In the present study, we observed a significant decrease in the respiratory activity of rat liver mitochondria with aging, and an increase in the advanced gly-cation endproduct-modified pr
Trang 1rat liver mitochondrial matrix during aging
Maud Hamelin, Jean Mary, Michal Vostry, Bertrand Friguet* and Hilaire Bakala*
Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Universite´ Paris 7-Denis Diderot, France
Aging is characterized by gradual deterioration of
cel-lular functions [1] associated with cumulative damage
to intracellular macromolecules, particularly proteins
[2] Several lines of evidence suggest that mitochondria
play a key role in aging, both by producing
intracellu-lar reactive oxygen species (ROS) and by being the
most adversely affected organelles during aging [3]
The electron transport chain located in the inner
mito-chondrial membrane is implicated in production of
ATP via oxidative phosphorylation, and is also known
to be the major intracellular site of free radical
genera-tion, such as that of superoxide anions and, sub-sequently, other potentially deleterious ROS [4] Numerous data have indicated an age-related increase
in the rate of mitochondrial free radical generation and in the extent of oxidative damage to mitochondrial macromolecules [5–9], especially enzymes involved in the respiratory chain, leading to impairment of respira-tory activity [7,9–12]
Although direct oxidation of proteins and other macromolecules is believed to be the main type of endogenous damage during aging [3,13,14], ROS can
Keywords
aging; glycation; liver mitochondria;
proteomics; urea cycle enzymes
Correspondence
H Bakala, Laboratoire de Biologie et
Biochimie Cellulaire du Vieillissement,
EA3106 ⁄ IFR117, Universite´ Paris 7-Denis
Diderot, 2 place Jussieu, 75251 Paris,
Cedex 05, France
Fax: +33 1 44 27 39 25
Tel: +33 1 44 27 82 27
E-mail: bakala@paris7.jussieu.fr
*These authors contributed equally to this
work
(Received 22 May 2007, revised 21
Septem-ber 2007, accepted 25 SeptemSeptem-ber 2007)
doi:10.1111/j.1742-4658.2007.06118.x
Aging is accompanied by gradual cellular dysfunction associated with an accumulation of damaged proteins, particularly via oxidative processes This cellular dysfunction has been attributed, at least in part, to impair-ment of mitochondrial function as this organelle is both a major source of oxidants and a target for their damaging effects, which can result in a reduction of energy production, thereby compromising cell function In the present study, we observed a significant decrease in the respiratory activity
of rat liver mitochondria with aging, and an increase in the advanced gly-cation endproduct-modified protein level in the mitochondrial matrix Wes-tern blot analysis of the glycated protein patWes-tern after 2D electrophoresis revealed that only a restricted set of proteins was modified Within this set,
we identified, by mass spectrometry, proteins connected with the urea cycle, and especially glutamate dehydrogenase, which is markedly modified in older animals Moreover, mitochondrial matrix extracts exhibited a signifi-cant decrease in glutamate dehydrogenase activity and altered allosteric regulation with age Therefore, the effect of the glycating agent methylgly-oxal on glutamate dehydrogenase activity and its allosteric regulation was analyzed The treated enzyme showed inactivation with time by altering both catalytic properties and allosteric regulation Altogether, these results showed that advanced glycation endproduct modifications selectively affect mitochondrial matrix proteins, particularly glutamate dehydrogenase, a crucial enzyme at the interface between tricarboxylic acid and urea cycles Thus, it is proposed that glycated glutamate dehydrogenase could be used
as a biomarker of cellular aging Furthermore, these results suggest a role for such intracellular glycation in age-related dysfunction of mitochondria
Abbreviations
AGE, advanced glycation endproduct; CEL, N-(carboxyethyl)lysine; CML, carboxymethyl-lysine; GDH, glutamate dehydrogenase; GO, glyoxal; MGO, methylglyoxal; OCT, ornithine carbamoyl transferase; RCR, respiratory control ratio; ROS, reactive oxygen species; TCA, tricarboxylic acid.
Trang 2also affect protein function through either
lipoperoxi-dative production of reactive aldehydes [15] or
glycoxi-dation pathways [16] Indeed, there is a causal
relationship between hyperglycemia-induced ROS
generation and intracellular advanced glycation
end-product (AGE) formation [17] This rise in AGE was
shown to be primarily if not exclusively due to a rapid
increase in AGE-forming methylglyoxal concentration
[18]
Alpha-dicarbonyl compounds such as glyoxal (GO)
and methylglyoxal (MGO) are physiological, highly
reactive intermediates involved in the Maillard reaction
[19] Interestingly, MGO may originate from various
biochemical pathways, including dephosphorylation of
glycolytic intermediates, metabolites of the polyol
pathway and metabolism of aminoacetones [20] At
physiological concentrations, MGO primarily targets
the arginine residues of proteins, leading primarily to
AGE-imidazolone [21,22] and -lysine residues to form
AGE adducts: N-(carboxyethyl)lysine (CEL) and
meth-ylglyoxal lysine dimer [23]
In a previous study, using an immunochemical
method, we showed that glycated proteins accumulate
in the rat liver mitochondrial matrix with aging [24]
In the present study, we show that glycation targets a
limited set of proteins, the most severely affected being
glutamate dehydrogenase, a crucial enzyme at the
interface between the tricarboxylic acid (TCA) and
urea cycles, indicating the interference of matrix
enzymes in mitochondrial impairment function with
aging
Results
Age-related changes in the respiratory function
of isolated mitochondria
Mitochondria were isolated from the livers of young
(3-month-old) and old (27-month-old) Wistar rats and
their respiratory parameters were measured As shown
in Table 1, there was a significant decrease in the rates
of mitochondrial oxygen consumption between 3-month-old and 27-month-old rats with gluta-mate⁄ malate or succinate as substrates The ADP ⁄ O ratios (P⁄ O) obtained whatever the substrate used indi-cated no change in coupling efficiency Although the respiratory control ratio (RCR) slightly decreased with glutamate⁄ malate substrate during aging, no change was observed using succinate In the presence of 2,4-dinitrophenol, oxygen consumption rates were the same in the presence of ADP (state 3) at each age Together, these results indicated age-related impair-ment of mitochondrial function
Determination of 6D12-detectable AGE content
in mitochondrial matrix proteins
We next investigated the levels of AGE-modified pro-teins to determine whether dysfunction of the organelle parallels the accumulation of modified mitochondrial matrix proteins For this purpose, we used monoclonal anti-AGE IgG (6D12) in a competitive ELISA using carboxymethyl-lysine (CML)-modified BSA as standard to evaluate AGE-modified protein content (Fig 1) AGE content increased significantly, by 48% from 12.01 ± 0.71 AUÆlg protein)1 (n¼ 8) in 3-month-old rats to 17.81 ± 1.83 AUÆlg protein)1 (n¼ 9) in 27-month-old rats (P < 0.01) These data indicated age-associated accumulation of AGE adducts
in mitochondrial matrix proteins
Identification of glycated mitochondrial matrix proteins by LC-MS⁄ MS after 2D gel
electrophoresis and western blotting 2D gel electrophoresis and western blotting Standard 2D gel electrophoresis of liver mitochondrial matrix proteins from young and old rats was run in parallel (Fig 2) Electrophoregrams showed a typical pattern of total protein, and more than a thousand
Table 1 Biochemical respiratory parameters in rat liver mitochondria from 3-month-old and 27-month-old rats Oxygen consumption rates were measured polarographically in the presence of either 10 m M succinate or 5 m M glutamate ⁄ 5 m M malate State 3 respiration was deter-mined after adding 310 nmol of ADP State 4 respiration is the rate of O 2 consumption after depletion of ADP Data represent the mean ± SEM n, number of animals *P < 0.05; **P < 0.01 versus 3-month-old rats.
Trang 3spots were detected using 2D Elite Master Software.
Western blot analysis using anti-AGE IgG (Fig 3) in
samples from both young and old rats revealed that
only a small number of proteins among the thousand
protein spots observed on gel stained with colloidal
Coomassie blue were targeted for glycation Moreover,
AGE-modified proteins were already present, although
to a lesser extent, in samples from young rats We
noted individual variations both in the nature of
modi-fied proteins and in their extent of labelling within
Fig 1 Determination of 6D12-detectable AGE protein content in
liver mitochondrial matrix with aging The AGE adduct content in
mitochondrial matrix proteins from 3-month-old and 27-month-old
rats was assayed by competitive ELISA using monoclonal anti-AGE
IgG (clone 6D12) The results are expressed as AUÆlg protein)1and
represent the mean ± SEM **P < 0.01 versus 3-month-old rats.
Number of animals is given in parentheses above the bar graphs.
A
250
-150
-100
-75
-50
-37
-25
-pI
250
150
100
75
50
37
25
pI
Fig 2 2D gel electrophoresis profile of liver
mitochondrial matrix proteins Liver
mito-chondrial matrix proteins (150 lg) were
sub-jected to isoelectrofocusing and subsequent
SDS ⁄ PAGE electrophoresis under reducing
conditions Gels containing samples from
(A) 3-month-old and (B) 27-month-old rats
were stained with colloidal Coomassie blue.
A
B
Fig 3 2D gel electrophoresis and western blotting analysis of liver mitochondrial matrix proteins; identification of AGE-modified pro-teins with aging Samples (150 lg) were subjected to isoelectrofo-cusing and subsequent SDS ⁄ PAGE under reducing conditions Gels were subjected to western blotting using monoclonal anti-AGE IgG (clone 6D12) to detect AGE-modified proteins in samples from (A) 3-month-old and (B) 27-month-old rats Matched proteins were identified by tandem LC-MS ⁄ MS mass spectrometry as: (1) gluta-mate dehydrogenase; (2) catalase; and (3) ornithine carbamoyl transferase.
Trang 4each age group Furthermore, we observed that
anio-nic isoforms appeared to be modified with age for
trails 1 and 3 (Fig 3B) Only spots from trails of
iso-forms numbered 1–3 that exhibited a significantly
increased yield of modifications with aging in a
repro-ducible way were retained for further analysis
(Fig 3A,B) and were matched with colloidal
Coomas-sie blue staining spots (Fig 2B), which were subjected
to LC-MS⁄ MS protein identification
Protein identification Proteins from selected spots were identified automati-cally by a computer program Three series of MS⁄ MS spectra were obtained (data not shown) At least two spots for each trail of isoforms were identified with sufficient peptide coverage (13–32%), and analysis led
to the characterization of three proteins in a narrow pI range (7.15–9.12), with a molecular mass in the range
Table 2 Identification of proteins located in spots 1–3 Tryptic peptides from in-gel digestion were subjected to LC ⁄ MS ⁄ MS analysis as described in the Experimental procedures Peptide identification was evaluated using Xcorr scores that measure similarities between mass-to-charge (m ⁄ z) ratios for fragment ions predicted from amino acid sequences and fragment ions observed in the MS ⁄ MS spectrum Xcorr, cross correlation scores; z, charge of the precursor ion; M*, oxidized methionine, C@, alkylated cysteine.
Spot No.
Protein UniProt
accession No.
Mass
dehydrogenase 1,
mitochondrial precursor,
P10860
K.KGFIGPGIDVPAPDM*STGER.E 212–231 2060.01 0.64 3 2.72 K.GFIGPGIDVPAPDM*STGER.E 213–231 1931.9 0.43 2 3.04
carbamoyltransferase,
mitochondrial, P00481
DM z Xcorr
Trang 539.9–61.4 kDa (Table 2) The number of peptides
lead-ing to the identification of each protein varied between
six and 12 The proteins identified corresponded to
glutamate dehydrogenase (GDH) for trail 1, catalase
for trail 2 and ornithine carbamoyl transferase (OCT)
for trail 3 By contrast to catalase, a potent enzyme in
antioxidant defense, the other two enzymes (GDH and
OCT) involved in the mitochondrial urea cycle
exhib-ited a wide extent of modification at 27 months
compared to 3 months Further investigations were
performed with GDH because this enzyme is relevant
due to its essential role in between the urea and TCA
cycles
Age-associated increased glycation and
decreased activity of GDH
Using anti-GDH serum, we first checked by western
blotting that GDH content within the mitochondrial
matrix was unchanged with age (data not shown)
GDH was then immunoprecipitated from matrix
sam-ples and western blotting was carried out to investigate
the increased GDH glycation rate with aging (Fig 4)
Spots of GDH protein exhibited identical intensity in
3-month-old (49.86 ± 3.10 AU) and 23-month-old
rats (54.98 ± 2.39 AU), whereas AGE-labelling
inten-sity significantly increased by 45%, from 32.71 ±
2.65 AU (n¼ 4) in 3-month-old rats to 47.29 ±
3.67 AU (n¼ 3) in 23-month-old rats (P < 0.01)
(Fig 4B) The rate of glycation expressed as the ratio
of AGE⁄ GDH intensity was significantly increased by 30%, from 0.66 ± 0.05 (n¼ 4) in 3-month-old rats to 0.86 ± 0.03 (n¼ 3) in 23-month-old rats (P < 0.01) (Fig 4B) These data indicate that GDH underwent increased glycation with aging
We next examined whether the glycation modifica-tion demonstrated above was associated with impair-ment of GDH function For this purpose, we measured GDH activity in mitochondrial matrix extracts (50 lg of total protein) in the absence and presence of allosteric effectors (ADP and GTP) (Table 3) The results obtained revealed a significant decrease (23%) in GDH activity with aging, from 2.47 ± 0.19 UÆmg)1 in 3-month-old rats to 1.89 ± 0.07 UÆmg)1 in 23-month-old rats (n¼ 4) (P ¼ 0.02)
In the presence of the ADP activator, this activity was enhanced by 1.20-fold in young samples, although slightly less (1.14-fold) in old samples However, this GDH activity markedly collapsed with the GTP inhibi-tor, with residual activity barely representing 8% in young samples compared to 12% in old samples (P < 0.01) These data suggest an inhibitory effect of glycation on GDH activity, in addition to a slightly altered response to allosteric regulation with aging
Effect of MGO on purified GDH activity and its allosteric regulation
To ascertain whether the glycation impact upon GDH activity was due to modifications in lysine⁄ arginine
A
B
Fig 4 Immunochemical identification of glycated GDH from mitochondrial matrix GDH from matrix samples (500 lg) was
immunoprecipitat-ed and samples resolvimmunoprecipitat-ed by SDS ⁄ PAGE electrophoresis Two identical gels from each sample run in parallel were subjected to western blotting using either monoclonal anti-AGE IgG or GDH polyclonal antibody to detect AGE modifications or GDH antigen content, respectively,
in samples from 3-month-old and 23-month-old rats (A) Blots were semiquantified by densitometry scanning and density expressed in arbitrary units (AU) Results are presented as the ratio of the AGE ⁄ GDH rate (B) Values are the means ± SEM **P < 0.01 versus 3-month-old rats.
Trang 6residues, we used MGO as a potent glycating agent
generated intracellularly to investigate the effect on
purified GDH
In vitro MGO modification of GDH
A purified GDH sample (10 lg) was incubated with
varying concentrations of MGO for up to 24 h and
subjected to western blotting (Fig 5) with monoclonal
anti-AGE IgG or polyclonal anti-GDH serum After
24 h of incubation, the GDH protein was
MGO-derived AGE-modified and the extent of modification
increased with the concentration of MGO (Fig 5A),
whereas the GDH immunolabelling intensity
concur-rently decreased (Fig 5B) These results clearly
demon-strated that glycated enzyme partially lost its
antigenicity, indicating that some antigenic sites of
GDH could be masked by glycation adducts
Effect of MGO on GDH activity and kinetic parameters
In vitro MGO treatment of purified GDH resulted in decreased activity with time of incubation and an increasing MGO concentration (Table 4) With 1 mm MGO, GDH activity significantly decreased at 30 min
of incubation, and markedly dropped by 37% within
5 h compared to control This MGO effect on GDH activity was compared with that induced by GO, an a-dicarbonyl metabolite presumed to possess noxious specificity like MGO Treatment with GO led to a sig-nificant inhibition effect with as little as 50 lm (38%), whereas a high concentration (1 mm) led to strong inhibition (67%), providing evidence that GDH activ-ity is altered by glycation modification Using amino acid analysis, we noted that both arginine (24 : 30) and lysine (22 : 32) residues were damaged in MGO-treated GDH, whereas 22 and eight residues, respectively, were damaged with GO, indicating that MGO was equally noxious to lysine and arginine residues, leading to the loss of part of its activity In addition, analysis of enzymatic parameters (Vmax and
Km) of GDH modified by 1 mm MGO for 24 h using a-ketoglutarate as substrate (Fig 6) showed that MGO treatment altered only the maximum velocity (Vmax), whereas the apparent Kmvalue did not change
Effect of MGO on GDH allosteric regulation
We next analyzed the allosteric regulator effects on purified GDH activity before and after treatment with MGO At indicated times, the activity of the enzyme incubated with 1 mm MGO was measured in the pres-ence of constant concentrations of allosteric effectors (activators: 250 lm ADP or 10 mm leucine; inhibitor:
30 lm GTP) GDH activity at t0 in the absence of ef-fectors (28.6 UÆmg enzyme)1) was set at 100% activity
As shown in Fig 7, GDH activity spontaneously decreased with time of incubation This decline was accentuated when GDH was incubated with 1 mm MGO (Fig 7A) and the activity ratio shifted from 0.85-fold to 0.71-fold at t240 versus t0 In the presence
of an ADP effector (Fig 7B), native GDH activity exhibited a significant 1.66-fold increase (Fig 7B, left panel), whereas this increase fell to 1.44-fold when GDH was preincubated with 1 mm MGO (Fig 7B, right panel) With leucine as an effector, we observed a similar phenomenon (Fig 7C); the activity increased
by 1.55-fold at 240 min with native enzyme (Fig 7B, left panel) and by 1.24-fold when the enzyme was preincubated with MGO (Fig 7B, right panel) These
Table 3 Age-related changes in GDH activity and allosteric effector
sensitivity Liver mitochondrial matrix extracts (50 lg total protein)
from young (3-month-old) and old (23-month-old) rats were
sub-jected to the GDH activity assay in the direction of a-ketoglutarate
amination, in the absence (control) or presence of constant
concen-trations of allosteric effectors (activator: 250 l M ADP; inhibitor:
10 l M GTP) Values are expressed as specific activity (UÆmg)1)
(control) and activity determined in the presence of effectors is
given as a percentage of control in the absence of effectors, at
each age Data represented the mean ± SEM (n ¼ 4) *P ¼ 0.02;
**P < 0.01 versus 3-month-old rats.
GDH activity
Age
3 months (n ¼ 4)
23 months (n ¼ 4) GDH activity (no effector) 2.47 ± 0.19 1.89 ± 0.07*
% of GDH activity (+ ADP) 120.88 ± 4.75 114.71 ± 16.71
% of GDH activity (+ GTP) 8.23 ± 0.75 11.57 ± 0.59**
4 3 2
1
E
G
A
A
4 3 2 1 H D G
B
Fig 5 Western blot detection of purified GDH modified by MGO.
Purified GDH (Sigma) samples (10 lg) were incubated with or
with-out varying MGO concentrations for 5 h and aliquots (1 lg) were
subjected to SDS ⁄ PAGE under reducing conditions Gels were
sub-mitted to western blotting using either monoclonal anti-AGE IgG
(clone 6D12) to detect MGO-derived AGE in GDH samples (A) or
GDH polyclonal antibody to determine whether the GDH load was
preserved (B) Lane 1, control, 0 l M MGO; lane 2, 50 l M MGO;
lane 3, 200 l M MGO; lane 4, 1 m M MGO.
Trang 7results indicate that GDH stimulation induced by
allo-steric effectors was partly abolished when the enzyme
was previously treated with MGO On the other hand,
the GTP inhibitor (Fig 7D) exhibited an efficient
effect on GDH activity; the activity, which was barely
0.44-fold at 240 min (Fig 7D, left panel), rose to
0.71-fold when GDH was pretreated with MGO (Fig 7D,
right panel), indicating the capacity of MGO to
abro-gate the inhibitory effect of GTP on GDH activity
Together, these results clearly demonstrate the
inhibi-tory effect of MGO on GDH activity, which
rein-forced the decline of its activity with time In addition,
this MGO modification deeply altered the
responsive-ness of GDH to its respective allosteric effectors, in
agreement with data observed in vivo
Discussion
In the present study, we have demonstrated a decline in mitochondrial respiratory chain activity upon aging concomitant with an accumulation of AGE-modified proteins in the mitochondrial matrix These modifica-tions affect several proteins that are targets for glyca-tion damage and, although some of these proteins are already modified at a young age (3-month-old rats), the level of damage significantly increased with increasing age (27-month-old rats) Among the glycated proteins, trails of isoforms appeared, in agreement with modifi-cations targeting the basic amino acids arginine and lysine, subsequently turning off their ionic charge In support of this assertion, recent studies have shown that glycation was associated with both loss of basic groups and shifts in pK of the acidic groups, consistent with a reduction in effective anionic charge [23,25] The identification of these proteins, which are increasingly glycated with aging, revealed three enzymes, GDH, OCT and catalase Catalase, a crucial antioxidant defense enzyme highly expressed in peroxisomes, and also constitutively present in the heart and liver mito-chondria [26–28], is maintained with GDH and OCT, which belong to the urea cycle GDH, in particular, which markedly emerged as being modified at
27 months, plays a key role in connecting TCA to the urea cycles, and exhibited a loss of activity and altera-tions in its allosteric properties with aging Interest-ingly, all these modified proteins are different from those preferentially found to be oxidized during aging
in the mitochondrial matrix, as previously identified (i.e aconitase [29] and adenine nucleotide translocase [30]) To determine whether the age-related inhibition
of GDH activity demonstrated here was related to glycation modification, purified GDH was treated with the a-dicarbonyl metabolite MGO Incubation of GDH with this compound resulted in time-dependent inacti-vation of the enzyme, consistent with the damaging
Table 4 Effect of MGO and GO concentrations on GDH activity Purified GDH samples (10 lg) were incubated in 100 m M TEA-HCl buffer
pH 7.3 with varying concentrations (0.05, 0.200 and 1 m M ) of either MGO or GO as glycating agents for variable times At indicated times, enzyme activity was determined spectrophotometrically on aliquots (1 lg) Data are expressed as a percentage of GDH activity control at t0for each concentration and represent the mean ± SEM (n, number of animals) *P < 0.05; **P < 0.01; ***P < 0.001 traited versus control at t0.
MGO [m M ]
GDH activity (n ¼ 4) Incubation time
GO [m M ]
Incubation time
Fig 6 Effect of MGO modification on GDH parameters Vmaxand
K m Kinetic analyses were performed to determine the effect of
MGO (1 m M ) on GDH enzymatic parameters V max and K m Purified
GDH (Sigma) samples (10 lg) were incubated with or without
MGO for 24 h and aliquots (1 lg) were taken for enzymatic assay.
Lineweaver–Burk plots were used with a-ketoglutarate as substrate
(S) in native (1) or MGO-treated (2) GDH.
Trang 8effect of glycation Interestingly, kinetic analysis of modified GDH showed that treatment with MGO reduced only the maximum velocity without affecting
Km, indicating that MGO-modified GDH is inacti-vated In addition, amino acid analysis performed on MGO- and GO-treated GDH revealed that lysine resi-dues were more sensitive to MGO than to GO modifi-cations, whereas arginine was equally sensitive to both dicarbonyl compounds, suggesting that GDH inactiva-tion was at least partly due to MGO-lysine⁄ arginine modifications, in accordance with the observed trails of isoforms in the 2D electrophoregram Indeed, numer-ous data have claimed that MGO modifications of criti-cal arginine⁄ lysine residues cause structural distortion, leading to enzyme inactivation [23,31] In addition, recent data on GDH studies indicate that among
33 lysine residues constitutive of its primary sequence, the prominent lysine 126 directly interacts with the a-carbon constituent on the substrate [32], suggesting that MGO modification affects enzymatic activity In support of this assertion, a lysine residue involved in inactivation of brain GDH isoproteins by O-phthal-aldehyde has been identified [33] Moreover, loss of GDH activity was reported under multiple system atro-phy conditions in which GDH activity was decreased
to a greater extent than other mitochondrial enzymes [34] indicating that GDH is more sensitive to insults Interestingly, a recent in vitro study showed that incu-bation of mitochondria with MGO led to rapid inhibi-tion of mitochondrial respiratory rates through particular protein target modifications [35] Both the TCA cycle and the electron respiratory chain were inhibited, indicating a link between mitochondrial MGO modified enzymes and altered function Mamma-lian GDH is allosterically regulated by a number of small molecules [36] and its regulation is of particular biological importance, as exemplified by the observa-tion that some regulatory mutaobserva-tions of the gene for GDH are associated with severe clinical manifestation
in children [32] In addition, as shown in a recent study
Control
0
20
40
60
80
100
120
140
160
180
0 1 MGO (m M )
0 15 120 240
ADP 250 µM
0
20
40
60
80
100
120
140
160
180
Leu 10 mM
0
20
40
60
80
100
120
140
160
180
GTP 30 µM
0
20
40
60
80
100
120
140
160
180
B
C
D
A
1
1
1
** **
****
***
# # #
# # #
**
**
# # #
# # # #
****
****
# # # #
# # # #
# # # #
# # # #
# # #
# # #
Fig 7 MGO effect on GDH activity and allosteric regulation Puri-fied GDH samples (10 lg; Sigma) was incubated with 1 m M MGO for up to 240 min and aliquots (1 lg) were subjected to the GDH activity assay in the absence (A) or presence of constant concentra-tions of allosteric activators ADP (B) and leucine (C), or inhibitor GTP (D) Values expressed as specific activity (UÆmg)1) are given as percentage of control (activity determined in the absence or pres-ence of MGO at t 0 ; left and right panels, respectively) in each range Data represented the mean ± SEM (n ¼ 6) *t-test: effector versus control at each time; #t-test: MGO 1 m M -treated versus nontreated MGO at each time Equivalent to * or #; P < 0.05;
P < 0.01; P < 0.005; P < 0.001.
Trang 9[37], the arginine side chain at position 463 of GDH is
thought to be involved in ADP allosteric activation
because the R463A mutant form of this enzyme is
insensitive to ADP stimulation In the present study,
MGO modifications altered the allosteric regulation
properties of the enzyme, suggesting that these effects
are not only due to a change in charge profile, but also
in the conformation of the molecule resulting from
gly-cation of the charged arginine⁄ lysine side-chain
resi-dues The fact that arginine⁄ lysine residues are targets
of glycation [38,39] suggests that the modifications
observed in the present study involve some of these
res-idues, leading to an impairment of allosteric regulation
and the catalytic properties of the enzyme
GDH is important in converting free ammonia and
a-ketoglutatrate to glutamate; it utilizes nicotinamide
nucleotide cofactor NAD+for nitrogen liberation and
NADP+ for nitrogen incorporation; however, it
should be recognized that the reverse reaction is a key
anapleurotic process linking amino acid metabolism
with TCA cycle activity In a reverse reaction, GDH
provides an oxidizable carbon source for the
produc-tion of energy, as well as the reduced electron carrier
NADH Thus, GDH is considered to be significant not
only because it catalyzes a reaction directly connected
to the TCA cycle, but also because of the pivotal
posi-tion in metabolism occupied by both glutamate and
a-ketoglutarate as a result of their ability to enter into
many metabolic pathways [40] Accordingly, the
build-up of an inactive form of GDH demonstrated in the
present study could contribute to a decreased
produc-tion of a-ketoglutarate and a diminished flux through
the TCA cycle, which might be at least partly be
responsible for impairment of mitochondrial function
with advanced age, as demonstrated by the decrease in
respiration driven by the glutamate–malate substrate
In summary, the results obtained in the present study
demonstrate that age-related impairment of
mitochon-drial respiration runs parallel to an accumulation of
AGE-modified matrix proteins Identification of
selec-tively glycated proteins revealed that two of these are
key urea cycle enzymes, among which GDH was the
main target protein and showed a loss of both activity
and sensitivity to allosteric effectors with aging In vitro
alterations in both allosteric regulation and catalytic
properties of this enzyme by the glycating agent MGO
during short-term incubation support the notion of the
dysfunctional power of intracellular glycation In line
with the central role played by this enzyme in cellular
metabolism and energy homeostasis, we hypothesize
that AGE modifications of GDH may contribute, at
least in part, to a defect in mitochondria with aging and
could be used as a biomarker of cellular aging
Experimental procedures
Animals
Experiments were performed on male Wistar rats (WAG⁄ Rij) born and raised in the animal care facilities of the Commissar-iat a` l’Energie Atomique (CEA, Gif-sur-Yvette, France) This strain remains lean even when fed ad libitum and does not suffer from age-associated nephropathy, hypertension or diabetes [41] Cohorts were constituted of young adult (3-month-old) and senescent (27-month-old) animals All stu-dies were conducted in accordance with the animal care policy
of national and European regulations
Chemicals
GDH (bovine liver; EC 1.4.1.3) and horseradish peroxidase-conjugated anti-(mouse IgG) or anti-(rabbit IgG) sera were purchased from Sigma Chemicals (Saint Quentin Fallavier, France) GDH was dissolved in 100 mm of triethanolamine,
pH 7.3 Monoclonal antibody to AGE (clone no 6D12) from Trans Genic Inc (Kumamoto, Japan) shows cross-reaction both to CEL and CML [42] Polyclonal antibody against GDH was obtained from Interchim (Montluc¸on, France) and pro-tein G-agarose bed (ImmunoPure immobilized propro-teinG Plus) from Pierce (Perbio Science Company, Brebie`res, France)
Isolation of mitochondria
A 10% (w⁄ v) tissue homogenate was prepared using a Pot-ter apparatus in an ice-cold medium containing 220 mm mannitol, 70 mm sucrose, 0.1 mm EDTA and 2 mm Hepes,
pH 7.4, supplemented with 0.5% BSA (w⁄ v) Nuclei and cellular debris were pelleted by centrifugation for 10 min at
800 g and 4C Supernatant was centrifuged at 8000 g for
10 min at 4C The mitochondrial pellet was then washed three times with the homogenization medium and used for polarographic measurements
To prepare mitochondrial matrix extract, mitochondria were suspended in 50 mm Tris⁄ HCl, pH 7.9, then dis-rupted by sonication (four times for 10 s) The resulting suspension was centrifuged at 15 000 g for 10 min and then at 100 000 g for 45 min at 4C The supernatant (containing matrix proteins) was stored at )80 C for fur-ther analysis of AGE-modified proteins The protein con-centration was assessed using a Bradford protein assay (Biorad, Mu¨nchen, Germany)
To estimate contamination of mitochondrial preparation with lysosomes, we used acid phosphatase activity as a marker
Measurements of mitochondrial respiration
Oxygen consumption was measured polarographically with
a Clark electrode in the sample, as described by Aprille
Trang 10and Asimakis [43], in a thermostatically controlled closed
2 mL chamber (30C) The rate of oxygen consumption
was measured in the presence of 310 nmol of ADP and
10 mm of succinate or 5 mm glutamate⁄ 5 mm malate
(state 3) and after all ADP had been consumed (state 4 or
resting state) Oxygen consumption rates are expressed as
ng atoms of oxygen consumedÆmin)1Æmg protein)1 The
rate of oxygen consumption in state 3 and in state 4,
RCR (the ratio of state 3 to state 4 respiration), an index
of electron transport chain activity, and the ADP⁄ O2ratio
were calculated Oxygen consumption in the presence of
40 lm of dinitrophenol (uncoupled state) was also
checked
Determination of 6D12-detectable AGE content in
mitochondrial matrix proteins by competitive
ELISA
The ELISA assay was conducted as previously described
[24] Briefly a 96-well microtiter Nunc-immuno plate
(Nunc, Roskilde Denmark) was coated with 100 lL of
CML-BSA (6.4 nmol CMLÆmL)1) by incubation overnight
at 4C Wells were washed with NaCl⁄ Pi)0.05%
Tween 20 (v⁄ v) (buffer A) and free binding sites were
blocked by incubation for 1 h at room temperature with
100 lL of NaCl⁄ Pi)6% skimmed milk or NaCl ⁄ Pi)1%
BSA (w⁄ v) After washing with buffer A, 50 lL of
com-peting antigen (test samples at 0.1 lgÆlL)1 or serial
dilu-tions of standard CML-BSA from 0.64 mm to 128 mm)
was added, followed by 50 lL of monoclonal anti-AGE
IgG (clone 6D12) (dilution 1 : 3000) The plate was
incu-bated for 2 h at room temperature, washed and then
incubated with 50 lL horseradish peroxidase-conjugated
anti-(mouse IgG) (dilution 1 : 10 000) for 2 h at room
temperature The wells were washed, then 100 lL of
sub-strate solution (40 mm ABTS and 200 lL of 30%
hydro-gen peroxide in 20 mL sodium acetate-phosphate buffer,
pH 7.2) were added per well and incubated Absorbance
(A) was measured at 405 nm on a micro-ELISA plate
reader (Spectra Rainbow, SLT Labinstruments, Salzburg,
Austria) Results are expressed as the ratio B⁄ Bo (bound ⁄
total), calculated as: experimental A) background A (no
antibody)⁄ total A (no competitor)) background A, versus
CML added, as pmol CMLÆlg protein)1 Finally, data
were expressed as arbitrary unitsÆlg protein)1 (AUÆlg
pro-tein)1) because anti-AGE IgG recognizes both CML and
CEL
The use of 6% skimmed milk (w⁄ v) as an alternative to
1% BSA (w⁄ v) as a blocking agent in the immunochemical
assay introduced an increment of less than 10% at the
CML level, with GO-modified BSA used as standard (data
not shown) We took advantage of these results and used
skimmed milk in all further immunochemical assays
(ELISA and western blotting)
1D and 2D gel electrophoresis of mitochondrial matrix proteins and western blotting
Mitochondrial matrix protein samples (150 lg) from 3-month-old and 27-3-month-old rats were mixed with 200 lL
of 2D sample buffer (7 m urea, 2 m thiourea, 4% Chaps, 1% dithithreitol, 2% Pharmalytes, Amersham Biosciences, Saclay, France; pH 3.0–10.0) The strips were allowed to rehydrate overnight 1D isoelectric focusing was performed
on Immobiline Drystrips (Amersham Biosciences; pH 3.0– 10.0, 13 cm) in a Multiphor II device (Amersham Bio-sciences) for 49 325 Vh After electrofocusing, immobilines were prepared for SDS⁄ PAGE and 2D SDS ⁄ PAGE was run vertically on a 12% polyacrylamide gel using the cool-ing Protean II system (Bio-Rad, Marne La-Coquette, France) The gels were either fixed and stained with colloi-dal Coomassie blue for total protein pattern and
LC-MS⁄ MS analysis, or western blotted onto a nitrocellulose membrane (Bio-Rad) overnight at 30 V The membrane was saturated with NaCl⁄ Pi, pH 7.4, 0.1% Tween 20 (v⁄ v), 5% skimmed milk (w⁄ v) overnight at 4 C, followed by four washes (10 min each) with NaCl⁄ Pi, pH 7.4, 0.2% Tween 20 (washing buffer) The membrane was then incu-bated for 2 h at room temperature with monoclonal anti-AGE IgG clone 6D12 (dilution 1 : 3000) in NaCl⁄ Pi, 0.1% Tween 20 (v⁄ v), washed four times, incubated for 1 h with anti-(mouse IgG) coupled to horseradish peroxidase (dilu-tion 1 : 3000) and given a final wash The proteins were revealed with a SuperSignal West Pico chemiluminescent reagent (Perbio Science Company, Brebie`res, France)
Protein identification by LC-MS⁄ MS
Colloidal Coomassie blue-stained spots matching with bands immunolabelled by monoclonal anti-AGE IgG were excised from gel, cut into 1 mm pieces and then treated for LC-MS⁄ MS analysis Gel pieces were washed twice in
100 mm ammonium bicarbonate buffer pH 8.8 and then dehydrated with acetonitrile The gel pieces were rehydrated
in 10 mm dithithreitol⁄ ammonium bicarbonate solution and proteins alkylated with 50 mm iodoacetamide After dehy-dratation with acetonitrile, gel pieces were rehydrated on ice for 10 min in 20 lL of 20 mm ammonium bicarbonate containing 50 ngÆlL)1 of sequence-grade modified porcine trypsin (Promega, Madison, WI, USA); then supernatants were replaced by 20 lL of 20 mm ammonium bicarbonate, and in-gel digestion was performed for 15 h at 37C The resulting peptides were extracted twice with 20 lL of
20 mm ammonium bicarbonate and then three times in
20 lL of 0.5% trifluoroacetic acid in 50% acetonitrile The peptide extracts were concentrated to 20 lL using an RC 10.22 evaporator concentrator (Jouan, Saint Herblain, France) Samples were then subjected to mass spectrometry analysis