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Avocado oil induces long-term alleviation of oxidative damage
in kidney mitochondria from type 2 diabetic rats by improving
glutathione status
Omar Ortiz-Avila
1
&María del Consuelo Figueroa-García
2
&
Claudia Isabel García-Berumen
1
&Elizabeth Calderón-Cortés
3
&
Jorge A. Mejía-Barajas
1
&Alain R. Rodriguez-Orozco
4
&Ricardo Mejía-Zepeda
2
&
Alfredo Saavedra-Molina
1
&Christian Cortés-Rojo
1
Received: 27 September 2016 / Accepted: 6 February 2017 /Published online: 18 February 2017
#Springer Science+Business Media New York 2017
Abstract Hyperglycemia and mitochondrial ROS overpro-
duction have been identified as key factors involved in the
development of diabetic nephropathy. This has encouraged
the search for strategies decreasing glucose levels and long-
term improvement of redox status of glutathione, the main
antioxidant counteracting mitochondrial damage. Previously,
we have shown that avocado oil improves redox status of
glutathione in liver and brain mitochondria from
streptozotocin-induced diabetic rats; however, the long-term
effects of avocado oil and its hypoglycemic effect cannot be
evaluated because this model displays low survival and insu-
lin depletion. Therefore, we tested during 1 year the effects of
avocado oil on glycemia, ROS levels, lipid peroxidation and
glutathione status in kidney mitochondria from type 2 diabetic
Goto-Kakizaki rats. Diabetic rats exhibited glycemia of 120–
186 mg/dL the first 9 months with a further increase to 250–
300 mg/dL. Avocado oil decreased hyperglycemia at interme-
diate levels between diabetic and control rats. Diabetic rats
displayed augmented lipid peroxidation and depletion of re-
duced glutathione throughout the study, while increased ROS
generation was observed at the 3rd and 12th months along
with diminished content of total glutathione at the 6th and
12th months. Avocado oil ameliorated all these defects and
augmented the mitochondrial content of oleic acid. The ben-
eficial effects of avocado oil are discussed in terms of the
hypoglycemic effect of oleic acid and the probable depen-
dence of glutathione transport on lipid peroxidation and thiol
oxidation of mitochondrial carriers.
Keywords Diabetic nephropathy .Reactive oxygen species .
Diabetes .Oleic acid .Hypoglycemic .Goto-Kakizaki rats
Introduction
Mitochondrial ROS overproduction has a predominant role in
the development of diabetic renal damage, as revealed by the
prevention of glomerular injury, tubulointerstitial fibrosis and
mesangial expansion through the use of mitochondria targeted
antioxidants, which blocks the signaling pathways involved in
the development of these renal abnormalities (Chacko et al.
2010; Hou et al. 2016). Mitochondrial ROS generation is ex-
acerbated during diabetes either by alterations in oxidative
phosphorylation, by antioxidants depletion or both (Moreira
et al. 2006;Coughlanetal.2009;Razaetal.2011; Sourris
et al. 2012). In turn, antioxidant depletion, particularly of
Electronic supplementary material The online version of this article
(doi:10.1007/s10863-017-9697-9) contains supplementary material,
which is available to authorized users.
*Christian Cortés-Rojo
christiancortesrojo@gmail.com
1
Instituto de Investigaciones Químico-Biológicas, Universidad
Michoacana de San Nicolás de Hidalgo, Edificio B-3, Ciudad
Universitaria, Av. Fco. J. Mújica S/N, 58030 Morelia, Mich, México
2
Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala,
Universidad Nacional Autónoma de México, Avenida de los Barrios
# 1, Los Reyes-Iztacala, 54090 Tlalnepantla, Estado de México,
México
3
Facultad de Enfermería, Universidad Michoacana de San Nicolás de
Hidalgo, Av. Ventura Puente 115, 58000 Morelia, Mich, México
4
Facultad de Ciencias Médicas y Biológicas BDr. Ignacio Chávez^,
Universidad Michoacana de San Nicolás de Hidalgo, Av. Dr. Rafael
Carrillo S/N, 58020 Morelia, Mich, México
J Bioenerg Biomembr (2017) 49:205–214
DOI 10.1007/s10863-017-9697-9
glutathione, may favor peroxidative damage in lipids from
mitochondrial membranes (Maddaiah 1990; Madrigal et al.
2001), leading to impaired mitochondrial electron transfer
and enhanced electron leak responsible for ROS generation,
creating a vicious circle of antioxidant depletion and ROS
production.
Previously, we have shown that avocado oil, which con-
tains a wide variety of lipophilic antioxidants and impor-
tant amounts of oleic acid, counteracts some defects re-
sponsible for high ROS production in kidney mitochondria
from streptozotocin (STZ) - induced diabetes rats, such as
loss of cytochrome c+c
1
and defective electron transport
at complex III, besides it augments the resistance of that
complex to the deleterious effects of in vitro oxidative
stress induced by Fe
2+
(Ortiz-Avila et al. 2013). Although
these data suggest that avocado oil has a beneficial role
against the deleterious effects of diabetes over mitochon-
dria, the induction of diabetes with STZ raises some exper-
imental issues that impede to evaluate properly other im-
portant aspects that may support the beneficial effects of
avocado oil during diabetes. For example, STZ provokes a
decrease in PUFA due to inhibited conversion of C18:2
into C20:4, lowering in this way the susceptibility of mi-
tochondrial membranes of diabetic animals to lipid perox-
idation, which is contradictory to other findings about an
increase in this parameter in renal mitochondria during
diabetes (Çelıketal.2012). Thus, the protective effect of
avocado oil against renal mitochondrial lipid peroxidation
cannot be properly evaluated in this model. Another issue
arises from the difficulty to evaluate the hypoglycemic ef-
fect of avocado oil on STZ-induced rats as this drug fully
depletes pancreatic insulin production due to β-cells death
(Szkudelski 2001). This is an important issue to be ad-
dressed since it has been reported that glucose control de-
creases the development of diabetic nephropathy by more
than 50% in diabetic patients (Nathan et al. 2013), al-
though the benefits of hypoglycemic therapy on the treat-
ment of diabetic nephropathy have been put in doubt by
other studies (Gross et al. 2005 and references therein). On
the other hand, a glycemic control in type 2 diabetic pa-
tients has been achieved using a diet rich in oleic acid from
avocado consumption (Lerman-Garber et al. 1994); there-
fore, the possibility remains that beneficial effects of avo-
cado oil on kidney mitochondria might be also related to
decreased concentrations of blood glucose. Besides, it is
difficult to evaluate the long-lasting effects of avocado oil
in the STZ model because the extremely high levels of
blood glucose reached in these animals (~370 mg/dL in
our previous studies) lead to decreased rat lifespan. This
is also crucial for the evaluation of the long lasting effects
of avocado oil on the mitochondrial content of glutathione,
since it has been described that increased mitochondrial
concentrations of this antioxidant can be achieved only
transitorily through other experimental manipulations
(Lash 2015).
The above issues may be addressed by studying the effects
of avocado oil in a model of non-insulin dependent diabetes
allowing analyzing the effects of avocado oil on prolonged
hyperglycemia. In this regard, Goto-Kakizaki rats, a lean mod-
el of type 2 diabetes, is a more proper experimental model
since these animals can live for more than 1 year (Moreira
et al. 2003). Besides, it has been pointed out that this model
resembles in a good degree the renal alterations seen in dia-
betic patients with prolonged hyperglycemia (Phillips et al.
2001), besides these animals do not exhibit full depletion of
insulin synthesis but insulin resistance (Dadke et al. 2000).
For all these reasons, we consider Goto-kakizaki rats as an
adequate model to evaluate whether the mitochondrial bene-
fits of avocado oil are long lasting and its relationship with an
improved glycemic control.
Given the importance of glutathione on the mainte-
nance of mitochondrial redox state and membrane integ-
rity, the participation of mitochondrial ROS in the devel-
opment of diabetic renal complications and the short-term
mitochondrial protective effects of avocado oil on STZ-
induced diabetic rats, we decided to test in type 2 diabetic
Goto Kakizaki rats whether avocado oil supplementation
decreases ROS levels in kidney mitochondria during
prolonged diabetes and its relationship with the redox
state and content of glutathione, the peroxidative damage
to membrane lipids, the fatty acid composition of mem-
branes and the levels of blood glucose.
Materials and methods
Animals and experimental design Male diabetic Goto-
Kakizaki rats were obtained from Charles River Laboratories
International, Inc. (Wilmington, MA, USA). Control animals
were non-diabetic male Wistar rats of similar age, obtained
from our local colony. Animals were kept under controlled
light and humidity conditions and with free access to standard
rodent chow (Laboratory Rodent Diet 5001, LabDiet) and
water. For animal management, we followed the recommen-
dations of the Federal Regulation for the Use and Care of
Animals (NOM-062-ZOO-1999) from the Mexican Ministry
of Agriculture. This research was also approved by the
Institutional Committee for Use of Animals of the
Universidad Michoacana de San Nicolás de Hidalgo.
Two months - old rats were randomly divided in four
groups: 1) Wistar normoglycemic rats (control); 2) Wistar
normoglycemic rats plus avocado oil (control + AO); 3)
Goto-Kakizaki rats (diabetes); 4) Goto-Kakizaki rats plus av-
ocado oil (diabetes + AO). The dose of avocado oil was 1 mL/
250 g weight and the oral administration was performed daily
for a period of 3, 6 and 12 months. A commercial presentation
206 J Bioenerg Biomembr (2017) 49:205–214
of avocado oil was used in the study (Ahuacatlan, DIRICOM,
S.A. de C.V., México), purchased from a local grocery.
Isolation of mitochondria Kidney mitochondria were isolat-
ed by differential centrifugation (Saavedra-Molina and Devlin
1997) from a homogenate of the whole kidney. The mitochon-
drial pellet was re-suspended in a buffer with 220 mM man-
nitol, 70 mM sucrose and 2 mM MOPS (pH 7.4).
Mitochondrial protein concentration was measured by a mod-
ification of the Biuret method (Gornall et al. 1949)calibrated
with bovine serum albumin.
Analysis of mitochondrial fatty acids composition Fatty
acids from kidney mitochondria were derivatized for its anal-
ysis using boron trifluoride (BF
3
)ina14%methanolsolution
according to the method of Morrison and Smith (1964).
Subsequently, the methyl-esters of fatty acids were analyzed
by gas chromatography under the conditions previously de-
scribed (Ortiz-Avila et al. 2013).
Evaluation of lipid peroxidation levels This determination
was carried out in 0.1 mg/mL kidney mitochondria protein by
measuring the levels of thiobarbituric acid reactive substances
(TBARS) (Buege and Aust 1978). Absorbance was measured
at 532 nm with a Perkin Elmer Lambda 18 UV/VIS spectro-
photometer. Data were expressed as nanomoles of TBA reac-
tive species (TBARS) per mg of mitochondrial protein.
Glutathione determinations Mitochondria samples (0.3 mg/
mL) were treated with 5% (v/v) sulfosalicilic acid and centri-
fuged at 7800 g for 10 min to remove denatured proteins.
Reduced (GSH) and oxidized (GSSG) glutathione were deter-
mined in the supernatant by an enzymatic method (Akerboom
and Sies 1981). The content of total glutathione (GSH +
GSSG) was assayed in a cuvette containing 90 μl of the su-
pernatant, 3 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)
and 0.115 unit/ml glutathione reductase in a final volume of
1 mL of 0.1 M sodium phosphate buffer (pH 7.5). After 5 min
of incubation at room temperature, 2 mM NADPH was added
and the kinetics of the reaction was monitored for 5 min. The
increment in absorbance at 412 nm was converted to GSH
concentration using a standard curve with known amounts
of GSH. For determination of GSSG, the same DTNB
recycling assay was performed after GSH derivatization by
incubating at room temperature with 3% (v/v) 4-
vinylpyridine for 1 h before starting DTNB assay. The con-
centration of GSH was calculated by subtracting the concen-
tration of GSH + GSSG minus the concentration of GSSG.
Measurement of ROS levels ROS were estimated by measur-
ing the oxidation rate of the fluorescent probe 2′,7′-
dichlorodihydrofluorescein (H
2
DCF). 0.5 mg/mL intact mito-
chondria and 1.25 mM H
2
DCF diacetate were incubated at
4 °C under constant shaking during 20 min in a buffer contain-
ing 10 mM HEPES, 100 mM KCl, 3 mM MgCl
2
and 3 mM
KH
2
PO
4
(pH 7.4). Then, the mitochondrial suspension was
placed in a quartz cuvette and the assay was started by record-
ing the basal fluorescence; 1 min later, 10 mM glutamate/
malate was added and the changes in H
2
DCF fluorescence
were followed by 20 min. Fluorescence changes were detected
in a Shimadzu RF-5301PC spectrofluorophotometer (λ
ex
485 nm; λ
em
520 nm).
Data analysis The results are expressed as the mean ± standard
error from at least three independent experiments using sam-
ples from different animals. Statistical differences of the data
were determined by 2-way ANOVA with Bonferroni post-hoc
test. Statistically significant differences were defined as
P<0.05.
Results
Effects of avocado oil on plasma glucose concentrations
Glucose levels are depicted in the Fig. 1. In control rats (white
circles), glucose concentrations fluctuated between 80 and
90 mg/dL throughout the experimental period. Importantly,
avocado oil (black circles) did not modify this trend in control
rats. As expected, diabetic rats (white triangles) exhibited
higher glucose levels ranging from 120 mg/dL to 186 mg/dL
during the first 9 months of the experiment, after which, a rise
to 250–300 mg/dL was observed during the last three months.
Fig. 1 Time-course of blood glucose levels in rats from control (white
circles), diabetic (white triangles), control plus avocado oil (black circles)
and diabetic plus avocado oil (black triangles) groups. Fasting glucose
concentrations were monitored each month after the beginning of the
treatment with avocado oil (i.e. month 0), which was daily administered
as described in Materials and Methods. Data are presented as the
mean ± standard error of n≥5. *P< 0.05 when compared to control
group;
#
P< 0.05 when compared to diabetic group (2-way ANOVA with
Bonferroni post-hoc test)
J Bioenerg Biomembr (2017) 49:205–214 207
Avocado oil had a hypoglycemic effect in diabetic rats (black
triangles) as glucose levels were in an intermediate level be-
tween the diabetic and the control animals, although glucose
concentrations also increased in the last three months of the
study parallel to diabetic rats but never reached the levels
observed in those animals.
Effects of diabetes and avocado oil on ROS generation
and lipid peroxidation
It can be observed that ROS levels at the 3rd month were
1.6-fold higher in mitochondria from diabetic rats in com-
parison to control mitochondria (Fig. 2), with avocado oil
fully preventing this effect without altering ROS levels in
the control group. In contrast, at the 6th month, ROS
levels were similar in mitochondria from control and dia-
betic groups, but is worth noting that ROS levels in these
groups were 23- and 13-fold higher, respectively, at this
time when compared to the levels of the same groups at
the 3rd month. Despite this exacerbation, avocado oil still
decreases ROS levels in 75% in mitochondria from the
diabetic group, while a non-statistically significant dimi-
nution of 27% was observed in the control group. At the
12th month, an augment of ~90% in ROS was detected in
diabetic rats with respect to control and control plus avo-
cado oil groups. Although in a partial way, the ability of
avocado oil to decrease ROS levels in mitochondria from
diabetic rats remained at this stage, exhibiting these mito-
chondria a 21% diminution in comparison to mitochon-
dria from diabetic rats.
The levels of lipid peroxidation are shown in the Fig. 3.
Lipid peroxidation increased in mitochondria from diabetic
rats in comparison to control mitochondria as the study
progressed, with increments of 48.4%, 70.3% and 93.1% at
3, 6 and 12 months, respectively. Avocado oil diminished lipid
peroxidation in diabetic rats at 3 and 12 months, while a non-
significant trend towards decreased lipid peroxidation was
detected at 6 months. Lipid peroxidation did not undergo
any change in mitochondria from control rats plus avocado
oil group at any stage.
Effects of avocado oil on mitochondrial glutathione
The content of glutathione and its redox status are
displayed in the Fig. 4. As observed in the panel a, dia-
betes consistently decreased the concentration of reduced
glutathione (GSH), with decrements of ~3-fold at 3 and
6 months and 4-fold at 12 months. Avocado oil fully
prevented the fall in GSH at 3 and 6 months, while a
partial prevention was observed at the 12th month, al-
though GSH levels remained 2.6 fold-higher than in the
diabetic group. In contrast to GSH, no significant changes
in oxidized glutathione (GSSG) were observed throughout
the experimental period (Fig. 4b), except by a two-fold
decrease in the diabetic group at the 12th month.
Regarding to total glutathione content (GSH + GSSG),
diabetes induced a decay at the 6th and 12th months, with
avocado oil restoring glutathione pool in a full way at the
6th month and only partially at the 12th month (Fig. 4c).
Influence of diabetes and avocado oil on mitochondrial
fatty acid profile
Mitochondrial fatty acid profile was determined in order
to assess probable changes induced by avocado oil con-
tributing to a higher resistance to oxidative damage via
Fig. 2 ROS levels of kidney mitochondria from rats supplemented with
avocado oil (AO) during 3, 6 and 12 months. The changes in the fluores-
cence of H
2
DCF (ΔF) in response to glutamate-malate addition were
quantified during 20 min and expressed as arbitrary units. Data are pre-
sented as the mean ± standard error of n≥4. Different letters indicate
statistically significant differences between groups (P< 0.05) (2-way
ANOVA with Bonferroni post-hoc test)
Fig. 3 Lipid peroxidation levels of kidney mitochondria from rats
supplemented with avocado oil (AO) during 3, 6 and 12 months. The
results are expressed as nanomoles of thiobarbituric acid reactive sub-
stances (TBARS) per milligram of mitochondrial protein. Data are pre-
sented as the mean ± standard error of n≥4. Different letters indicate
statistically significant differences between groups (P< 0.05) (2-way
ANOVA with Bonferroni post-hoc test)
208 J Bioenerg Biomembr (2017) 49:205–214
decreased peroxidizability of mitochondrial membranes.
The only change observed in the percentage of saturated
fatty acids was an increase in the control plus avocado oil
group at the 12th month (Fig. 5a). Regarding to monoun-
saturated fatty acids (MUFA), significant increments in
this fatty acid were observed in mitochondria from dia-
betic rats supplemented with avocado oil at any stage of
the protocol when compared to the diabetic group. In
control rats, MUFA percentage increased only at the
12 month (Fig. 5b). A moderate diminution of this fatty
acid was observed solely in diabetic rats at the 3rd month.
The only effect produced by avocado oil on polyunsatu-
rated fatty acids (PUFA) content was a decrease in control
mitochondria at the 12th month, while diabetes did not
induced any modification (Fig. 5c). On the other hand,
the peroxidizability index (PI), which estimates the sus-
ceptibility of a membrane to undergo lipid peroxidation
based on its fatty acid composition, was calculated from
the data of mitochondrial fatty acids depicted in the
Tab le S1. No significant changes among all the experi-
mental groups were observed at the 3rd and the 6th
months (Fig. 5d). Supplementation with avocado oil only
decreased the PI of mitochondria from control rats at the
12th month.
Discussion
The results of the present study show that, in kidney mito-
chondria from diabetic rats, avocado oil decreases ROS levels
at all the stages of the study (Fig. 2), diminishes lipid perox-
idation at the 3rd and 12th months (Fig. 3), prevents the de-
pletion of GSH (Fig. 4a), replenishes the pool of total gluta-
thione (Fig. 4c) and increases the content of MUFA (Fig. 5b),
although without decreasing the peroxidizability index
(Fig. 5d). Importantly, avocado oil displays a partial hypogly-
cemic effect, which was not statistically significant at the 3rd,
5th and 6th months of the study (Fig. 1). Regarding the later
effect, a glycemic control has been achieved in type 2 diabetic
individuals with diets enriched with MUFA from sources of
C18:1 like avocado (Lerman-Garber et al. 1994) or olive oil
administered up to 1-year (Brehm et al. 2009). The benefits of
Fig. 4 Concentrations of reduced glutathione (GSH, panel a), oxidized
glutathione (GSSG, panel b) and total glutathione (panel c) in kidney
mitochondria from rats treated during 3, 6 and 12 months with avocado
oil (AO). Data are presented as the mean ± standard error of
n≥3. Different letters indicate statistically significant differences
between groups (P< 0.05) (2-way ANOVA with Bonferroni post-hoc
test)
J Bioenerg Biomembr (2017) 49:205–214 209
C18:1 in glucose levels have been linked to enhanced release
of glucagon-like peptide-1 (GLP-1), which improves both the
secretion and sensitivity to insulin, inhibits the secretion of
glucagon and delays gastric absorption of nutrients (Rocca
et al. 2001). In addition, oxidative stress has been demonstrat-
ed to disrupt GLUT4 translocation from internal pools to plas-
ma membrane via disruption of the trafficking of proteins
needed for that process (Tirosh et al. 1999). Thus, the high
oleic acid content in avocado oil and its antioxidant capacity
demonstrated in this and other studies might be involved in the
diminution of blood glucose concentrations by modulating the
effects of GLP-1 and GLUT4 on glucose transport and
metabolism.
Glucose absorption in the kidney is primarily mediated by
non-insulin dependent transporters like SLGT-2 and GLUT-2
(Bakris et al. 2009). Thus, glucose overload occurs in renal
cells during diabetes, leading to increased glycolytic flux, en-
hanced pyruvate formation and high NADH/NAD
+
ratios
(Forbes et al. 2008). In turn, elevated NADH/NAD
+
ratios
lead to augmented ROS production in the complex I in
forward mode (Kareyeva et al. 2012). Alternatively, inhibition
of ROS production by substrate has been observed in complex
I at high NADH concentrations, leading to NADH oxidation
and ROS production by soluble matrix dehydrogenases
(Grivennikova and Vinogradov 2006). Accordingly, at the
3rd month of treatment, the parallel increase in blood glucose
and ROS levels in diabetic rats, as well as the decrease in-
duced by avocado oil in these parameters fits well with this
picture (Figs 1and 2), although the effect of avocado oil lacks
of statistical significance. However, when diabetes progress
up to 6 months, it seems that there are additional factors in-
volved in ROS production, as there is no a clear relation
among blood glucose and ROS levels at that time.
Meanwhile, at 12 months, blood glucose and ROS levels
shows again some degree of concordance, with diabetes in-
creasing both glucose and ROS levels, although not in a sim-
ilar proportion, and avocado oil having the contrary effect
except in the control plus avocado oil group. Considering that
mitochondrial ROS levels are central in the development of
diabetic nephropathy (Chacko et al. 2010; Hou et al. 2016),
Fig. 5 Fatty acid indexes of kidney mitochondria from rats treated during
3, 6 and 12 months with avocado oil (AO). The percentages of saturated
fatty acids (SFA, panel a), monounsaturated fatty acids (MUFA, panel b),
polyunsaturated fatty acids (PUFA, panel c) and the peroxidizability
indexes (PI, panel d) were determined from the data of mitochondrial
fatty acids displayed in the Table S1. PI were calculated as reported by
Pamplona et al. (1998). Data are presented as the mean ± standard error of
n≥4. Different letters indicate statistically significant differences between
groups (P< 0.05) (2-way ANOVA with Bonferroni post-hoc test)
210 J Bioenerg Biomembr (2017) 49:205–214
the lack of a clear correlation between glucose levels and ROS
generation during diabetes progression is in agreement to the
controversial role of hypoglycemic therapy against this com-
plication in diabetic patients (Gross et al. 2005). Indeed, a
more effective therapy against diabetic nephropathy than hy-
poglycemic agents is the blocking of angiotensin II actions by
antagonizing the activation of AT
1
receptors (Brenner et al.
2001). It has been described that AT
1
blocking decrease mito-
chondrial ROS production by inhibiting NADPH oxidase,
whose activity drives to oxidative damage in the electron
transport chain, enhanced ROS generation, and impaired kid-
ney function (de Cavanagh et al. 2006; Dikalov and
Nazarewicz 2013). These findings supports the idea that de-
creasing mitochondrial ROS generation may be more benefi-
cial for kidney disease attenuation than the decrease of blood
glucose levels. In this regard, avocado oil decreased ROS
levels (Fig. 2) and lipid peroxidation (Fig. 3)inmitochondria
from the diabetic rats even at the 12th month of treatment,
with a concomitant augment of GSH concentrations
(Fig. 4a). This suggest that at long term, avocado oil decreases
oxidative stress in diabetic mitochondria by boosting the an-
tioxidant capacity of mitochondrial glutathione system rather
than by improving blood glucose levels, the former occurring
by augmenting the pool of total glutathione (Fig. 4c)andby
enhancing its reduced state (Fig. 4a). Given the central role of
mitochondrial glutathione on the defense against oxidative
stress and other toxicants (Marí et al. 2009), this has led to
the suggestion that modulation of the redox state of mitochon-
drial glutathione may be a therapeutic target to treat diabetic
kidney disease (Lash 2015). Accordingly, the dietary supple-
mentation during two months with GSH decreases renal oxi-
dative stress and normalizes urinary markers of kidney dam-
age in STZ-induced diabetic rats (Ueno et al. 2002).
Nevertheless, it has been argued that supplementation with
GSH only provokes transient increments of mitochondrial
GSH concentrations, conferring only short-term improvement
of mitochondrial antioxidant status (Lash 2015), in contrast to
the sustained improvement of both GSH levels and total glu-
tathione in mitochondria from diabetic rats observed up to
12 months in this study (Figs 4a and c).
A probable factor explaining the maintenance of glutathi-
one in its reduced form by avocado oil might be the presence
of β-sitosterol, the main phytosterol present in the edible por-
tion of avocado (Dreher and Davenport 2013), which we have
also detected in a preliminary characterization of the oil used
in this study along with other bioactive compounds like
campesterol and squalene (unpublished data). β-sitosterol in-
creases the amount of GSH in stressed macrophages via the
activation of the estrogen/phosphatidylinositol 3-kinase path-
way (Vivancos and Moreno 2005), being this pathway active
also in the kidney (Satake et al. 2008). The improvement on
GSH by β-sitosterol was attributed to a higher activity of
glutathione peroxidase (GPX); however, in our case, we
thought that increased availability of GSH might be also me-
diated by increased GSSG reduction by glutathione reductase,
being this possibility currently explored in our laboratory.
There is an apparent lack of correlation among the levels of
ROS and the extent of lipid peroxidation at the 6th month of
the study, as it should not be expected a rise in lipid peroxida-
tion in mitochondria from diabetic rats (Fig. 3)becausenota
parallel augment was seen in the ROS levels of that group in
comparison to control mitochondria (Fig. 2). This controversy
can be explained considering that there was a more clear in-
verse relationship between the levels lipid peroxidation
(Fig. 3)andGSHlevels(Fig.4a) in mitochondria from dia-
betic rats, in such way that lipid peroxidation may be more
determined by the extent of glutathione reduction than by the
levels of ROS. This makes sense considering that lipid perox-
idation is counteracted in mitochondrial membranes by gluta-
thione peroxidase 4 (Gpx4) in a process requiring the redox
cycling of glutathione by glutathione reductase (Nomura et al.
1999). Therefore, the drastic drop in GSH content in the dia-
betic group may drive to a decreased ability of Gpx4 to repair
peroxidized phospholipids, making membrane phospholipids
more prone to be peroxidized even by unaltered levels of ROS
as observed at 6 months in diabetic rats.
Total glutathione content was decreased in mitochondria
from diabetic rats at the 6th and the 12th months (Fig. 4c),
suggesting that diabetes impairs the transport of this antioxi-
dant into mitochondria. Glutathione is transported into kidney
mitochondria by the dicarboxylate and 2-oxoglutarate carriers
(Lash 2012),which contain reactive –SH groups whose chem-
ical modification inhibits its activity by interfering with sub-
strate binding (Palmieri et al. 1974;Capobiancoetal.1996).
In turn, there are proteins with exposed cysteines that react
with ROS to yield oxidized thiol species like sulfenic acids,
producing inactivated proteins that can be reactivated by en-
zymatic reduction of oxidized thiols at expense of GSH (Hurd
et al. 2008). Based on these antecedents, it is feasible to hy-
pothesize that decreased total glutathione content in mitochon-
dria from diabetic rats might proceed by oxidation of reactive
–SH groups from glutathione transporters (i.e. dicarboxylate
and 2-oxoglutarate carriers) due to enhanced ROS generation
and/or decreased GSH availability, inhibiting in this way the
binding of glutathione and its internalization into mitochon-
drial matrix. Moreover, the increase in GSH concentration, the
lower levels of ROS, and the higher content of total glutathi-
one elicited by avocado oil fits well with the idea that the oil
increases mitochondrial glutathione content by generating a
more reduced redox status that allows better glutathione trans-
port by maintaining the thiols from mitochondrial carriers in a
more reduced form.
Membrane fatty acid composition determines the physical
properties of biomembranes, as increased content of PUFA
with 4, 5 and 6 double bonds augments membrane fluidity
(Yang et al. 2011). It has been shown that mitochondrial
J Bioenerg Biomembr (2017) 49:205–214 211
glutathione transport is affected by decreased inner membrane
fluidity (Lluis et al. 2003). According to our results, it would
not be expected an influence of membrane fatty acid compo-
sition overglutathione content, since not consistent changes in
these fatty acids were detected throughout the study
(Table S1). The more remarkable change in fatty acids was
an increase in C18:1 but this fatty acid does not promote
changes in membrane fluidity (Yang et al. 2011). According
to several studies (Bruch and Thayer 1983; Dobretsov et al.
1977; Sevanian et al. 1988;ChenandYu1994), another factor
decreasing membrane fluidity is lipid peroxidation.
Consistently, lipid peroxidation in mitochondria from diabetic
rats increased as the study progress, until reaching twice the
levels observed in control rats (Fig. 3). Parallelly, total gluta-
thione levels dropped during the progress of the study up to
less than 35% of the levels of control mitochondria (Fig. 4c).
Therefore, the inverse correlation between lipid peroxidation
levels and total glutathione content suggest that lipid peroxi-
dation leads to a decrease in membrane fluidity that impairs
glutathione transport, resulting in lower levels of total gluta-
thione in mitochondria from diabetic rats. Conversely, in mi-
tochondria from diabetic rats treated with avocado oil, the
decrease of lipid peroxidation levels correlates with an in-
crease in total glutathione content that reach at 12 months
twice the concentrations with respect to mitochondria from
diabetic rats (Figs 4and 5, respectively). This suggest that
avocado oil improves at the long term the total content of
mitochondrial glutathione also by decreasing lipid peroxida-
tion levels, which, in turn, it might be augmenting membrane
fluidity and enhancing the activity of glutathione transporters,
although measurements of membrane viscosity are required to
further validate this hypothesis. In summary, avocado oil
might be augmenting the pool of mitochondrial glutathione
by a mechanism involving attenuation of thiol oxidation in
glutathione transporters and improvement of membrane fluid-
ity by decreasing lipid peroxidation. More importantly, the
augment of GSH levels by avocado oil observed in this study
and the same outcome obtained in previous studies from our
group in liver and brain mitochondria from type 1 diabetic rats
(Ortiz-Avila et al. 2015a; Ortiz-Avila et al. 2015b), suggest
that avocado oil is an efficient Benhancer^of mitochondrial
GSH levels, which leads to an improvement of mitochondrial
redox status that it is translated into a lower levels of ROS and
attenuated oxidative damage even at long term (i.e. 1 year) as
observed in the present study. Despite avocado oil increases
the mitochondrial content of C18:1 and the amount of MUFA
(Table S1 and Fig. 5b, respectively), this does not seems to
contribute to augmented resistance to oxidative stress as
peroxidizability index remained constant in the diabetic group
throughout the study (Fig. 5d). This disagree with an study
showing the benefits of the mitochondrial enrichment with
MUFA in aged rats consuming olive oil (Quiles et al. 2002),
but this discrepancy may be attributed to the differences in the
type and content of antioxidants and the concentration of
C18:1 among these oils.
The pathogenesis of diabetic nephropathy involves glomer-
ular mesangial expansion and tubulointerstitital fibrosis due to
remodeling of extracellular matrix, the latter event being trig-
gered by upregulation of TGF-β1 and PAI-1. This occurs
through ROS-induced activation of transcription factors like
NF-κB, AP-1 and SP1 in a process mediated by PKC (Lee
et al. 2003). Mitochondrial ROS produced at the electron
transport chain have been identified as the main PKC activa-
tors during hyperglycemia since the inhibition of complex II,
the uncoupling of respiration and MnSOD overexpression
fully abolished PKC activation and other pathways of kidney
damage (Nishikawa et al. 2000). Given the predominant role
of excessive mitochondrial ROS generation in the etiology of
diabetic renal damage, the search for strategies to scavenging
ROS or enhancing mitochondrial function has been encour-
aged for the treatment of this diabetic complication (Che et al.
2014). On this basis, our results about the prevention of ex-
cessive ROS generation and the improvement of GSH in kid-
ney mitochondria, provide promising evidence about the po-
tential of avocado oil to prevent and/or delay the development
of kidney nephropathy, which is intensively investigated in
our laboratory to obtain further data supporting the value of
avocado oil as a functional food to aid to mitigate the cata-
strophic consequences of diabetes, one of the main health
challenges facing the mankind in the current century given
the pandemic character of this disease (van Dieren et al. 2010).
In conclusion, in renal mitochondria from type 2 diabetic
rats, avocado oil counteracts increased ROS levels and exac-
erbated lipid peroxidation in a sustained way, which is attrib-
uted rather to an increase in total glutathione pool and en-
hanced reduction of this molecule than to substantial changes
in mitochondrial membranes or to the hypoglycemic effect of
the oil.
Acknowledgements This study was supported by a grant from
Programa de Investigación 2016-2017 de la Coordinación de la
Investigación Científica, Universidad Michoacana de San Nicolás de
Hidalgo, Morelia, Mich., México (1821440 to CCR).
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