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Biochem. J. (2012) 445, 247–254 (Printed in Great Britain)
247
doi:10.1042/BJ20112142
Mitochondrial stress causes increased succination of proteins in adipocytes
in response to glucotoxicity
Norma FRIZZELL*1 , Sonia A. THOMAS†, James A. CARSON† and John W. BAYNES*
*Department of Pharmacology, Physiology and Neuroscience, School of Medicine, University of South Carolina, Columbia, SC 29208, U.S.A., and †Department of Exercise Science,
Arnold School of Public Health, University of South Carolina, Columbia, SC 29208, U.S.A.
2SC [S-(2-succino)-cysteine] is a chemical modification formed
by a Michael addition reaction of fumarate with cysteine
residues in proteins. Formation of 2SC, termed ‘succination’
of proteins, increases in adipocytes grown in high-glucose
medium and in adipose tissues of Type 2 diabetic mice.
However, the metabolic mechanisms leading to increased
fumarate and succination of protein in the adipocyte are unknown.
Treatment of 3T3 cells with high glucose (30 mM compared
with 5 mM) caused a significant increase in cellular ATP/ADP,
NADH/NAD + and ψ m (mitochondrial membrane potential).
There was also a significant increase in the cellular fumarate
concentration and succination of proteins, which may be attributed
to the increase in NADH/NAD + and subsequent inhibition
of tricarboxylic acid cycle NAD + -dependent dehydrogenases.
Chemical uncouplers, which dissipated ψ m and reduced the
NADH/NAD + ratio, also decreased the fumarate concentration
and protein succination. High glucose plus metformin, an inhibitor
of complex I in the electron transport chain, caused an increase
in fumarate and succination of protein. Thus excess fuel supply
(glucotoxicity) appears to create a pseudohypoxic environment
(high NADH/NAD + without hypoxia), which drives the increase
in succination of protein. We propose that increased succination
of proteins is an early marker of glucotoxicity and mitochondrial
stress in adipose tissue in diabetes.
INTRODUCTION
cysteine residue (Cys39 ). This cysteine residue can participate in
disulfide bonding to generate the higher-order secreted oligomers
(primarily trimers, hexamers and octodecamers), that regulate
lipid and glucose metabolism in muscle and liver [5]. We showed
that Cys39 near the N-terminus is succinated and that succination
prevented the incorporation of the adiponectin monomer into
high-molecular-mass isoforms [3]. As ∼ 8 % of total adiponectin
is succinated and is no longer secreted from the adipocyte [3], we
have proposed that succination contributes in part to the decrease
in circulating adiponectin oligomers in Type 2 diabetes [6,7].
Further pathological relevance of increased succination is evident
in type II PRCCs (papillary renal cell carcinomas) derived from
a mutation in the fumarate hydratase gene. The elevated intracellular fumarate levels in the tumour tissue result in extensive
protein succination, and the modification of regulatory cysteine
residues within the KEAP1 [Kelch-like ECH (erythroid cellderived protein with cap‘n’ collar homology)-associated protein
1] lead to altered Nrf2 (nuclear factor 2) signalling, suggesting that
succination may indirectly increase the expression of genes under
the control of the ARE (antioxidant-response element) [8,9].
We have shown previously that the increase in protein
succination in adipocytes grown in high-glucose medium and
in adipose tissue of db/db mice is the direct result of an increase in
the intracellular fumarate concentration [2,3]. Succination is also
increased in adipose tissue of ob/ob diabetic mice and to a lesser
extent in insulin-resistant DIO (diet-induced obese) mice [10].
Nutrient excess has been documented to result in mitochondrial
damage in the expanding adipocyte [11,12], suggesting that an
increase in mitochondrial stress occurs as a result of fuel excess
[13,14]. Intra-adipocyte organelle stress [15,16] may culminate
in adipocyte hypertrophy and cell death, in addition to lipid
We have previously described 2SC [S-(2-succino)-cysteine],
a natural chemical modification of proteins [1,2], formed by
a Michael addition reaction of the tricarboxylic acid cycle
metabolite, fumarate, with low pK a cysteine residues on proteins
[1,2]. Succination of protein, which produces an acid-stable
thioether linkage to protein, is distinct from succinylation, in
which an acid-labile ester, thioester or amide bond is formed with
cysteine or lysine residues in protein. Succination of proteins
increases 5–10-fold in 3T3-L1 adipocytes cultured in highglucose medium, in adipose tissue of obese Type 2 (db/db)
diabetic mice [2,3] and in skeletal muscle of streptozotocininduced Type 1 diabetic rats after 6 months [4]. Approximately
20 % of the glycolytic enzyme GAPDH (glyceraldehyde-3phosphate dehydrogenase) is succinated in skeletal muscle,
consistent with a ∼ 25 % decrease in specific activity of the
enzyme in this tissue. Of the four cysteine residues per subunit
of GAPDH, only two (Cys149 and Cys244 ) reacted measurably
with fumarate, indicating the specificity of succination [4]. We
have identified 14 other succinated proteins in adipocytes [2,3],
including the molecular chaperones GRP78 (glucose-regulated
protein of 78 kDa) and PDI (protein disulfide-isomerase) [2],
whose succination on critical thiols may alter the trafficking
and folding of other intracellular proteins. Several cytoskeletal
proteins are also succinated (actin, tropomyosin and vimentin)
[2], which may contribute to alterations in adipocyte dynamics
and structure in obesity and diabetes.
To understand the pathophysiological significance of protein
succination in more detail, we examined the insulin-sensitizing
hormone adiponectin, which contains a structurally important
Key words: adipocyte, cysteine, fumarate, mitochondrial
hyperpolarization, pseudohypoxia, succination.
Abbreviations used: m , mitochondrial membrane potential; CCCP, carbonyl cyanide m-chlorophenylhydrazone; 2D, two-dimensional; DNP, 2,4dinitrophenol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JC-1, 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine iodide;
ROS, reactive oxygen species; 2SC, S -(2-succino)-cysteine; SA, salicylic acid; TFA, trifluoroacetic acid; UCP, uncoupling protein.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
248
N. Frizzell and others
accumulation in other tissues, such as muscle and liver [17–19].
Therefore understanding the events that contribute to adipocyte
metabolic stress are central to understanding the mechanisms
underlying increased succination in diabetes.
Both glucotoxicity and lipotoxicity are associated with the
development of insulin resistance and diabetes in several
tissues, including the adipocyte [20–25]. However, fatty acid
oxidation is a minor source of energy in the white adipocyte
[26,27]. Accordingly, we hypothesized that glucotoxicity is the
primary source of mitochondrial stress and increased fumarate
concentration in the adipocyte during diabetes. In the present
study, we examined the metabolic events which contribute to
increased protein succination in adipocytes cultured in high
glucose. We show that a high glucose concentration leads to an
increase in total cellular ATP/ADP and NADH/NAD + ratios,
alongside the hyperpolarization of the IMM (inner mitochondrial
membrane), and ultimately the accumulation of fumarate and
succination of proteins. In support of this hypothesis we also show
that chemical uncouplers, which dissipate ψ m (mitochondrial
membrane potential), also lower the NADH/NAD + ratio,
decrease fumarate concentration and decrease succination of
proteins. Consequently, we establish a mechanistic link between
fuel excess, mitochondrial stress and succination of protein in
adipocytes in Type 2 diabetes.
for 10 min and removal of the acetone, the protein pellet was
resuspended in 500 μl of RIPA buffer. The protein content
was determined by the Lowry assay [28].
Determination of the NADH/NAD + and ATP/ADP ratio
Analysis of both NADH and NAD + was performed in cell
lysates after removal of protein and lipids by Folch extraction
[29]. Briefly, 3-cm-diameter plates containing adipocytes were
washed and scraped into ice-cold RIPA buffer, followed by
the immediate addition of 20 vol. ice-cold chloroform/methanol
(2:1, v/v). The samples were vortex-mixed and allowed to stand
on ice for 10 min with intermittent vortex-mixing prior to the
addition of 0.2 vol. H2 O. The samples were vortex-mixed and
allowed to stand on ice for an additional 2 min, followed by
centrifugation at 2000 g for 10 min at 4 ◦ C. The supernatant
was removed and dried in vacuo, then resuspended in H2 O.
Aliquots (100 μl) were removed for analysis of NADH and
NAD + (NAD + /NADH Quantification Kit, Biovision) according
to the manufacturer’s instructions. ATP, ADP and AMP levels
were measured immediately after nucleotide extraction using a
bioluminescence assay (ApoSENSORTM , Biovision).
Measurement of ψ m
EXPERIMENTAL
Chemicals
Unless otherwise noted, all chemicals were purchased from
Sigma–Aldrich. Criterion polyacrylamide gels and Precision
Plus protein ladder were purchased from Bio-Rad Laboratories.
PVDF membrane and ECL Plus chemiluminescent substrate were
from GE Healthcare. The synthesis of 2-succinocysteamine and
preparation of a polyclonal anti-2SC antibody have been described
previously [2].
3T3-L1 adipocyte experiments
Murine 3T3-L1 fibroblasts were obtained from the laboratory
of Dr Howard Green (Harvard Medical School, Boston, MA,
U.S.A.). The cells were maintained and differentiated into adipocytes as described previously [2]. After differentiation, the
adipocytes were cultured in DMEM (Dulbecco’s modified Eagle’s
medium) containing 5 μg/ml (800 nM) insulin and either 5 mM
D-glucose, 30 mM D-glucose or 30 mM D-glucose containing drug
treatments [CCCP (carbonyl cyanide m-chlorophenylhydrazone),
DNP (2,4-dinitrophenol), SA (salicylic acid) or metformin
(1,1-dimethylbiguanide hydrochloride)], for a further 2–8 days
(maturation period); during this time lipid droplets accumulated
in the cytoplasm. Preliminary experiments using L-glucose as
an osmotic control indicated that it had no effect on protein
succination (results not shown). Culture medium was changed
every 48 h, but glucose was supplemented into the media at
24 h intervals to maintain glucose levels. Cells were harvested
in 500 μl of RIPA lysis buffer [50 mM Tris/HCl, 150 mM
NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % SDS and 0.5 %
sodium deoxycholate, pH 7.4, with the addition of 2 mM DTPA
(diethylenetriaminepenta-acetic acid) and a protease inhibitor
cocktail (P8340, Sigma–Aldrich)]. The cell lysate was pulsesonicated at 2 W RMS using a Model 100 sonic dismembrator
(Fisher Scientific) for 1 min prior to resting on ice for 30 min
in lysis buffer. The protein was precipitated with 9 vol. of icecold acetone for 10 min on ice. After centrifugation at 3000 g
c The Authors Journal compilation c 2012 Biochemical Society
ψ m was measured using the fluorescent potentiometric
dye
JC-1
(5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine iodide) (Molecular Probes). Briefly, 3T3
adipocytes were cultured in 24-well plates and treatments in
maturation medium were continued for 2 days, which was the
optimal time point for measurement of ψ m , as increased
lipid accumulation at later times interfered with fluorescence
measurements. After 2 days in various treatments, the cells were
incubated with 10 μg/ml JC-1 dye for 20 min at 37 ◦ C. The cells
were then washed and resuspended in 200 μl of PBS, and JC-1
fluorescence was measured on a Tecan Safire2 microplate reader
(Tecan). The fluorescence of the JC-1 monomer was measured
at λex = 485 nm/λem = 535 nm and the fluorescence of the JC-1
aggregate was measured at λex = 550 nm/λem = 600 nm. The
aggregate/monomer ratio (600:535) was used to assess ψ m .
Cells treated with 100 μM CCCP for 20 min were used as a
positive control to demonstrate complete depolarization of the
mitochondrial membrane.
Cell viability assay
The CellTiter-Blue® Cell Viability Assay (Promega) was used to
determine the effects of various compounds on 3T3-L1 adipocyte
viability during prolonged incubations. Cells were also monitored
daily using light microscopy to observe the effects on cell number
and morphology, which were unaffected in any of the experiments.
Prior to measurement of cell viability, the treatment medium was
removed and replaced with 200 μl of fresh treatment medium
and 40 μl of CellTiter-Blue Cell Viability reagent (Promega).
The cells were then incubated in the dark for 1 h at 37 ◦ C and
the fluorescent signal generated upon reduction of resazurin to
resorufin was measured on a Tecan Safire2 microplate reader at
λex = 560 nm/λem = 590 nm, as described by the manufacturer.
Triacylglycerol analysis
The triacylglycerol concentration was measured in 5 μl aliquots
of cell lysates using the Thermo DMA enzymatic triacylglycerol
assay kit and standard (Thermo Fisher Scientific), according to
Mitochondrial stress and protein succination in adipocytes
249
the manufacturer’s instructions. The results were normalized
to the protein content of the cells.
Measurement of intracellular malate and fumarate
Malate and fumarate were measured in adipocyte lysates using
an adaptation of the procedure described by Hatch [30]. Briefly,
adipocyte lysates harvested in RIPA buffer from confluent
100 mm×20 mm plates were immediately acidified with 500 μl
of 3 M hydrochloric acid, then extracted twice with 2 ml of
ethyl acetate. The extracts were evaporated under nitrogen,
redissolved in 1 % TFA (trifluoroacetic acid) and applied to
a 1 ml C-18 Sep Pak column (Waters) to remove endogenous
fluorophores, including NADH and NADPH. The columns were
washed with 1 % TFA, and the eluate was collected and dried
in vacuo. The dried extracts were resuspended in assay buffer
(25 mM Hepes/KOH, pH 7.5, 4 mM NADP + , 4 mM MgCl2
and 5 mM potassium phosphate) and aliquots of each sample
corresponding to ∼ 30 μg of protein in the original cell extract
were added to the wells of a 96-well plate. The formation of
NADPH (λex = 340 nm/λem = 450 nm) from malate was measured
at the 20 min endpoint after the addition of 1 unit of malic enzyme
(Sigma–Aldrich; M1567). After this time, fumarate was measured
by the addition of 1 unit of fumarase (Sigma–Aldrich; F1757)
and NADPH fluorescence was recorded after another 20 min.
Background fluorescence (samples without added enzymes) was
subtracted from each measurement. A standard curve of NADPH
fluorescence with increasing amounts of fumarate was used to
calculate the amount of malate and fumarate present in each
sample.
2D (two-dimensional) gel electrophoresis and Western
immunoblotting
Isoelectric focusing on pI 4–7 strips and 2D gel electrophoresis
were performed as described previously [3]. The 2D gels
were stained for protein using Sypro Ruby total protein stain
(Invitrogen) [3] or transferred on to PVDF membrane to detect
protein succination by Western blotting. Western blotting to probe
for protein succination was performed as described previously
[2]. ImageJ software (NIH; http://rsbweb.nih.gov/ij/) was used to
quantify band intensity by densitometry.
Statistical analysis
Results are summarized throughout as means +
− S.E.M. and are
plotted using Sigma Plot 11 software (Systat Software). Statistical
analyses were performed using SigmaPlot 11 and Prism 4
(GraphPad Software). Differences between groups were analysed
using the unpaired two-tailed t test or one-way ANOVA with the
Holm–Sidak post-test.
RESULTS
ATP/ADP and NADH/NAD + ratios and protein succination are
increased in adipocytes grown in high-glucose medium
To assess the effects of high-glucose medium on adipocyte
metabolism, adipocytes were cultured in 5 mM compared with
30 mM glucose concentration. As shown in Figure 1, there
was a ∼ 4.6-fold increase in the total cellular ATP/ADP
ratio (12.26 compared with 2.69, P < 0.001, Figure 1A) and
a ∼ 6-fold increase in the NADH/NAD + ratio (2.69 +
− 0.33
compared with 0.446 +
− 0.076, P < 0.001, Figure 1B) in
adipocytes cultured in 30 mM glucose compared with 5 mM
Figure 1 ATP/ADP, NADH/NAD + and 2SC are increased during maturation
of adipocytes in high glucose
(A) The ATP/ADP ratio in adipocytes grown in 5 mM compared with 30 mM glucose was
measured after 2 days growth in maturation medium, as described in the Experimental section.
Results are expressed as means +
− S.E.M., n = 4, ***P < 0.001 for 30 mM compared with
5 mM. The ATP concentration in control cells was 0.8 mM. (B) The NADH/NAD + ratio was
measured in adipocyte lysates after 2 days growth in maturation medium, as described in
the Experimental section. Results are expressed as means +
− S.E.M., n = 4, ***P < 0.001 for
30 mM compared with 5 mM. The control (5 mM) level of NAD + was ≈214 pmol/mg of protein
and NADH was ≈69 pmol/mg of protein. (C) 2SC increases on adipocyte protein in association
with an increased ratio of both ATP/ADP and NADH/NAD + . Total cell lysates (30 μg of protein)
from adipocytes cultured in 5 or 30 mM glucose for 4 days were separated by SDS/PAGE.
2SC-modified proteins were detected using a polyclonal anti-2SC antibody, as described in the
Experimental section. M indicates the marker lane and molecular masses of marker proteins
are indicated on the right-hand side. (D–G) Protein (150 μg) from adipocytes grown in 5 mM
or 30 mM glucose was analysed by 2D gel electrophoresis across a 4–7 pH range. Duplicate
gels were either stained with Sypro Ruby to observe total protein (D and E) or transferred on to
PVDF and immunoblotted with an anti-2SC antibody to detect succinated proteins (F and G).
Protein succination was increased in adipocytes cultured in 30 mM glucose (G) compared with
5 mM glucose (F), although there were no major changes in the total protein profile (D and E
respectively).
glucose. Protein succination was also increased, even at
an early stage (∼ 3 days) during adipocyte maturation in
high glucose (Figure 1C). Although only one major protein
(previously identified as tubulin; ∼ 55 kDa) was modified
in the adipocytes cultured in 5 mM glucose (lane 1), an increase in
succinated proteins was detected in adipocytes cultured in 30 mM
glucose (lane 2), confirming that succination increases in
adipocytes in concert with the increase in the ATP/ADP and
NADH/NAD + ratios in response to high glucose. A duplicate 2D
gel analysis of equal amounts of protein from 5 mM and 30 mM
glucose-treated adipocytes (8 days maturation) was performed
after isoelectric focusing across pI 4–7. As shown in Figures 1(D)
and 1(E), there was no significant difference in total proteins
with the different glucose treatments. However, Figures 1(F) and
1(G) demonstrate that protein succination was increased only in
30 mM glucose (Figure 1G), confirming that increased protein
succination is not due to large changes in protein abundance, but
is a result of increased post-translational modification.
c The Authors Journal compilation c 2012 Biochemical Society
250
N. Frizzell and others
Figure 2 Chemical uncoupling lowers ψ m and NADH/NAD + ratio in
adipocytes cultured in high glucose
(A) Adipocytes cultured in 5 mM glucose, 30 mM glucose or 30 mM glucose plus 10 μM
CCCP, 10 μM DNP or 1 mM SA for 2 days were loaded with the dye JC-1 to measure the ψ m .
After 20 min, the cells were washed and fluorescence was measured at λex /λem 550/600 nm
and λex /λem 485/535 nm, as described in the Experimental section. The ψ m is a ratio of
fluorescence at 600/535 nm and the results are expressed as a percentage of the control
(5 mM). The results are representative of n = 4 measurements expressed as means +
− S.E.M.;
###
P < 0.001 for 30 mM compared with 5 mM glucose, **P < 0.01 for 30 mM glucose
compared with 30 mM glucose plus DNP, and ***P < 0.001 for 30 mM glucose compared
with 30 mM glucose plus CCCP or SA. (B) NADH/NAD + levels were measured in adipocyte
lysates after culture in 5 mM glucose, 30 mM glucose or 30 mM glucose plus 10 μM CCCP,
10 μM DNP or 1 mM SA for 2 days. Before expressing the data as a ratio, the control (5 mM
glucose) levels of NAD + was measured as ≈182 pmol/mg of protein and NADH was measured
as ≈78 pmol/mg of protein. The results are expressed as means +
− S.E.M., n = 4; ***P < 0.001
for 30 mM glucose compared with 30 mM glucose plus CCCP, DNP or SA.
Effect of glucose concentration and uncouplers on ψ m and the
NADH/NAD + ratio
The overall increase in the NADH/NAD + ratio is consistent with
feedback inhibition of the electron transport chain by respiratory
control at a high ATP/ADP ratio. To test this hypothesis, we
examined the effects of a high glucose concentration on ψ m
and also the effects of uncouplers of oxidative phosphorylation.
As shown in Figure 2(A), there was a significant increase
in the ψ m in adipocytes cultured in 30 mM compared with
5 mM glucose (fluorescence ratio = 0.757 +
− 0.44 compared
with 0.369 +
0.03,
P
<
0.001).
The
uncouplers
CCCP and DNP at
−
10 μM concentration reversed the increase in ψ m in adipocytes
grown in 30 mM glucose (P < 0.001 and P < 0.01 for CCCP and
DNP respectively). SA, at a concentration of 1 mM, was also an
effective uncoupler in cells grown in 30 mM glucose (P < 0.001).
Treatment with CCCP, DNP and SA also caused a 46 %, 58 %
and 82 % decrease in the NADH/NAD + ratio in adipocytes
grown in high-glucose medium (P < 0.001, Figure 2B), consistent
with uncoupling of oxidative phosphorylation and activation of
the electron transport chain. The toxicity of the uncouplers was
assessed by a cell viability assay. The adipocytes were cultured
for 8 days (to determine viability at the time when succination was
measured) in concentrations ranging from 1 to 100 μM CCCP and
DNP and 1 to 10 mM SA. After 8 days’ exposure, cells cultured in
10 μM CCCP or DNP showed no significant cell death compared
with untreated control cells (Supplementary Figures S1A and S1B
at http://www.BiochemJ.org/bj/445/bj4450247add.htm), indicating that these low concentrations were well tolerated and did not
c The Authors Journal compilation c 2012 Biochemical Society
Figure 3 Malate and fumarate are reduced after treatment with chemical
uncouplers
Adipocytes were matured in 5 mM or 30 mM glucose or 30 mM glucose plus 10 μM CCCP,
10 μM DNP or 1 mM SA for 2 days. Malate and fumarate levels were measured in an enzymatic
assay as described in the Experimental section. The results are expressed as nmol/mg of
protein calculated from a standard curve of NADPH production. The results are expressed as
means +
− S.E.M., n = 3; ***P < 0.001, **P < 0.01 or *P < 0.05 for 30 mM glucose compared
with 30 mM glucose plus CCCP, DNP or SA or ## P < 0.001 for 5 mM compared with 30 mM
glucose.
contribute to cell death. However, there was a significant decrease
in cell viability in both 25 and 50 μM concentrations of CCCP
and DNP against untreated cells; and at 100 μM concentrations,
no cells survived. SA appeared to be well tolerated at all
concentrations used; however, a significant ∼ 33 % reduction in
cell viability was observed at 10 mM SA compared with untreated
cells in 30 mM glucose (P = 0.015, Supplementary Figure S1C).
CCCP and DNP, used at 10 μM concentrations, had a minimal
effect on triacylglycerol biosynthesis in cells grown in 30 mM
glucose, whereas SA reduced lipogenesis by approximately 50 %,
to levels observed in cells grown in 5 mM glucose (P = 0.012,
Supplementary Figure S1D). Consequently, CCCP and DNP were
used in all experiments at 10 μM and SA was used at 1 mM.
Effect of uncouplers on malate and fumarate concentrations
To validate the role of NADH as the inhibitor of tricarboxylic acid
cycle activity in adipocytes grown in high-glucose medium, we
examined the effects of uncouplers on fumarate concentration.
Since the enzymatic assay also measures malate concentration
(see the Experimental section), both results are reported. Both the
malate and fumarate concentration were significantly elevated in
adipocytes grown in high- compared with low-glucose medium
(Figures 3A and 3B, 30 mM compared with 5 mM: 28.3 +
− 3.24
compared with 6.7 +
− 0.47 nmol/mg of protein, P < 0.01, for
malate; and 19.9 +
− 5.03 compared with 2.4 +
− 0.91 nmol/mg
of protein, P = 0.001 for fumarate). Although the malate
concentration in high glucose was slightly greater than the
fumarate concentration, the fold increase between 30 mM and
5 mM glucose was greater for fumarate than for malate (∼ 7.6fold compared with ∼ 4-fold). Malate levels were significantly
decreased by chemical uncoupling (68 % and 84 % for DNP
and SA respectively, P < 0.01; and 43 % for CCCP, P < 0.05;
Mitochondrial stress and protein succination in adipocytes
Figure 4
251
2SC levels are reduced after treatment with CCCP, DNP and SA
Adipocytes were matured in 5 mM or 30 mM glucose, or 30 mM glucose plus 10 μM CCCP,
10 μM DNP or 1 mM SA for 8 days. (A) Total protein was harvested and 30 μg of protein was
electrophoresed and blotted with the anti-2SC polyclonal antibody to detect protein succination.
(B) Blots were reprobed with an anti-β-tubulin antibody to detect equal loading. (C) The levels of
2SC-modified proteins were normalized to the tubulin content after densitometry analysis. The results are representative of those obtained after performing three independent experiments. The
###
values are expressed as means +
− S.E.M., n = 3; P < 0.001 for 5 mM compared with 30 mM
glucose or ***P < 0.001 for 30 mM glucose compared with 30 mM glucose plus CCCP, DNP
or SA. (D) Adipocytes were matured in 5 mM glucose for 8 days, 30 mM glucose for 8 days or
30 mM glucose for 4 days and then switched to 5 mM glucose for 4 days (30→5), after which
30 μg of protein was analysed to detect succinated proteins. A representative blot is shown.
Figure 3A). Fumarate levels were reduced by all uncoupling
treatments compared with 30 mM glucose alone. The decrease in
fumarate was statistically significant for both DNP and SA, a 68 %
and 74 % reduction compared with 30 mM glucose respectively
(P < 0.01, Figure 3B). However, although CCCP resulted in
a ∼ 40 % decrease in fumarate, this change did not achieve
statistical significance, because of the variance of measurements
at 30 mM glucose.
Protein succination is reduced by uncouplers of oxidative
phosphorylation
To determine whether the effects of uncouplers on the
NADH/NAD + ratio and fumarate concentration ultimately
affected protein succination, we examined succination of proteins
by Western blotting with an anti-2SC antibody. As can be observed
in Figure 4(A), there were multiple succinated proteins in the
adipocytes cultured in 30 mM compared with 5 mM glucose.
However, when the adipocytes were treated with the chemical
uncouplers, a significant reduction in succination of a range of
proteins was detected (Figure 4A), consistent with the decrease
in ψ m and NADH/NAD + ratio (Figure 2). The gel was stripped
and reprobed with an anti-β-tubulin antibody to assess protein
loading (Figure 4B), and the densitometry obtained for the
2SC staining was normalized to the tubulin content of each
lane. There was a statistically significant reduction in protein
succination with all uncouplers (Figure 4C; P < 0.001). Under
Figure 5
Protein succination is increased after treatment with metformin
Adipocytes were matured in 5 mM or 30 mM glucose +
− 1 mM or 2 mM metformin (Met) for
4 days. (A and B) Intracellular malate and fumarate levels were measured enzymatically after Folch
extraction as described in the Experimental section. (C) Protein (30 μg) was electrophoresed
and blotted with the anti-2SC polyclonal antibody to detect protein succination. Blots were
reprobed with an anti-β-tubulin antibody to detect equal loading (bottom panel). (D) The levels
of the 2SC-modified proteins were normalized to the tubulin content by densitometric analysis.
The results are representative of three independent experiments. The values are means +
− S.E.M.,
***P < 0.001 or **P < 0.01 for 30 mM glucose compared with 30 mM glucose plus metformin
or ## P < 0.01 for 5 mM compared with 30 mM glucose.
normal conditions, protein succination is directly related to the
high glucose environment. When adipocytes have been matured
in 30 mM glucose for 4 days, and are then switched to 5 mM
glucose for a further 4 days, succinated proteins revert to levels
seen in cells grown in 5 mM glucose for 8 days (Figure 4D).
In contrast with the mitochondrial uncouplers, the anti-diabetic
drug metformin, which at suprapharmacological concentrations is
an inhibitor of mitochondrial complex I [31], caused an increase in
both fumarate and malate concentrations (P < 0.001, Figures 5A
and 5B), as well as an increase in protein succination after 4 days
treatment in maturation medium (P < 0.01, Figures 5C and 5D).
The increase in protein succination was most evident for tubulin
(∼ 55 kDa) at the 4 day time point when cells were harvested.
Longer incubations at these concentrations of metformin (1 mM
and 2 mM) resulted in toxicity.
DISCUSSION
The results from the present study outline our current
understanding of the mechanisms effecting the increase in
succination of proteins in adipocytes in high-glucose medium
and in diabetes. Elevated carbohydrate fuel supply exceeds the
c The Authors Journal compilation c 2012 Biochemical Society
252
N. Frizzell and others
metabolic needs of the cell and drives mitochondrial stress
due to the accumulation of ATP in adipocytes, which in turn
leads to feedback inhibition of oxidative phosphorylation. The
corresponding decrease in ADP concentration inhibits the F1 Fo
ATP synthase (respiratory control) and produces a high ψ m .
Accordingly, electron transport is inhibited and the cellular redox
status shifts towards an increase in NADH concentration. The
increase in the NADH/NAD + ratio in diabetes, also known
as reductive stress, was first described by Williamson and coworkers as ‘pseudohypoxia’ [32,33], i.e. an increase in the
NADH/NAD + ratio in the absence of hypoxia. The increase in
NADH causes an increase in fumarate and malate, consistent
with feedback inhibition of the NAD + -dependent dehydrogenases
of the tricarboxylic acid cycle. Fumarate then reacts with
cysteine residues on proteins within the mitochondrion, or it
may be transported into the cytosol and other organelles via
dicarboxylate carriers [34], leading to succination of a wide range
of intracellular proteins. In collaboration with Dr Thomas Metz at
the Pacific Northwest National Laboratories, we have identified
∼ 70 succinated proteins in adipocytes grown in high-glucose
medium (N. Frizzell, T. O. Metz and J. W. Baynes, unpublished
work). This sequence of events leading to increased succination
of proteins requires the synergistic effects of both high glucose
and high insulin concentrations: succination is not significantly
increased when the adipocytes are matured in fasting insulin
(0.05 nM) concentrations (A. Manuel, S. A. Thomas, J. W.
Baynes and N. Frizzell, unpublished work). Thus the combined
effects of elevated insulin and high glucose concentrations are
necessary to induce mitochondrial stress and protein succination
in the 3T3 adipocyte [20,21], providing an excellent model to
understand how succination is also increased in the db/db mouse
adipose tissue [3].
Protein succination can be reduced by chemical uncouplers
The use of chemical uncouplers in the present study has
demonstrated the mechanistic link between high glucose
concentration and elevated ψ m , the increase in the
NADH/NAD + ratio, the accumulation of fumarate and increase
in succination of protein – all of these events may be viewed
as a consequence of glucotoxicity. Uncouplers, such as CCCP
and DNP, decrease ψ m and stimulate mitochondrial fuel
metabolism. At high concentrations, they completely uncouple
mitochondria and induce cell death; however, at a 10 μM
concentration, adipocytes could be cultured for 8 days without
evidence of toxicity (Supplementary Figures S1A and S1B).
Under these conditions, the high glucose-induced increase in
ψ m was normalized (Figure 2A) and the accumulation of
tricarboxylic acid cycle intermediates and protein succination
was significantly reduced (Figures 3 and 4). We also observed
that sodium SA, a much weaker uncoupler [35], typically used at
1000-fold higher concentrations than DNP or CCCP, significantly
lowered the ψ m , the NADH/NAD + ratio and protein succination
without evidence of toxicity. The recently published results
of the TINSAL-T2D [Targeting Inflammation using Salsalate
(salicysalicylate) in Type 2 Diabetes] study suggests that the
treatment of Type 2 diabetes with salsalate, which is hydrolysed
to SA (t1 /2 ∼ 60 min) offers improved therapeutic potential
with significant reductions in HbA1c (glycated haemoglobin)
levels [36]. The beneficial effect of salsalate therapy is
generally attributed to its anti-inflammatory activity; however,
the results of the present study suggest that mild mitochondrial
uncoupling by SA, with the consequent increase in fuel (glucose)
utilization, may assist in the reduction of the metabolic stresses
induced by hyperglycaemia in diabetes. Although reductions in
c The Authors Journal compilation c 2012 Biochemical Society
triacylglycerol content in 3T3 adipocytes have been reported
both with chemical uncoupling and UCP (uncoupling protein)
overexpression [37], we did not observe any significant decrease
in triacylglycerol content in the uncoupled adipocytes compared
with high glucose controls, with the exception of SA treatment
(Supplementary Figure S1D). Whether SA affects adiposity in
Type 2 diabetic patients remains to be explored.
Although protein succination can be lowered by chemical
uncouplers, Figure 4(D) demonstrates that a reduction in glucose
to 5 mM after 4 days exposure to 30 mM glucose limits the
accumulation of succinated proteins over the next 4 days and
permits the turnover of existing succinated proteins. The factors
that determine the stability and turnover of succinated proteins
are currently under further investigation in our laboratory.
However, these results suggest that chronic high-glucose
exposure is analogous to a sedentary individual who maintains
hyperglycaemia through sustained feeding. Healthy individuals
may also have a transient increase in protein succination with a
large meal; however, glucose utilization due to physical activity
and/or caloric restriction should lower succination due to protein
turnover. The results suggest that the succinated protein load
may be highest and most pathogenic in the adipocytes of earlystage diabetics who are not receiving hyperglycaemia-lowering
treatments or reducing their calorie intake.
If the sequence of events we have proposed is correct,
electron transport chain inhibitors should also increase both
fumarate levels and protein succination by reducing electron
flow. We evaluated the effects of rotenone, sodium amobarbital
and oligomycin; however, all of these compounds caused
significant cytotoxicity during the long-term incubations, which
are necessary to detect protein succination, even at submicromolar
concentrations (results not shown). In contrast, the biguanide
drug metformin has been documented to act as a weak inhibitor
of complex I at millimolar concentrations [31,38,39]. Although
the doses used in the present study (1–2 mM) are relatively
high compared with peak plasma concentrations in metformintreated diabetics (∼ 10 μM), this drug is known to bioaccumulate
in mitochondria to ∼ 100–500 μM [40,41]. More specifically,
Wang et al. [41] have demonstrated that the EC50 for blood
lactate accumulation in rats is 734 μM, and this is sufficient
to lower oxygen consumption and promote lactic acidosis in
rat hepatocytes. This indicates that, at concentrations 734 μM,
metformin is acting as an inhibitor of oxidative phosphorylation,
and the concentrations used in the present study (1–2 mM)
are a model of acute electron transport chain inhibition, as
demonstrated previously [42]. At physiological pH, metformin
is positively charged and accumulates slowly at a membrane
potential-driven rate that requires prolonged exposure, such as
the 4 day treatment in the present study. Metformin treatment
also increases glucose uptake via GLUT4 (glucose transporter 4)
in white subcutaneous adipocytes [43], which in combination with
complex I inhibition might be expected to lead to an increased
fumarate concentration.
The present study demonstrates that exposure to elevated
concentrations of metformin increases the concentration of the
tricarboxylic acid cycle intermediates malate and fumarate,
and increases protein succination at high glucose concentration
(Figures 5A–5C), consistent with its action as a complex
I inhibitor, and indirectly as an inhibitor of tricarboxylic
acid cycle activity. Although micromolar concentrations of
metformin in clinical use are clearly beneficial in Type 2 diabetes
treatment, elevated concentrations of metformin are known to
be deleterious, given the clinical association with lactic acidosis,
particularly during renal injury or metformin intoxication suicide
attempts [44–51]. The results from the present study suggest that
Mitochondrial stress and protein succination in adipocytes
metformin-associated lactic acidosis and hepatorenal damage
in a clinical setting may be associated with increased protein
succination.
253
FUNDING
This work was supported by the National Institutes of Diabetes and Digestive and Kidney
Diseases [grant number DK-19971 (to J.W.B. and N.F.)] and the American Diabetes
Association [grant number 1-11-JF-13 (to N.F.)].
Role of pseudohypoxia in succination of proteins
The increase in NADH/NAD + in adipocytes grown in highglucose medium implies a critical role for hypoxia or
pseudohypoxia (increased NADH/NAD + in the absence of
hypoxia) in the increased succination of proteins. However, there
is no evidence that oxygen supply is limiting to adipocytes in vitro,
since cellular maturation and lipogenesis proceed efficiently.
When the oxygen tension is altered either by growing the
adipocytes in variable depths of medium (from 8 to 12 ml
of medium on a 100-mm-diameter plate), or in 2 % oxygen,
we observe no change in succination in vitro (S. A. Thomas,
J. W. Baynes and N. Frizzell, unpublished work). Thus, when the
energy supply exceeds the requirements of cellular metabolism,
the overfed adipocyte appears to employ the pseudohypoxic
response (accumulation of NADH) for feedback regulation of
the tricarboxylic acid cycle, which sets the stage for increased
succination of proteins. At the same time, there is strong evidence
for true hypoxia in adipose tissue in obesity and diabetes [52–55],
so that both true hypoxia and pseudohypoxia may be relevant in
understanding changes in adipose tissue metabolism in vivo. It
remains to be determined whether the (pseudo)hypoxic response
and increased succination of proteins is a common response to
glucotoxicity in other cell types, such as the β-cell, or specific
retinal, renal and vascular cells, which are subject to glucotoxicity
in Type 2 diabetes.
Conclusions
Although the sequence of events documented in the present
study illustrates how protein succination occurs as a result
of pseudohypoxia, alternative or complementary mechanisms
may also be involved. Increased oxidative stress in the diabetic
adipocyte has been documented extensively [56–60], and recent
studies by Wang et al. [60] suggest that elevated ROS (reactive
oxygen species) production in adipocytes can also inhibit
respiration. Lin et al. [61] have shown that ROS production in
high-glucose treated adipocytes is related to decreases in insulin
sensitivity; interestingly, the ‘rescue’ strategies employed by
Lin et al. [61] included overexpression of UCP2 and treatment
with CCCP; both of which should simultaneously lower protein
succination. Although these studies [56–61] demonstrate that
ROS and markers of oxidative damage are produced, the adipocyte
is well equipped with antioxidant defence systems. The most
abundant of these, glutathione, rapidly reverses the oxidation
of cysteine residues to sulfenic acid, regenerating the protein
thiol. In contrast, the succination of cysteine residues cannot
be reversed by known cellular antioxidant systems and appears
to be a cumulative irreversible biomarker of protein damage
until the protein is turned over intracellularly. The interplay
between oxidative stress and protein succination remains to be
elucidated. Further investigations on specific succinated proteins
should provide a novel insight into how succination contributes
to adipose tissue damage during the development of diabetes.
AUTHOR CONTRIBUTION
Norma Frizzell designed and performed the experiments, contributed to the discussion
and wrote the paper. Sonia Thomas performed the experiments and contributed to the
discussion. James Carson reviewed/edited the paper prior to submission. John Baynes
designed the experiments, contributed to the discussion and reviewed/edited the paper
prior to submission.
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doi:10.1042/BJ20112142
SUPPLEMENTARY ONLINE DATA
Mitochondrial stress causes increased succination of proteins in adipocytes
in response to glucotoxicity
Norma FRIZZELL*1 , Sonia A. THOMAS†, James A. CARSON† and John W. BAYNES*
*Department of Pharmacology, Physiology and Neuroscience, School of Medicine, University of South Carolina, Columbia, SC 29208, U.S.A., and †Department of Exercise Science,
Arnold School of Public Health, University of South Carolina, Columbia, SC 29208, U.S.A.
Figure S1
Cell viability and triacylglycerol accumulation
(A–C) Cell viability was measured as described in the Experimental section of the main text in adipocytes cultured with the following drug concentrations; 1–100 μM CCCP (A), 1–100 μM DNP
(B) or 1–10 mM SA (C). ***P < 0.001 and **P < 0.01 for CCCP- or DNP-treated cells compared with untreated controls (0). (D) Triacylglycerols were measured in an aliquot of cell lysate as
described in the Experimental section of the main text. The measurements were normalized to the protein content of the cells, and the results are expressed as means+
−S.E.M., n = 3, *P = 0.012
for 30 mM glucose compared with SA.
Received 12 December 2011/3 April 2012; accepted 24 April 2012
Published as BJ Immediate Publication 24 April 2012, doi:10.1042/BJ20112142
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society