatty acids were utilized as acyl CoA derivatives (octanoyl-CoA,
palmitoyl-CoA) in the presence of carnitine, and in another series, palmitoyl-L-carnitine
was used. The oxygen uptake due to oxidation of these substrates was measured in the
presence of 2,4-dinitrophenol (DNP); under these conditions, the rate of β-oxidation
occurs at its maximum speed. Thus, the effects of estrogen deficiency on fatty acid
transport and the oxidizing capacity of intra-mitochondrial enzymes could be evaluated.
[Fig. 1] shows that the β-oxidation was inhibited irrespective of the fatty acid used. The
oxidation of octanoyl-CoA was inhibited in OVX mice by 56.2%, and palmitoyl-CoA by
45.4%, as well as palmitoyl-carnitine, whose oxidation was inhibited in OVX mice by
∼ 45%.
Fig. 1 Oxygen uptake due to fatty acid oxidation in intact mitochondria uncoupled with 2,4-dinitrophenol
(DNP). The oxidation of fatty acids was measured polarographically in an incubation
medium, in the presence of DNP. The reaction was initiated by the addition of palmitoyl-L-carnitine,
palmitoyl-CoA + L-carnitine, or octanoyl-CoA + L-carnitine. The vertical bars represent
standard errors of the mean. The asterisks indicate significant differences between
the groups, SHAM (n = 6) and ovariectomized (OVX) (n = 6), as revealed by analysis of variance with Student t-test set at *p < 0.05.
The β-oxidation inhibition irrespective of the fatty acid used could be a result of
alterations in the electron transport chain. In this way, the effects of estrogen
deficiency on the activities of membrane-bound enzymes, NADH-oxidase and succinate-oxidase
were analyzed. However, the activities of these enzymes were unaffected in the livers
of OVX mice. So, the reduction in the mitochondrial β-oxidation capacity could not
be secondary to an inhibition in the respiratory chain ([Fig. 2]).
Fig. 2 Nonalcoholic steatohepatitis (NADH)-oxidase and succinate-oxidase activities in freeze-thawing
disrupted mitochondria. The vertical bars represent standard errors of the mean. The
asterisks indicate significant differences between the values in SHAM (n = 8) and ovariectomized (OVX) (n = 8) conditions as revealed by analysis of variance with Student t-test set at *p < 0.05.
Oxygen Uptake, 14CO2 Production and Ketogenesis in the Liver of Fasted Mice
To determine whether the changes observed on mitochondrial functions were reproduced
in intact livers, metabolic fluxes due to exogenous octanoate oxidation were measured
in livers from SHAM and OVX mice. In the effluent perfusion fluid, the levels of ketone
bodies (acetoacetate and β-hydroxybutyrate), CO2 production and oxygen uptake due to octanoate oxidation were measured. [Fig. 3] illustrates the time course of these experiments. After the stabilization of oxygen
uptake, samples of the outflowing perfusate were collected for the measurement of
acetoacetate and β-hydroxybutyrate. After a period of 12 minutes, the livers were
perfused with 0.3 mM of octanoate plus [1-14C]octanoate (0.01 μCi/ml) and 50 μM of fatty acid-free bovine serum albumin for 24
minutes. The onset of octanoate infusion caused increases in oxygen uptake, β-hydroxybutyrate,
acetoacetate and 14CO2 production.
Fig. 3 Time course of the changes in metabolic flux due to oxidation of octanoate in perfused
livers from fasted mice. 0.3 mM octanoate, [1-14C]octanoate (0.01 μCi/ml) plus 50 μM fatty acid-free bovine serum albumin were infused
at 12–36 min of perfusion. Oxygen uptake and 14CO2 production (panel A) and β-hydroxybutyrate and acetoacetate production (panel B)
are reduced in the ovariectomized group (n = 4) compared with SHAM group (n = 4). Vertical bars represent mean standard errors. Asterisks indicate significant
differences between the values as revealed by variance analysis with post hoc Newman-Keuls
testing (p < 0.05).
[Fig. 3A] shows that oxygen uptake and 14CO2 production were decreased in OVX mice. At minute 36, the oxygen consumption of OVX
mice were reduced by 38.5%, and the 14CO2 production of this group was decreased by 48.7% when compared with SHAM mice. The
production of β-hydroxybutyrate and acetoacetate was also reduced; the OVX mice presented
a reduction of 46.8% in the total amount of β-hydroxybutyrate and acetoacetate ([Fig. 3B]).
General Liver Redox State
In [Table 3], the total and mitochondrial GSH and MDA contents from these animals are also presented.
from the livers of OVX mice exhibited total GSH contents 50% lower than those found
in the control mice. Similarly, in the isolated mitochondria, the GSH content also
was significantly lower in the OVX group (-21%). As expected, the MDA contents were
extensively increased in homogenate and mitochondria from OVX mice by 250% and 186%,
respectively.
Table 3
Reduced glutathione and malondialdehyde contents
|
Variable
|
SHAM (n = 5)
|
OVX (n = 5)
|
|
Homogenate GSH
|
3.19 ± 0.24
|
1.59 ± 0.64***
|
|
Mitochondrial GSH
|
0.94 ± 0.02
|
0.74 ± 0.03**
|
|
Homogenate MDA
|
4.03 ± 0.23
|
14.08 ± 1.89***
|
|
Mitochondrial MDA
|
1.50 ± 0.15
|
4.29 ± 0.45***
|
Abbreviations: GSH, reduced glutathione; MDA, malondialdehyde; OVX, ovariectomized.
Reduced GSH and MDA contents are expressed as means ± standard error of the mean (SEM)
(μg GSH/mg protein or nmol MDA/mg protein, respectively).
**p < 0.01; ***p < 0.001 vs the control. Asterisks indicate significant differences between the groups
as revealed by Student t-test.
Mitochondrial and Peroxisomal H2O2 Generation
The results of the experiments of measurements of H2O2 production by isolated liver mitochondria and peroxisomes of OVX and control mice
are presented in [Fig. 4]. As it can be seen, the mitochondrial H2O2 generation was ∼ 68% higher in OVX mice than in the control group. Similarly, the
peroxisomal H2O2 production, during the oxidation of palmitoyl-CoA, was significantly higher in OVX
mice, by 14%, as compared with the control group.
Fig. 4 Mitochondrial and peroxisomal hydrogen peroxide (H2O2) generation. Mitochondrial and peroxisomal H2O2 generation from SHAM (n = 6) and OVX (n = 6) mice was measured fluorometrically. The results are expressed as pmol 2,4-dinitrophenol
(DCF) produced/min × mg protein. The results are means ± standard error of the mean
(SEM) and asterisks indicate significant differences between the groups as revealed
by analysis of variance with Student t-test set at *p < 0.05.
Antioxidant Enzyme Activities
Considering the high levels of lipoperoxidation and the restricted GSH levels found
in the livers of OVX mice, the activity of several antioxidant enzymes was also evaluated,
and the results are presented in [Table 4].
Table 4
Antioxidant enzyme activities
|
Antioxidant Enzyme (per mg protein)
|
SHAM (n = 6)
|
OVX (n = 6)
|
|
SOD1 (U)
|
1.20 ± 0.22
|
0.80 ± 0.17*
|
|
SOD2 (U)
|
1.57 ± 0.13
|
0.92 ± 0.07***
|
|
CT (µmol/min)
|
18.35 ± 1.26
|
10.7 ± 0.92**
|
|
GSSG-Red (nmol/min)
|
53.4 ± 4.29
|
32.55 ± 2.86**
|
|
GSH-Px (nmol/min)
|
173.4 ± 14.51
|
293.15 ± 30.62*
|
|
G6PD (nmol/min)
|
6.18 ± 0.56
|
4.15 ± 0.48*
|
Abbreviations: CT, chymotrypsin; G6PD, glucose-6-phosphate dehydrogenase activity; GSH-Px, glutathione peroxidase; GSSG-Red, glutathione reductase; OVX, ovariectomized;
SOD1, cytosolic superoxidase dismutase; SOD2, mitochondrial superoxidase dismutase.
The results represent the means ± standard error of the mean (SEM).
*p < 0.05; **p < 0.01; ***p < 0.001 vs the control.
Asterisks indicate significant differences between the groups as revealed by the Student
t-test.
First, the activities of cytosolic and mitochondrial SOD (SOD1 and SOD2, respectively)
were assessed in the livers of OVX and control mice. This enzyme serves as a first
antioxidant defense in the cell by catalyzing the dismutation of superoxide anions
to hydrogen peroxide. When compared with the control group, the activities of SOD1
and SOD2 in OVX mice were reduced significantly, by 33% and 41%, respectively.
The peroxisomal CT activity was reduced by ∼ 42% in the OVX group as compared with
the control mice. The activity of another H2O2-scavenger enzyme, GSH-Px, exhibited a significant increase in the livers of OVX mice,
of ∼ 40%. The activities of the two enzymes responsible for restoring the GSH system,
GSSG-Red and G6PD, are also presented in [Table 4]. As it can be seen, the activities of these enzymes were reduced in the livers of
OVX mice by 40% and 33%, respectively, as compared with the control group.
Discussion
The results of the present study revealed that OVX mice fed with a standard diet during
10 weeks after the surgery reproduce a situation comparable to a physiological state
of menopause that occurs in women in midlife. The levels of total cholesterol and
LDL-cholesterol were higher in the OVX mice than in the SHAM mice, and these animals
also exhibited increased body weight and adiposity, features that are reported in
both women and in rodents with estrogen deficiency.[26]
[27]
[28]
[29] The increased body weight could be a result of reduced energy expenditure. Rogers
et al[30] demonstrated that loss of ovarian function in mice after 12 weeks of ovariectomy
decreases energy expenditure and promotes less spontaneous physical activity during
the dark phase. Oliveira et al[29] also revealed decreased energy expenditure during light and dark periods in mice
after five weeks of ovariectomy associated with a high-fat diet.
As expected and demonstrated in the literature,[28] the livers of OVX animals exhibited a higher content of lipids than the mice from
the SHAM group, which was confirmed by gravimetry measurements. This lipid accumulation
could be the result of several factors, such as excessive food intake, alterations
in the uptake or exportation of fatty acids, changes in mitochondrial oxidation of
fatty acids and a reduction in the export of TGs. The average daily intake of food
was not different between the SHAM and OVX groups. In fact, the normalization of the
data by expressing food intake per kg of body weight revealed that OVX mice ingested
lower amounts of food than SHAM mice, indicating that lipid accumulation in OVX mice
is not a result of hyperphagia. This result also corroborates with the literature,[27]
[28]
[30]
[31] in which authors found that OVX mice gained more weight than SHAM mice despite eating
similar amounts of food, indicating an increased propensity to store energy as body
weight.
The livers of OVX mice that were fed a standard diet for 10 weeks presented a clear
modification in the fatty acid oxidative capacity. When compared with the mitochondria
isolated from the SHAM group, fatty acid oxidation was reduced irrespective of the
chain length. Moreover, no differences were found in the degree of inhibition between
the oxidation of the acyl- or carnityl derivatives of fatty acids. These results support
the view that the inhibition of fatty acid oxidation is not due to an alteration in
a distinct step of the β-oxidation of medium-chain and long-chain fatty acids. A reduction
in a common step, namely the enzymes of mitochondrial β-oxidation, the citric acid
cycle and the respiratory chain, seems to be, thus, a plausible explanation.
The lack of significant differences between the oxidation of NADH or succinate oxidation
in mitochondria from the SHAM and OVX mice groups excluded the hypothesis of a reduced
activity of the electron transport chain components from complexes I and II to IV
as a contributing factor to the inhibition of fatty acid oxidation.
The experiments performed in the perfused livers show the metabolic fluxes due to
exogenous fatty acid oxidation, and this can reveal more details not only of mitochondrial
oxidation but of other metabolic pathways linked to fatty acid oxidation in intact
cells. The reduction found in 14CO2 production from [1-14C]octanoate indicated that the activity of the citric acid cycle due to oxidation
of exogenous octanoate was decreased in the perfused livers of OVX mice. Under normal
conditions, the rate of the citric acid cycle is strictly dependent on NADH reoxidation
via the mitochondrial respiratory chain and, in accordance, a parallel decrease in
the oxygen consumption by the livers of OVX mice was observed.
The finding that the production of ketone bodies was reduced without significant change
in the β-hydroxybutyrate/acetoacetate ratio in the perfused liver suggested that the
reduced flow through citric acid cycle was not a consequence of an impairment of specific
enzymes of the cycle. Usually, in this case, an increase in the ketone bodies production
is observed, as the rate of ketone bodies production is dependent on the rates of
acetyl-CoA oxidation in the citric acid cycle, being the latter saturated at a lower
concentration of acetyl-CoA than that of ketone bodies production.[32] A reduced production of acetyl-CoA derived from octanoate oxidation seems to be,
thus, the most plausible explanation for the decrease in both ketogenesis and 14CO2 production from [1-14C]octanoate in perfused livers.
Since the contribution of mitochondria to fatty acid oxidation is nearly 90% of the
overall fatty acid oxidation in isolated hepatocytes,[33] it seems, thus, reasonable to suggest that a reduced rate of mitochondrial β-oxidation
has accounted for most of the changes found in the parameters of fatty acid oxidation
measured in both isolated mitochondria and perfused rat liver.
The exact mechanisms by which the livers of OVX mice reduced their capacity of fatty
acid oxidation after a prolonged time of estrogen deficiency cannot be inferred from
these data. However, a response to the insulin resistance state is a possibility to
be considered, since suppression of estrogen production has demonstrated to impair
both insulin sensitivity and glucose metabolism.[34] There is evidence that under insulin resistance condition, the hepatic fatty acid
synthesis is usually activated, and mitochondrial β-oxidation of fatty acid oxidation
is impaired.[35]
Even though the reduced mitochondrial fatty acid oxidative capacity may be a consequence
of the insulin resistance state, it may be also an adaptive or protective mechanism
of hepatocytes to steatosis. It should also be commented that the reduced fatty acid
capacity is probably only one of the metabolic disturbances of the livers resulting
from a long-term menopausal condition.
The results of the present study also show the suitability of the animal model to
compare the redox alterations involved in the postmenopausal NAFLD in women. The fat
liver accumulation in the OVX mice was associated with a general worsening of the
liver redox status.
In this work, the mitochondrial and peroxisomal ROS generations, which contribute
to the majority of cellular ROS, were evaluated. Both mitochondria and peroxisomes
of OVX mice produced considerably larger amounts of ROS than control mice and this,
per se, could produce an imbalance in the liver redox status. As mitochondria are
the main targets of the harmful effects of these species, a vicious cycle is established
and the more mitochondrial ROS generation, the more mitochondrial damage and ROS generation.[36]
Besides the higher mitochondrial and peroxisomal ROS, the current study revealed that
several antioxidant defenses against oxidative stress were impaired. The liver GSH
contents of OVX mice were decreased both in cytosol and mitochondria and this was
accompanied by an increase in the lipoperoxidation, indicating that some oxidative
damage had already occurred.
The activity of several antioxidant enzymes was also assessed in this work. The results
revealed that both SOD1 and SOD2 had their activity reduced in OVX mice. This could
be explained, at least in part, as a consequence of the reduction in the CT activity,
also found in these animals.[37]
In this way, a decrease in CT activity may compromise the overall antioxidant enzyme
defense system, since H2O2 is shown to be a potent inhibitor of SOD activity. In fact, SOD and CT have their
activities reduced in toxic or pathological conditions associated with severe liver
oxidative status.[37] Thus, CT inactivation would lead to oxidative damage not only directly through H2O2 and its derivatives, but also indirectly through inhibition of SODs, hence leading
to increased levels of superoxide radicals. Because of this and several other vicious
cycles, it would be expected that the more oxidative damage the more ROS accumulation.
The results demonstrated that the GSH levels are decreased and GSH-Px activity is
increased in OVX mice. Glutathione peroxidase is the major ROS scavenger, eliminating the H2O2 that escapes from the mitochondria and peroxisomes. As this enzyme consumes GSH,
the elimination of hydrogen peroxide is another factor that contributes to the reductions
in GSH levels observed in OVX mice, in which mitochondrial ROS generation is increased.
The activities of GSSG-Red, a GSH-restoring enzyme, and the G6PD were reduced in OVX
mice, and this could also contribute to the lower levels of GSH in these animals.
The reduction in the activity of G6PD can be attributed to estrogen deficiency. It
has been known for a long time that G6PD is positively controlled by estrogen,[38] and this has been further corroborated in studies performed in our laboratory.[6]
[7]
Conclusion
In conclusion, OVX mice fed a standard diet present reduced capacity of mitochondrial
fatty acid oxidation along with dyslipidemia, alteration of liver redox state and
high fasting glucose when maintained for a long time period after ovaries removal.
Although the mechanisms underlying the reduced capacity of the livers to oxidize fatty
acid in livers of OVX mice remain to be elucidated, it seems clear that the livers
can develop more extensive metabolic disturbances under this endocrine status. Thus,
it is plausible that livers from women in a long-term postmenopausal condition are
more susceptible to metabolic dysfunctions, a possibility that deserves further experimental
investigation.
