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Diabetes-induced changes in glucose synthesis, intracellular glutathione status and hydroxyl free radical generation in rabbit kidney-cortex tubules

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Diabetes-induced changes in glucose formation, intracellular and mitochondrial glutathione redox states as well as hydroxyl free radicals (HFR) generation have been investigated in rabbit kidney-cortex tubules. In contrast to renal tubules of control animals, diabetes-evoked increase in glucose formation in the presence of either aspartate+glycerol+octanoate or malate as gluconeogenic precursors (for about 50%) was accompanied by a diminished intracellular glutathione reduced form (GSH)/glutathione oxidised one (GSSG) ratio by about 30-40%, while the mitochondrial GSH/GSSG ratio was not altered. However, a relationship between the rate of gluconeogenesis and the intracellular glutathione redox state was maintained in renal tubules of both control and diabetic rabbits, as concluded from measurements in the presence of various gluconeogenic precursors. Moreover, diabetes resulted in both elevation of the glutathione reductase activity in rabbit kidney-cortex and acceleration of renal HFR generation (by about 2-fold). On the addition of melatonin, the hormone exhibiting antioxidative properties, the control values of HFR production were restored, suggesting that this compound might be beneficial during diabetes therapy. In view of the data, it seems likely that diabetes-induced increase in HFR formation in renal tubules might be responsible for a diminished intracellular glutathione redox state despite elevated glutathione reductase activity and accelerated rate of gluconeogenesis, providing glucose-6-phosphate for NADPH generation via pentose phosphate pathway.
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Molecular and Cellular Biochemistry 261: 91–98, 2004.
c
2004 Kluwer Academic Publishers. Printed in the Netherlands.
Diabetes-induced changes in glucose synthesis,
intracellular glutathione status and hydroxyl free
radical generation in rabbit kidney-cortex
tubules
K. Winiarska, J. Drozak, M. Wegrzynowicz, T. Fraczyk and J. Bryla
Department of Metabolic Regulation, Institute of Biochemistry, Warsaw University, Poland
Abstract
Diabetes-induced changes in glucose formation, intracellular and mitochondrial glutathione redox states as well as hydroxyl free
radicals (HFR) generation have been investigated in rabbit kidney-cortex tubules. In contrast to renal tubules of control animals,
diabetes-evoked increase in glucose formation in the presence of either aspartate + glycerol + octanoate or malate as gluco-
neogenic precursors (for about 50%) was accompanied by a diminished intracellular glutathione reduced form (GSH)/glutathione
oxidised one (GSSG) ratio by about 30–40%, while the mitochondrial GSH/GSSG ratio was not altered. However, a relation-
ship between the rate of gluconeogenesis and the intracellular glutathione redox state was maintained in renal tubules of both
control and diabetic rabbits, as concluded from measurements in the presence of various gluconeogenic precursors. Moreover,
diabetes resulted in both elevation of the glutathione reductase activity in rabbit kidney-cortex and acceleration of renal HFR
generation (by about 2-fold). On the addition of melatonin, the hormone exhibiting antioxidative properties, the control values
of HFR production were restored, suggesting that this compound might be beneficial during diabetes therapy. In view of the
data, it seems likely that diabetes-induced increase in HFR formation in renal tubules might be responsible for a diminished
intracellular glutathione redox state despite elevated glutathione reductase activity and accelerated rate of gluconeogenesis,
providing glucose-6-phosphate for NADPH generation via pentose phosphate pathway. (Mol Cell Biochem 261: 91–98, 2004)
Key words: diabetes, gluconeogenesis, glutathione redox state, hydroxyl free radicals, melatonin, rabbit renal tubules
Introduction
Glutathione (L-γ -glutamyl-L-cysteinylglycine), the predom-
inant non-protein thiol in mammalian cells, plays a crucial
role in defense against oxidative stress [1]. Under physiolog-
ical conditions, more than 98% of intracellular glutathione
exists as the reduced thiol form (GSH), while the residue is
present mainly as either the oxidized disulfide form (GSSG)
or mixed disulfides. Reduction of GSSG to GSH is catalyzed
by glutathione reductase utilizing NADPH as the reducing
equivalent.
It is commonly accepted that hyperglycaemia can induce
oxidative stress, which is considered to be the main cause of
Address for offprints:J.Bryla, Department of Metabolic Regulation, Institute of Biochemistry, Warsaw University, ul. Miecznikowa 1, 02-096 Warszawa,
Poland (E-mail: bryla@biol.uw.edu.pl)
diabetic complications [2, 3]. To elucidate the mechanisms
of glucose-induced oxidative stress several hypotheses have
been proposed, including glucose auto-oxidation, glycation
of proteins and formation of advanced glycation end prod-
ucts (AGEs) (cf. [2] for review). Oxidative stress is accompa-
nied by a diminished intracellular GSH level [1]. However,
data concerning diabetes-evoked changes in glutathione re-
dox state are controversial. For example, although several
authors seem to agree that diabetes induces a decline in GSH
levels in erythrocytes [4–8], liver [9–11], pancreas [9–12],
heart [9] and aorta [13] in rat diabetic kidneys either dimin-
ished [11, 14], unchanged [10] or increased [15] GSH levels
have been reported. Moreover, high glucose has been shown
92
to decrease GSH levels in vascular smooth muscles [16] and
mesangial cells [17] as a result of limited expression of γ -
glutamylcysteine synthetase, the key enzyme of glutathione
synthesis. On the other hand, the intracellular GSH/GSSG ra-
tios are significantly elevated in rabbit kidney-cortex tubules
effectively synthesizing glucose de novo probably due to
an increased content of glucose-6-phosphate, which is uti-
lized for NADPH generation via pentose phosphate pathway
[18].
As (i) kidney, in addition to liver, makes a significant con-
tribution to glucose whole body metabolism [19], (ii) renal
glucose production in type 1 diabetic patients is increased
proportionately to systemic glucose appearance [20], and (iii)
the intracellular localization of renal gluconeogenic enzymes
in rabbit is similar to that in human [21], the aim of this study
wastoexamine diabetes-induced changes in the intracellular
glutathione status and hydroxyl free radicals (HFR) genera-
tion, as well as to estimate the relationship between the rate
of gluconeogenesis and the intracellular GSH/GSSG ratio in
kidney-cortex tubules of alloxan-diabetic rabbits.
Materials and methods
Animals
The experiments were performed with adult male white
Termond rabbits weighing approximately 2–2.5 kg. Ani-
mals were maintained on the standard rabbit chow with
free access to water and food. Diabetes was induced by
the single injection of alloxan (175 mg kg
1
body weight)
[22]. Only those alloxan-treated animals which exhibited de-
creased or stabilised body weight and blood glucose concen-
tration in excess of 300 mg/100 ml, 3 days after treatment
were considered diabetic and used for experiments. All an-
imal treatment procedures were approved by the First War-
saw Local Commission for the Ethics of Experimentation on
Animals.
Isolation of kidney-cortex tubules, mitochondrial
and cytosolic fractions
Rabbit kidney-cortex tubules were obtained according to
Jarzyna et al. [23]. Approximately 95% of renal tubules ex-
cluded Trypan blue.
Mitochondrial fractions for GSH and GSSG determination
were obtained from isolated kidney-cortex tubules incubated
for1hinthepresence of substrates, following treatment with
digitonin and centrifugation [24]. The pellets were extracted
with either 12% PCA or 50 mM NEM in 12% PCA for mea-
surements of reduced (GSH) and oxidised (GSSG) forms of
glutahione, respectively [18].
Cytosol for the measurement of glutathione reductase ac-
tivity was obtained from kidney-cortex homogenised in 0.9%
NaCl solution (5 ml per 1 g of tissue). The activity of the en-
zyme was assayed in the final supernatant fraction following
differential centrifugation.
Incubation of renal tubules
Isolated kidney-cortex tubules were incubated at 37
Cin25
ml Erlenmeyer flasks sealed with rubber stoppers in an at-
mosphere of O
2
:CO
2
(95%:5%) in Krebs–Ringer bicarbon-
ate buffer, pH 7.4, in the presence of substrates indicated in
the tables. The rates of gluconeogenesis under all conditions
studied were linear at least for 90 min of incubation. Reac-
tions were stopped following 60 min of incubation by either
the addition of 1 ml sample to 0.1 ml of 35% perchloric
acid (PCA) or centrifugation of kidney-cortex tubules sus-
pension through the silicone oil layer to 1 ml of 12% PCA
[25]. To avoid non-enzymatic GSH oxidation, samples used
for GSSG determinations were centrifuged into 50 mM N-
ethylmaleimide (NEM) in 12% PCA [18]. Excess NEM was
removed by hexane extraction.
Samples tested for GSH determination were stored as PCA
extracts, while the others were neutralised immediately after
deproteinisation.
Determination of hydroxyl free radicals
HFR were estimated as 2,3-dihydroxybenzoic acid (2,3-
DHBA) generated in the presence of sodium salicylate (SAL)
[26]. Incubation of isolated renal tubules was performed in
the presence of 1 mM SAL. After 60 min of incubation, 1 ml
samples were withdrawn and acidified with 0.1 ml of 35%
PCA containing 1 mM ethylenediaminetetraacetate (EDTA)
and 4 mM sodium metabisulphite (Na
2
S
2
O
5
). Following cen-
trifugation, supernatants were placed on ice and analysed on
the day of experiment.
2,3-DHBA assays were performed by HPLC using Beck-
man Ultrasphere ODS column. The mobile phase consisted of
50 mM NaH
2
PO
4
, 1.125 mM octanesulphonic acid, 0.2 mM
EDTA, 3% methanol and 5.5% acetonitrile (v/v). pH was
adjusted to 2.8 with 1 M orthophosphoric acid. 110B Sys-
tem Gold HPLC (Beckman, USA) was equipped with a Bio-
Rad 1640 electrochemical detector (Bio-Rad, USA) and a
glassy carbon working electrode operating at +0.75 V against
Ag/AgCl reference electrode and a detection range of 2 nA.
The flow rate was 1 ml min
1
and all separations were per-
formed at 30
C. Quantification was achieved using external
standard of 2,3-DHBA. Data from the detector were collected
and integrated by a PC equipped with appropriate interface
and chromatography software.
93
Analytical methods
Glutathione reductase activity was determined fluorimetri-
cally [27]. Glucose, pyruvate, malate, glucose-6-phosphate
and GSSG were estimated either spectrophotometrically or
fluorimetrically [27]. GSH levels were determined by
HPLC (Beckman, USA) after derivatization with N-(1-
pyrenyl)maleimide (NPM) [28]. Blood glucose was anal-
ysed with hexokinase and glucose-6-phosphate dehydroge-
nase [27].
Protein content of cytosolic fraction was determined spec-
trophotometrically according to Bradford [29].
Chemicals
Enzymes, co-enzymes and nucleotides for metabolite de-
terminations were purchased from Roche (Mannheim,
Germany). All other chemicals were from Sigma Chemicals
(St. Louis, MO, USA).
Expression of results
The significance of the observed differences was estimated
using ANOVA. When data for more than two groups were
compared Tukey’s post hoc test was applied following
ANOVA. Values are expressed as means ± S.D. for 5–7 sep-
arate experiments.
Results
Glucose formation
Rabbit kidney-cortex tubules were incubated in the presence
of various substrates, which differentiate in terms of their util-
isation for glucose synthesis. In agreement with the previous
Table 1. Glucose formation, intracellular GSH and GSSG contents and GSH/GSSG ratios in kidney-cortex tubules of control and diabetic rabbits
Glucose formation GSH GSSG
Substrates Rabbits (µmol·g
1
dry weight·h
1
)(µmol·g
1
dry weight) (µmol·g
1
dry weight) GSH/GSSG
Glycerol C 4.2 ± 0.3 1.84 ± 0.20 0.052 ± 0.008 36.8 ± 2.7
D 4.5 ± 0.3 2.26 ± 0.34 0.080 ± 0.012
∗∗
28.2 ± 2.3
∗∗
Aspartate C 3.3 ± 0.2 2.36 ± 0.30 0.103 ± 0.012 23.4 ± 3.5
D 39.0 ± 9.4
,∗∗
2.87 ± 0.28 0.078 ± 0.005
∗∗
37.4 ± 2.7
,∗∗
Aspartate + glycerol + octanoate C 90.0 ± 5.9
2.76 ± 0.30 0.047 ± 0.005 60.6 ± 8.5
D 140.0 ± 21.1
,∗∗
3.00 ± 0.28 0.076 ± 0.004
∗∗
39.3 ± 2.6
,∗∗
Alanine + glycerol + octanoate C 51.5 ± 6.9
2.51 ± 0.20 0.047 ± 0.006 47.1 ± 6.1
D 58.8 ± 8.8
2.64 ± 0.22 0.078 ± 0.010
∗∗
35.4 ± 3.7
,∗∗
Malate C 85.9 ± 6.4
2.09 ± 0.31 0.040 ± 0.007 55.8 ± 7.6
D 155.6 ± 14.2
,∗∗
2.04 ± 0.13 0.055 ± 0.007
∗∗
39.6 ± 5.4
,∗∗
Renal tubules isolated from control (C) and diabetic (D) rabbits were incubated for 60 min. Glycerol, amino acids, octanoate and malate were
added at 2, 2, 0.5 and 5 mM concentrations, respectively. Values are means ± S.D. for 5–7 experiments. Statistical significance:
p < 0.05 vs.
values for renal tubules exhibiting negligible glucose synthesis in the presence of either glycerol or aspartate alone,
∗∗
p < 0.05 vs. corresponding
values for renal tubules isolated from control rabbits.
report [30], aspartate and glycerol applied as sole substrates
were not used for glucose formation in kidney-cortex tubules
of control rabbits, while the rates of gluconeogenesis from
aspartate + glycerol + octanoate and malate were similar and
twice as high as those determined in the presence of alanine
+ glycerol + octanoate (Table 1). Under diabetic conditions
gluconeogenesis in the presence of either aspartate, aspartate
+ glycerol + octanoate or malate was potentiated whereas,
the rates of glucose formation from both glycerol and alanine
+ glycerol + octanoate were not significantly altered.
Intracellular and mitochondrial glutathione redox states
The intracellular GSH level varied depending on the substrate
added to the incubation medium. However, diabetes resulted
in no significant changes in the content of this metabolite
under all conditions studied. In contrast to the intracellu-
lar GSH content, GSSG levels in renal tubules of alloxan-
diabetic rabbits was increased by 50–60% when glycerol,
aspartate + glycerol + octanoate, alanine + glycerol + oc-
tanoate or malate were added to the incubation medium, while
in the presence of aspartate alone a decline in the intracellular
GSSG content was observed. Thus, diabetes diminished the
intracellular GSH/GSSG ratio in renal tubules incubated with
all tested substrates but aspartate. In the presence of the lat-
ter glucose precursor diabetes-induced gluconeogenesis was
accompanied by the increase in the intracellular GSH/GSSG
ratio by about 60%. It is worth noting that despite diabetes-
evoked disturbances in the intracellular GSH/GSSG ratio,
there is a relationship between the rate of gluconeogenesis
and glutathione redox state similar to that observed in con-
trol animals. In diabetic animals, the highest GSH/GSSG ratio
was determined in renal tubules intensively producing glu-
cose from either aspartate + glycerol + octanoate or malate,
whereas renal tubules synthesising negligible amounts of
94
Table 2. Mitochondrial GSH and GSSG contents as well as GSH/GSSG ratios in kidney-cortex tubules of control and diabetic
rabbits
Substrates Rabbits GSH (nmol·mg
1
dry weight) GSSG (nmol·mg
1
dry weight) GSH/GSSG
Glycerol C 0.175 ± 0.019 0.013 ± 0.002 13.6 ± 2.1
D 0.106 ± 0.020
∗∗
0.010 ± 0.002
∗∗
10.9 ± 2.0
Aspartate + glycerol + octanoate C 0.324 ± 0.074
0.016 ± 0.002 19.1 ± 3.3
D 0.183 ± 0.017
,∗∗
0.012 ± 0.002
∗∗
16.0 ± 3.0
Renal tubules of control (C) or diabetic (D) rabbits were incubated for 60 min with either 2 mM glycerol or 2 mM aspartate + 2
mM glycerol + 0.5 mM octanoate. Values are means ± S.D. for 4–5 experiments. Statistical significance:
p < 0.05 vs. values
for renal tubules incubated in the presence of glycerol, i.e. under conditions of negligible glucose synthesis (cf. Table 1),
∗∗
p <
0.05 vs. corresponding values for renal tubules isolated from control rabbits.
glucose in the presence of glycerol exhibited the lowest in-
tracellular GSH/GSSG ratio.
In both control and diabetic animals, the mitochondrial
GSH/GSSG ratios (Table 2) were significantly lower than
the corresponding intracellular values (cf. Table 1). How-
ever, when tubules were incubated with aspartate + glycerol
+ octanoate, i.e. under conditions of effective gluconeogene-
sis, values were about 40% higher than those measured in the
presence of glycerol. In mitochondria of alloxan-diabetic rab-
bits the content of the two glutathione forms was decreased,
consequently resulting in no changes in the glutathione redox
state.
Since under conditions of effective gluconeogenesis the
intracellular GSH/GSSG ratio might increase due to the ac-
celerated NADPH generation via pentose phosphate path-
way [18], the diabetes action on the intracellular glucose-6-
phosphate content and NADPH/NADP
+
ratio, estimated in
view of malate/pyruvate ratio measurements [31], has been
investigated. In renal tubules effectively synthesising glucose
from aspartate + glycerol + octanoate under both control and
diabetic conditions the intracellular glucose-6-phosphate lev-
els and malate/pyruvate ratios were much higher than in those
producing negligible glucose amounts in the presence of glyc-
erol. Surprisingly, despite increased gluconeogenic rates, di-
abetes changed neither the intracellular glucose-6-phosphate
level nor NADPH/NADP
+
ratio. On the contrary, in kidney-
cortex of diabetic animals the activity of glutathione reduc-
tase, which utilises NADPH for GSH formation, was twice
higher (157.0 ± 16.8 nmol·mg
1
protein·min
1
) than that in
control ones (76.5 ± 11.5 nmol·mg
1
protein·min
1
).
HFR generation
As shown in Table 4, renal tubules of diabetic rabbits exhib-
ited significantly increased rates of HFR synthesis (at least
2-fold), as concluded from 2,3-DHBA generation following
the addition of 1 mM salicylate into the incubation mixtures
containing various gluconeogenic precursors. It is also inter-
esting that melatonin, a potent antioxidant [32], markedly
attenuated the level of HFR produced by diabetic renal
tubules incubated under various conditions.
Discussion
Although oxidative stress is commonly considered as the
main reason of diabetic complications, the data concerning
diabetes-induced alterations in glutathione status are full of
discrepancies. Most of the authors seem to agree that GSH
level is decreased in erythrocytes [4–8] and serum [33, 34]
of diabetic patients. However, both increased [4, 33] and un-
changed [35] GSSG contents have been observed under dia-
betic conditions.
The data for animals with experimental diabetes are even
more controversial. A decline in GSH level in kidneys of rats
with long-term streptozotocin diabetes has been reported [11,
14]. On the other hand, Bastar et al. [10] demonstrated no
changes in GSH content in kidneys of rats with 33-h strepto-
zotocin diabetes, confirming our findings (cf. Table 1). Sur-
prisingly, there are also reports indicating that long-term ex-
perimental diabetes might lead to an increase in renal GSH
[15] and total glutathione content [9]. In rabbit kidney-cortex
tubules alloxan-diabetes does not alter the intracellular GSH
level, while GSSG content is increased, resulting in a signifi-
cant decline in the intracellular GSH/GSSG ratio (cf. Table 1).
Similarly, in kidneys of 5-week streptozotocin-diabetic rats, a
decreased GSH/GSSG ratio has been reported [36], resulting
however from a diminished GSH content rather than from an
elevated GSSG level.
The discrepancies in the reports concerning renal glu-
tathione status might be caused by the different periods of
diabetes treatment. It is very likely that changes in GSH level
under conditions of prolonged diabetes would also be ob-
served in contrast to renal tubules of 72-h diabetic rabbits
(cf. Table 1). However, it is worth noting that the period of 3
days after alloxan treatment was sufficient to induce oxidative
stress in rabbit renal tubules, manifested in both a decreased
GSH/GSSG ratio (cf. Table 1) and elevated hydroxyl free
radicals formation (cf. Table 4).
95
Among reactive oxygen species, HFR are considered to be
the most reactive and dangerous [37]. Acceleration of HFR
formation in diabetic state might result from hyperketone-
mia, as observed in human erythrocytes in vitro [38]. Ac-
cording to Pennathur et al. [39], hyperglycaemia promotes
protein oxidation by hydroxyl radicals in arterial tissue and
in consequence leads to diabetic vascular disease. Treatment
with dimethylthiourea, HFR scavenger, significantly corrects
nerve and vascular dysfunction in diabetic neuropathy [40],
while prevention of HFR generation in the presence of de-
feroxamine (iron chelator) improves coronary arteries func-
tion in patients with type 1 diabetes mellitus [41], indicating
importance of HFR for pathological disturbances during di-
abetes.
It is necessary to point out, that even though diabetes sig-
nificantly diminishes the intracellular GSH/GSSG ratio in
renal tubules incubated with all tested substrates but aspar-
tate (cf. Table 1), the relationship between the rate of glucose
synthesis and glutathione redox state, that has been described
for renal tubules of control rabbits [18], is also maintained
under diabetic conditions (cf. Fig. 1). Moreover, diabetes-
induced gluconeogenesis from aspartate, occurring probably
due to elevated concentration of endogenous glycerol, ketone
bodies and fatty acids [42], is accompanied by an increased
intracellular GSH/GSSG ratio in comparison with the value
estimated for renal tubules of control animals. However, in
view of the data for control animals, diminished intracel-
lular GSH/GSSG ratios in renal tubules of diabetic rabbits
despite accelerated glucose synthesis from either aspartate +
glycerol + octanoate or malate is surprising. Increase in the
intracellular glucose-6-phosphate level resulting in elevated
NADPH/NADP
+
ratio has been suggested as a mechanism of
gluconeogenesis-induced rise in the intracellular GSH/GSSG
ratio in kidney-cortex tubules of control rabbits [18]. Further-
more, in the presence of high glucose concentrations (10–25
mM) the activity of glucose-6-phosphate dehydrogenase, an
enzyme responsible for NADPH delivery for GSH regenera-
tion, is decreased probably due to its phosphorylation by pro-
tein kinase A [43]. However, in our hands, no diabetes-evoked
changes in either the intracellular glucose-6-phosphate level
or NADPH/NADP
+
ratio have been observed in renal tubules
of diabetic rabbits (cf. Table 3). Thus, a decline in the intra-
cellular GSH/GSSG ratio under diabetic conditions might
result rather from an increased NADPH utilisation than
from its impaired generation via the pentose phosphate
pathway.
An elevated glutathione reductase activity in rabbit kidney-
cortex is in agreement with data for kidneys of streptozotocin-
diabetic rats [11, 36]. According to Atalay et al. [44], who
examined glutathione reductase activity in erythrocytes of
diabetic humans, an increase in the activity of this enzyme
might represent a mechanism of compensatory up-regulation
of glutathione homeostasis in response to oxidative stress. As
Fig. 1. Relationship between the rates of glucose synthesis and the intracel-
lular GSH/GSSG ratios in kidney-cortex tubules isolated from both control
and diabetic rabbits. Each symbol represents data of a separate experiment
with the use of kidney-cortex tubules incubated in the presence of following
metabolites: aspartate (open triangle), glycerol (solid triangle), aspartate +
glycerol + octanoate (solid squares), alanine + glycerol + octanoate (solid
circles), malate (open circles).
previously postulated by Davis et al. [45], diabetes-induced
alterations in activities of antioxidative enzymes seem to be
tissue-dependent. Thus, in organs such as retina [46], brain
and heart [47], diabetes is accompanied by a diminished glu-
tathione reductase activity. The most confusing are data con-
cerning erythrocytes of diabetic patients, in which both ele-
vated [44] and decreased [4, 7, 8] glutathione reductase ac-
tivities have been observed.
In view of these data, it seems likely that the diabetes
induced oxidative stress, as manifested in an accelerated HFR
generation (cf. Table 4), is responsible for a decline in the in-
tracellular GSH/GSSG ratio in renal tubules of diabetic rab-
bits. Melatonin, probably due to its antioxidative properties
[32], attenuates diabetes-induced elevation of HFR synthe-
sis in renal tubules. Other authors have also postulated this
96
Table 3. Intracellular malate, pyruvate and glucose-6-phosphate contents as well as malate/pyruvate ratios in rabbit kidney-cortex tubules of
control and diabetic rabbits
Malate Pyruvate Glucose-6-phosphate
Substrates Rabbits (µmol·g
1
dry weight) (µmol·g
1
dry weight) (µmol·g
1
dry weight) Malate/pyruvate
Glycerol C 0.25 ± 0.04 0.29 ± 0.04 0.030 ± 0.005 0.86 ± 0.15
D 0.30 ± 0.05 0.26 ± 0.05 0.034 ± 0.005 1.11 ± 0.20
Aspartate + glycerol + octanoate C 0.88 ± 0.14
0.33 ± 0.04 0.149 ± 0.030
2.57 ± 0.38
D 0.80 ± 0.11
0.31 ± 0.03 0.173 ± 0.036
2.69 ± 0.40
Renal tubules of control (C) and diabetic (D) rabbits were incubated for 60 min under conditions described in the legend to Table 2. Values are
means ± S.D. for 4–5 experiments. Statistical significance:
p < 0.05 vs. values for renal tubules incubated in the presence of glycerol, i.e.
under conditions of negligible glucose synthesis (cf. Table 1).
Table 4. Production of 2,3-DHBA in kidney-cortex tubules of control and diabetic rabbits
2,3-DHBA production (nmol·h
1
·g
1
dry weight)
Substrates Rabbits Melatonin +Melatonin (100 µM)
Glycerol C 13.1 ± 2.6 10.4 ± 1.8
D 29.4 ± 10.2
10.5 ± 2.8
∗∗
Aspartate C 15.4 ± 1.7 10.4 ± 2.0
D 26.8 ± 5.6
9.7 ± 0.7
∗∗
Aspartate + glycerol + octanoate C 14.2 ± 4.5 9.6 ± 2.3
D 32.8 ± 3.9
10.6 ± 3.2
∗∗
Alanine + glycerol + octanoate C 12.8 ± 2.9 10.8 ± 1.9
D 28.7 ± 6.4
11.6 ± 3.7
∗∗
Renal tubules isolated from control (C) and diabetic (D) rabbits were incubated in the presence of 1 mM SAL for
60 min. Glycerol, amino acids, octanoate and malate were added to incubation medium at 2, 2, 0.5 and 5 mM
concentrations, respectively. Values are means ± S.D. for 4–5 experiments. Statistical significance:
p < 0.05 vs. corre-
sponding values for renal tubules isolated from control rabbits,
∗∗
p < 0.05 vs. corresponding values without melatonin.
compound as a beneficient agent during diabetes therapy. At
100 and 1000 µM concentrations, melatonin has been demon-
strated to restore activities of antioxidative enzymes and GSH
levels in diabetic skin fibroblasts [48]. Similar observations
were made for liver, heart, kidney [49] and erythrocytes [50]
of diabetic rats. Moreover, it has been suggested that mela-
tonin might protect against diabetes-related nephropathy [51]
and attenuate hyperglycaemia [12, 52].
Acknowledgements
The technical assistance of Miss B. Dabrowska is acknowl-
edged. We are also very grateful to Dr. Z. Bartoszewicz (Med-
ical University of Warsaw) for making available the electro-
chemical detector for HFR measurements as well as to Mr.
L. Lipiec (Brenntag-Polska) for the generous gift of silicone
oil. This investigation was supported by grants of the Ministry
of Scientific Research and Information Technology (Nos. 3
P05A 049 25 and BW 1601/57).
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... Akita::Keap1 FA/FA mice display suppressed cast formation. serves as an abundant and important antioxidant in this situation [82,83]. In Akita mouse kidneys, we detected a decrease in GSH levels, which deteriorated upon the depletion of Nrf2. ...
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Oxidative stress is an essential component in the progression of diabetic kidney disease (DKD), and the transcription factor NF-E2-related factor-2 (Nrf2) plays critical roles in protecting the body against oxidative stress. To clarify the roles of Nrf2 in protecting against DKD, in this study, we prepared compound mutant mice with diabetes and loss of antioxidative defense. Specifically, we prepared compound Ins2Akita/+ (Akita) and Nrf2 knockout (Akita::Nrf2−/−) or Akita and Nrf2 induction (Akita::Keap1FA/FA) mutant mice. Eighteen-week-old Akita::Nrf2−/− mice showed more severe diabetic symptoms than Akita mice. In the Akita::Nrf2−/− mouse kidneys, the glomeruli showed distended capillary loops, suggesting enhanced mesangiolysis. Distal tubules showed dilation and an increase in 8-hydroxydeoxyguanosine-positive staining. In the Akita::Nrf2−/− mouse kidneys, the expression of glutathione (GSH) synthesis-related genes was decreased, and the actual GSH level was decreased in matrix-assisted laser desorption/ionization mass spectrometry imaging analysis. Akita::Nrf2−/− mice exhibited severe inflammation and enhancement of infiltrated macrophages in the kidney. To further examine the progression of DKD, we compared forty-week-old Akita mouse kidney compounds with those of Nrf2-knockout or Nrf2 mildly induced (Akita::Keap1FA/FA) mice. Nrf2-knockout Akita (Akita::Nrf2−/−) mice displayed severe medullary cast formation, but formation was ameliorated in Akita::Keap1FA/FA mice. Moreover, in Akita::Keap1FA/FA mice, tubule injury and inflammation-related gene expression were significantly suppressed, which was evident in Akita::Nrf2−/− mouse kidneys along with marked medullary cast formation. These results demonstrate that Nrf2 contributes to the protection of the kidneys against DKD by suppressing oxidative stress and inflammation.
... These findings are consistent with our previous observation that GSH content is significantly reduced if Lias gene expression level drops by 50%. 25 GSH is the predominant intracellular non-protein thiol compound, and renal GSH levels are decreased during Metabolism DN. 26 Other investigators also showed that the effect of LA on reno-protection was associated with reduced oxidative stress and restoration of renal cortical levels of GSH. 27 MDA is a peroxide formed by oxygen free radicals attacking unsaturated fatty acids in biomembrane, which can reflect the degree of lipid peroxidation in organism and the degree of damage of cells attacked by free radicals. ...
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Introduction Diabetic nephropathy (DN) develops in about 40% of patients with type 2 diabetes and remains the leading cause of end-stage renal disease. The mechanisms of DN remain to be elucidated. Oxidative stress is thought to be involved in the development of DN but antioxidant therapy has produced conflicting results. Therefore, we sought to define the role of antioxidant in retarding the development of DN in this study. Research design and methods We generated a new antioxidant/diabetes mouse model, Lias H/H Lepr db/db mice, by crossing db/db mice with Lias H/H mice, which have overexpressed Lias gene (~160%) compared with wild type, and also correspondingly increased endogenous antioxidant capacity. The new model was used to investigate whether predisposed increased endogenous antioxidant capacity was able to retard the development of DN. We systemically and dynamically examined main pathological alterations of DN and antioxidant biomarkers in blood and kidney mitochondria. Results Lias H/H Lepr db/db mice alleviated major pathological alterations in the early stage of DN, accompanied with significantly enhanced antioxidant defense. The model targets the main pathogenic factors by exerting multiple effects such as hypoglycemic, anti-inflammation, and antioxidant, especially protection of mitochondria. Conclusion The antioxidant animal model is not only very useful for elucidating the underlying mechanisms of DN but also brings insight into a new therapeutic strategy for clinical applications.
... Diabetic nephropathy (DN), referring to the deterioration of kidney function associated to both Type 1 and Type 2 diabetes, can progress to chronic kidney disease and, in fact, is a strong predictor of mortality in diabetic patients. Emerging evidence points to the oxidative and nitrosative stress as the underlying mechanism by which chronic hyperglycemia causes renal cellular damage [129][130][131][132]. Low levels of renal GSH have been described associated with DN [131,133,134]. In turn, dietary GSH supplementation has been shown to partially protect against many of the pathological changes due to DN [135]. ...
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... In addition, a marked upregulation of the cellular antioxidant factors, such as Glutathione peroxidase (GPx), Heme oxygenase-1 (HO-1), NAD (P)H Quinone Dehydrogenase 1) (NQO 1), and glutathione reductase (GR), was detected. Of note, melatonin possesses a potent direct and indirect antioxidant capacities as well as anti-inflammatory function, which are involved in cell membrane stabilization and cryoprotection against oxidative stress-related damage and inflammation [164][165][166][167][168]. Melatonin promotes the increased synthesis of glutathione and the production of the intrinsic antioxidant agents that quell the oxidative stress consequences [169][170][171]. [172]. ...
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... Furthermore, oxidative stress results in tissue damage causing protein oxidation, lipid peroxidation via formation of hydroxyl radical, which is considered as one of the most reactive and hazardous free radical species. 30,31 In the present study, superpulsed 904 nm laser PBM treatment markedly decreased both ROS and NO levels in the burn wound milieu. As LPO is usually ...
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A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
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Oxidative stress occurs in diabetic patients and experimental models of diabetes. We examined whether two antioxidants, melatonin and taurine, can ameliorate diabetic nephropathy. Enhanced expression of glomerular TGF-β1 and fibronectin mRNAs and proteinuria were employed as indices of diabetic nephropathy. Experimental diabetes was induced by intravenous injection of streptozotocin 50 mg/kg. Two days after streptozotocin, diabetic rats were assigned to one of the following groups: i) untreated; ii) melatonin supplement by 0.02% in drinking water; or iii) taurine supplement by 1% in drinking water. Four weeks after streptozotocin, diabetic rats (n = 6: plasma glucose 516 ± 12 mg/dl) exhibited 6.1 fold increase in urinary protein excretion, 1.4 fold increase in glomerular TGF-β1 mRNA, 1.7 fold increase in glomerular fibronectin mRNA, 2.2 fold increase in plasma lipid peroxides (LPO), and 44 fold increase in urinary LPO excretion above the values in control rats (n = 6: plasma glucose 188 ± 14 mg/dl). Chronic administration of melatonin (n = 6) and taurine (n = 6) prevented increases in glomerular TGF-β1 and fibronectin mRNAs and proteinuria without having effect on blood glucose. Both treatments reduced lipid peroxidation by nearly 50%. The present data demonstrate beneficial effects of melatonin and taurine on early changes in diabetic kidney and suggest that diabetic nephropathy associated with hyperglycemia is largely mediated by oxidative stress. Science
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A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
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Kidney International aims to inform the renal researcher and practicing nephrologists on all aspects of renal research. Clinical and basic renal research, commentaries, The Renal Consult, Nephrology sans Frontieres, minireviews, reviews, Nephrology Images, Journal Club. Published weekly online and twice a month in print.
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The metabolism of glutathione and activities of its related enzymes were studied in erythrocytes from patients with non-insulin-dependent diabetes mellitus (NIDDM). A decrease in the levels of the reduced form of glutathione and an increase in the levels of glutathione disulfide were found in erythrocytes of diabetics. To elucidate these changes in the levels of glutathione, synthetic and degradative processes were studied. The activity of gamma-glutamylcysteine synthetase was significantly lower in diabetics than in normal controls. The activity of glutathione synthetase of each group was the same. The rate of outward transport of glutathione disulfide in diabetics decreased to approximately 70% of that of normal controls. The activity of glutathione reductase decreased in diabetics. These data suggest that the decrease in the levels of reduced form of glutathione in erythrocytes of diabetics is brought about by impaired glutathione synthesis and that the increase in the levels of glutathione disulfide is brought about by the decreased transport activity of glutathione disulfide through the erythrocyte membrane together with a decrease in the activity of glutathione reductase. These data also suggest that the impairment of glutathione metabolism weakens the defense mechanism against oxidative stress in erythrocytes of diabetics.
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Effects of chronic ethanol consumption, withdrawal and fasting on the free cytosolic NADP+/NADPH ratio and NADPH-regenerating enzyme activities of rat liver were studied. Ethanol consumption was shown to decrease the NADP+/NADPH ratio in non-fasted rats, and both ethanol withdrawal and fasting in ethanol-fed animals appeared to increase the ratio to the normal or higher level. Any treatment of rats caused the complex interaction on hepatic NADPH-regenerating enzyme activities, none of the enzyme activity correlating with the free cytosolic NADP+/NADPH ratio. Relationship between free cytosolic NADP+/NADPH ratio and lipogenic capacity of withdrawn rat liver is discussed, and a hypothesis for development of the fatty liver is suggested.
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1.1. The intracellular distribution of phosphoenolpyruvate carboxykinase (EC 4.1.1.32, PEPCK-ase) varies among the livers of rabbit and rat. In rabbit liver, most of the PEPCK-ase is located in mitochondria; in rat liver most of the enzyme is in the soluble fraction.2.2. Fasting and hydrocortisone administration were without effect on rat and rabbit liver mitochondrial PEPCK-ase, although amounts of this enzyme in soluble fraction of these same livers were nearly doubled.3.3. Hydrocortisone administration also caused marked increases in rabbit kidney cortex soluble-fraction PEPCK-ase, but produced no significant changes in mitochondrial PEPCK-ase levels.4.4. In alloxan diabetes PEPCK-ase activity of rat kidney cortex extract displayed an average 2.2-fold rise over normal values.5.5. At early stages of embryonic development rabbit liver displayed no PEPCK-ase activity until the 25th day of embryogenesis when the activity was extremely low in both mitochondrial and soluble liver fractions. On the following days of fetal development PEPCK-ase activity grew gradually in both fractions. Within the first postnatal day the enzyme activity rose abruptly to values higher than those of adult animals. Variations in PEPCK-ase activity in rabbit liver have been followed for 30 days of their postnatal development.
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1.1. In hepatocytes isolated from both fed and fasted rabbits glucagon and dibutyryl 3',5'-cAMP resulted in an increase of the glucose synthesis from 23 to 72% depending on the glucose precursor.2.2. In contrast, neither glucagon nor dibutyryl 3',5'-cAMP affected the rate of glucose formation in hepatocytes isolated from alloxan-diabetic animals.3.3. Both glucagon and dibutyryl 3',5'-cAMP slightly shifted the NADH/NAD+ ratios, in the mitochondrial and cytosolic compartments, towards a more reduced state, as manifested by an increase in the 3-hydroxybutyrate/acetoacetate and lactate/pyruvate ratios, respectively.4.4. However, the contribution of both mitochondrial and cytosolic phosphoenolpyruvate carboxykinases to gluconeogenesis in hepatocytes isolated from starved animals seems to be unaltered by glucagon, as concluded from a similar inhibitory effect of quinolinate on glucose synthesis from lactate in either the presence or absence of glucagon.5.5. The stimulation of gluconeogenesis by glucagon, from lactate in hepatocytes from starved rabbits, was accompanied by elevated levels of malate, phosphoenolpyruvate, 3-phosphoglycerate plus 1,3-diphosphoglycerate, fructose 6-phosphate and glucose 6-phosphate, while the content of both pyruvate and fructose 1,6-bisphosphate was decreased.6.6. These changes are consistent with a facilitation by glucagon of reactions located in gluconeogenic pathway between pyruvate and phosphoenolpyruvate and between fructose 1,6-bisphosphate and glucose.7.7. A decreased rate of lactate production, from fructose in hepatocytes incubated with glucagon, may be correlated with the hormone-induced decline of glycolysis.8.8. The 2-fold increase of K0.5 value of pyruvate kinase for phosphoenolpyruvate in livers of glucagontreated animals is consistent with this suggestion.9.9. On the basis of presented results it is possible to conclude that despite differences in the intracellular localization of phosphoenolpyruvate carboxykinase in livers of rat and rabbit, the site of glucagon action on the gluconeogenic pathway in hepatocytes isolated from these two species is similar.