help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2006-0998
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/1/293    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rachek, L. I.
Right arrow Articles by Wilson, G. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rachek, L. I.
Right arrow Articles by Wilson, G. L.
Endocrinology Vol. 148, No. 1 293-299
Copyright © 2007 by The Endocrine Society

Palmitate Induced Mitochondrial Deoxyribonucleic Acid Damage and Apoptosis in L6 Rat Skeletal Muscle Cells

L. I. Rachek, S. I. Musiyenko, S. P. LeDoux and G. L. Wilson

Department of Cell Biology and Neuroscience, College of Medicine, University of South Alabama, Mobile, Alabama 36688

Address all correspondence and requests for reprints to: L. I. Rachek, Department of Cell Biology and Neuroscience, University of South Alabama, Mobile, Alabama 36688. E-mail: lrachek{at}jaguar1.usouthal.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A major characteristic of type 2 diabetes mellitus (T2DM) is insulin resistance in skeletal muscle. A growing body of evidence indicates that oxidative stress that results from increased production of reactive oxygen species and/or reactive nitrogen species leads to insulin resistance, tissue damage, and other complications observed in T2DM. It has been suggested that muscular free fatty acid accumulation might be responsible for the mitochondrial dysfunction and insulin resistance seen in T2DM, although the mechanisms by which increased levels of free fatty acid lead to insulin resistance are not well understood. To help resolve this situation, we report that saturated fatty acid palmitate stimulated the expression of inducible nitric oxide (NO) synthase and the production of reactive oxygen species and NO in L6 myotubes. Additionally, palmitate caused a significant dose-dependent increase in mitochondrial DNA (mtDNA) damage and a subsequent decrease in L6 myotube viability and ATP levels at concentrations as low as 0.5 mM. Furthermore, palmitate induced apoptosis, which was detected by DNA fragmentation, caspase-3 cleavage, and cytochrome c release. N-acetyl cysteine, a precursor compound for glutathione formation, aminoguanidine, an inducible NO synthase inhibitor, and 5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrinato iron (III), a peroxynitrite inhibitor, all prevented palmitate-induced mtDNA damage and diminished palmitate-induced cytotoxicity. We conclude that exposure of L6 myotubes to palmitate induced mtDNA damage and triggered mitochondrial dysfunction, which caused apoptosis. Additionally, our findings indicate that palmitate-induced mtDNA damage and cytotoxicity in skeletal muscle cells were caused by overproduction of peroxynitrite.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TYPE 2 DIABETES MELLITUS (T2DM) is characterized by insulin resistance primarily in skeletal muscle (1). The mechanisms responsible for the reduced sensitivity of muscle to insulin still remain unclear. However, insulin resistance has been correlated with elevated levels of free fatty acids (FFAs) (2), which can be explained by different mechanisms, including the Randle hypothesis (3) and a direct effect on the function of proteins in the insulin signaling pathway (4). Also, there is evidence that oxidative stress, which occurs in cells when the equilibrium between prooxidant and antioxidant species favors prooxidant stress, leads to insulin resistance (5), tissue damage, and other complications observed in T2DM (6). Oxidative stress results from increased production of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS). These species can directly oxidize and damage DNA, proteins, and lipids and are believed to play a direct key role in the multiple forms of insulin resistance (7) and pathogenesis of late-occurring diabetic complications (6). Skeletal muscle is particularly vulnerable to oxidative stress because it requires a large amount of oxygen for its action and is postmitotic and thus capable of accumulating oxidative damage over time.

Mitochondria are the primary site for fuel metabolism and ATP synthesis and a major source of ROS production (8). In vitro evidence indicates that elevated FFA levels have numerous deleterious effects on mitochondria, including the uncoupling of oxidative phosphorylation (9), increased production of ROS including superoxide (10), and impairment of endogenous antioxidant defenses by the reduction of intracellular glutathione (11, 12). A growing body of evidence has indicated that oxidative stress resulting from ROS-induced mitochondrial dysfunction (13 ; for review, see Ref. 14) contributes to diabetes complications. In addition to lipid accumulation, several studies have indicated that the obese/insulin-resistant phenotype is associated with decreased mitochondrial FFA oxidative capacity in skeletal muscle and is characterized by a deficiency of subsarcolemmal mitochondria, unusual mitochondrial morphology, and lower than normal levels of mitochondrial enzymes (15, 16). In agreement with the notion of a possible destructive role for ROS on mitochondria in T2DM is the finding that there are increased mitochondrial DNA (mtDNA) mutations in skeletal muscle from T2DM patients (17). Moreover, depletion of mtDNA causes impaired glucose use and insulin resistance in L6 skeletal muscle cells, suggesting a crucial role for mtDNA in the development of insulin resistance (18). Additionally, that mtDNA is a critical target for ROS damage is illustrated by the fact that increased mtDNA damage and deletions have been observed in a mouse with symptoms resembling the metabolic syndrome (19). This mouse was created by knocking out the gene encoding the DNA repair enzyme NEIL1 (19). Recently, mitochondrial localization of NEIL1 has been reported, thus linking NEIL1 to repair of oxidative damage in mtDNA (20). Furthermore, it has been revealed that the accumulation of mtDNA mutations increased ROS production (21). Therefore, it is likely that damage to mtDNA could further exacerbate the oxidative stress in mitochondria that originally was initiated by exposure to elevated levels of FFA.

DNA is a prime target for toxic agents because, although not invariably lethal, it does affect genetically encoded cellular responses. Of the two types of DNA in the cell, mtDNA is more vulnerable to damage because, unlike nuclear DNA, it has no introns and is not protected by histones. Although the exact mechanisms leading to mitochondrial damage in muscle in T2DM have yet to be fully elucidated, it has been proposed that mitochondrial FFA-derived lipid peroxides can cause injury to molecular targets in mitochondria, including mtDNA (22). Based on recent results from our laboratory, an additional cause of FFA-induced mtDNA damage may be nitric oxide (NO) and/or its derivative RNS. These studies have shown that FFA caused a rise in NO that damaged mtDNA and ultimately led to apoptosis in INS-1 cells (23). In support of this notion, an increase in NO production and expression of the inducible NO synthase (iNOS) has been observed in skeletal muscle from individuals with T2DM (24). The aim of the present study was to use a well-characterized in vitro model of skeletal muscle, rat L6 skeletal muscle cells, to establish whether FFAs cause mtDNA damage and consequently induce mitochondrial dysfunction and apoptosis in these muscle cells. The results described herein reveal that the saturated fatty acid palmitate caused a simultaneous rise in production of ROS and NO in myotubes that damaged mtDNA, diminished ATP production, and ultimately led to apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and treatment
Rat L6 skeletal muscle cells were obtained from ATCC (Manassas, VA). Cells were grown in DMEM supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and 50 µg/ml penicillin/streptomycin (Sigma, St. Louis, MO) in 5% CO2 at 37 C. For these studies, L6 myoblasts were plated in culture dishes or 24-well plates and used at the myotube stage of differentiation. Differentiation was achieved by culturing preconfluent cells in differentiation media (2% fetal bovine serum instead of 10% in the growth media) for 7 d. Reduction of serum allowed cell-to-cell fusion and formation of myotubes. A stock concentration of palmitate (Sigma) was dissolved in 50% ethanol and used for treatment of L6 myotubes after conjugating it with 2% FFA-free BSA (Sigma). Control cells were treated with drug diluent only (2% BSA and 0.4% ethanol in serum-free medium). In some experiments, L6 myotubes were incubated with palmitate and 5 mM N-acetyl cysteine (NAc), 1 mM aminoguanidine, or 200 µM 5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrinato iron (III) (FeTPPS; Calbiochem, San Diego, CA).

Measurements of ROS
Rat L6 myotubes were exposed to 2 mM palmitate and 10 µM dihydroethidium (DHE; Molecular Probes, Eugene, OR) for 10 or 30 min. Control cells were treated with drug diluent only and DHE. After treatment, cells were scraped in PBS, sonicated twice, and fluorescence was evaluated using a fluorometer (Photon Technology International, Lawrenceville, NJ; excitation, 470 nm; emission, 555 nm). Values were expressed per microgram of protein.

NO production
L6 myotubes were exposed to varying concentrations of palmitate for 6 h in serum-free culture medium. Control cells received drug diluent only. After treatment, aliquots of media were collected, and nitrite production was evaluated in duplicate using the Griess reaction (25). Nitrite values were determined using varying concentrations of sodium nitrite as standards. Additionally, nitrite levels were measured in palmitate-supplemented medium because the addition of FFA to medium causes a considerable increase in background nitrite levels (26, 27).

Preparation of cellular fractions and Western blot analysis
Cytosolic protein fractions were isolated from one 100-mm dish of L6 myotubes (23). The lysis buffer for isolating total cellular fractions contains 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, and 5 µl/ml of a mixture of protease inhibitors (Sigma). Protein concentrations were determined using the Bio-Rad protein dye microassay (Bio-Rad, Hercules, CA). SDS-PAGE and transfer of separated proteins to polyvinylidene fluoride membranes were performed as previously described (23). Blocking and antibody immunoblotting were performed in 5% nonfat dry milk and Tris-buffered saline with 0.1% Tween 20. Tris-buffered saline with 0.1% Tween 20 and Tris-buffered saline were used for washing. Antibodies used were cytochrome c (PharMingen, San Diego, CA), actin (Sigma), caspase-3 (Cell Signaling, Beverly, MA), and iNOS (BD Biosciences, San Jose, CA). Complexes formed were detected with horseradish peroxidase-conjugated antimouse IgG or antirabbit IgG antibodies (Promega, Madison, WI) using chemiluminescent reagents (SuperSignal, Pierce, Rockford, IL).

Assay for mtDNA damage
L6 myotubes were exposed to palmitate as described above. DNA isolation and quantitative Southern blots were performed as previously described (23, 27, 28, 29, 30, 31, 32). Cells were lysed in 10 mM Tris-HCI (pH 8.0), 1 mM EDTA (pH 8.0), 0.5% SDS, and 0.3 mg/ml proteinase K overnight at 37 C. High-molecular weight DNA was extracted with phenol, treated with ribonuclease (to a final concentration of 1 µg/ml), and digested to completion with BamHI (10 U/µg of DNA overnight). Digested samples were precipitated, resuspended in Tris-EDTA buffer, and quantified using a Hoefer TKO 100 minifluorometer and TKO standard kit. Samples containing 5 µg DNA were heated at 70 C for 15 min and cooled at room temperature for 20 min. A sodium hydroxide solution was added to a final concentration of 0.1 N, and samples were incubated for 15 min at 37 C. This alkali treatment produced single-strand breaks at all abasic or sugar-modified sites in the DNA. Gel electrophoresis and vacuum transfer were carried out as described previously (23, 27, 28, 29, 30, 31, 32, 33). After prehybridization, membranes were hybridized with a denatured PCR-generated mitochondrial probe (23), washed, and autoradiographed. The resultant band images were analyzed using Molecular Analyst (Bio-Rad) software. Break frequency was determined using the Poisson expression (s= –lnPo, where s is the number of breaks per fragment, and Po is the fraction of fragments free of breaks) (23, 27, 28, 29, 30, 31, 32, 33). Break frequency for nuclear DNA fragments was calculated by scanning similar-sized fragments (about 11 kb) from the ethidium bromide-stained gel photographs (34).

Cell viability
For viability studies, L6 myotubes cells were grown in 24-well culture plates, treated with varying concentrations of palmitate for 24 h. Control cultures were exposed to drug diluent only. Twenty-four hours later, trypan blue was added, and viable cells were counted using light microscopy. Data are expressed as a percentage of untreated control.

MTS assay
The CellTiter 96 assay (Promega), a colorimetric method for determining the number of viable cells by assessing mitochondrial function, was performed 24 h after exposure to different concentrations of palmitate. The reagent contains a tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), and an electron-coupling reagent (phenazine methosulfate). MTS is bioreduced by the dehydrogenase enzymes found in metabolically active cells into a formazan product soluble in tissue culture medium. The quantity of formazan product measured is directly proportional to the number of living cells in culture. The reagent was added to culture wells, and the cells were incubated for 2 h. OD was read at 490 nm in a microplate reader. Data are indicated as percentage of untreated controls.

DNA fragmentation assay
The presence of fragmented nuclear DNA in the cytoplasmic fraction of cell lysates was assessed by measuring DNA associated with nucleosomal histones using a specific two-site ELISA with an antihistone primary antibody and a secondary anti-DNA antibody (Roche Diagnostics Corporation, Indianapolis, IN). Briefly, cells were grown in 24-well culture plates, treated with varying concentrations of palmitate for 24 h, washed twice with PBS, and incubated with 0.5 ml lysis buffer for 20 min at room temperature. After centrifugation to remove nuclei and cellular debris, the supernatants were diluted 1:4 with lysis buffer and 20 µl from each sample were analyzed by ELISA. The intensity of the color which developed was determined by measuring the absorbance at 405 nm, whereas that at 490 nm was used as a blank. Each condition was assessed in duplicate, and experiments were repeated three times.

ATP level
Cells were grown in 24-well culture plates and treated with palmitate for 12 h. To determine the total cellular ATP level, an ATP bioluminescence assay kit (Roche, Mannheim, Germany) was used. This kit employs a well-established technique, which uses the ATP dependency of the light omitting luciferase catalyzed oxidation of luciferin for the measurement of extremely low concentrations of ATP (35). The emitted light is linearly related to the ATP concentration and is measured using a luminometer. Values were normalized per microgram of protein and shown as a percentage of untreated control.

Statistical analysis
Data are expressed as means ± SE. Statistical analyses were performed using paired Student’s t test or one-way ANOVA followed by Bonferroni analysis where appropriate. Statistical significance was determined at the 0.05 level.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Palmitate stimulated production of ROS, NO, and iNOS expression
Because there is evidence that FFA can stimulate the production of ROS (36), we measured superoxide levels in L6 myotubes using the superoxide-sensitive dye DHE. This probe is exquisitely sensitive to the superoxide anion, which oxidizes it to the fluorescent product ethidium (37). Ethidium fluorescence levels were elevated after 10 or 30 min of treatment with 2 mM palmitate (Fig. 1Go). Because NO and/or its derivative RNS, generated as a result of exposure to palmitate, could provide an additional source of injury to mtDNA, we measured nitrite levels in the culture medium after exposure to increasing concentrations of palmitate using the Griess reaction (25). Additionally, nitrite levels were measured in palmitate-supplemented medium because the addition of FFA to culture medium causes an increase in background nitrite levels (26, 27). Also, the nitrite content was measured in myotube cultures incubated with 2 mM palmitate and 1 mM aminoguanidine, an inhibitor of iNOS. Nitrite concentrations were elevated after 6 h treatment with 0.5–2 mM palmitate (Fig. 2AGo). Note that aminoguanidine inhibited NO production in palmitate-treated cultures. To establish that the production of NO was the result of the expression of iNOS, Western blot analysis was performed 6 h postexposure to 2 mM palmitate. iNOS expression was found to be markedly enhanced (Fig. 2BGo).


Figure 1
View larger version (38K):
[in this window]
[in a new window]

  		FIG. 1. Generation of superoxide after exposure to palmitate. L6 myotubes were exposed to 2 mM palmitate and 10 µM DHE for 10 or 30 min. Control cells were treated with drug diluent and DHE only. After treatment, cells were scraped in PBS and sonicated twice, and fluorescence was evaluated. Values were expressed per microgram of protein. The mean ± SE are shown (n = 3). *, P < 0.05.

 

Figure 2
View larger version (31K):
[in this window]
[in a new window]

  		FIG. 2. Palmitate induced NO production and iNOS expression in L6 myotubes. Nitrite production in L6 myotube cultures after treatment with palmitate A, L6 myotubes were incubated in serum-free culture media containing 2% BSA and various concentrations of palmitate (0.5, 1, or 2 mM) for 6 h. After treatment, aliquots of media were collected, and nitrite production was evaluated using the Griess reaction. Also, nitrite levels were measured in the palmitate-supplemented cell free medium to show the background level of nitrites at each concentration (C, untreated control). The mean ± SE are shown (n = 3). *, P < 0.05. B, Western blot analysis of iNOS protein expression after 6 h treatment with 2 mM palmitate (C, untreated control). Actin antibody was used to show equal loading.

 
Palmitate damaged mtDNA to a greater extent than nuclear DNA
To determine whether exposure to palmitate initiated deleterious processes that caused damage to mtDNA in L6 myotubes, dose and time response studies were performed. Myotubes were exposed to different concentrations of palmitate (0.5, 1, or 2 mM) for 1 or 6 h. Quantitative alkaline Southern blot analysis was performed using a mtDNA-specific probe. A progressive amount of mtDNA damage was observed at both 1 and 6 h after exposure to palmitate (Fig. 3AGo). The number of breaks for the control group (untreated control, zero palmitate concentration) was by definition zero (see Materials and Methods). Without a damaging agent, control mtDNA would have no breaks, and this is the standard against which mtDNA is compared when it has been exposed to a damaging agent (27, 28, 29, 30, 31, 32, 33). To establish that this damage was preferentially directed toward mtDNA, damage to nuclear DNA was assessed by performing a densitometric scan of the DNA in the ethidium bromide-stained alkaline agarose gel used for the Southern blot. Damage to nuclear DNA in similarly sized fragments was approximately 6-fold less than that detected in mtDNA after 6 h of palmitate treatment (Fig. 3BGo).


Figure 3
View larger version (26K):
[in this window]
[in a new window]

  		FIG. 3. Palmitate damaged mtDNA to a greater extent than nuclear DNA in L6 myotubes. L6 myotubes were incubated in serum-free culture media containing 2% BSA and various concentrations of palmitate (0.5, 1, and 2 mM) for 1 or 6 h. A, Break frequency per 10.8-kb fragment of mtDNA after 1 or 6 h of treatment with the indicated concentrations of palmitate (C, untreated control). B, Break frequency per 10.8-kb fragment of nuclear and mtDNA after 6-h treatment with the indicated concentrations of palmitate (C, untreated control). The mean ± SE are shown for both A and B (n = 3).

 
ROS/RNS scavengers and inhibitors attenuated palmitate-induced mtDNA damage and cell death
To examine the role of ROS/RNS in palmitate-induced mtDNA damage and cell viability, we used ROS scavengers and different inhibitors of NOS. For mtDNA damage studies, L6 myotubes were treated with 2 mM palmitate because this was the maximum concentration used in the dose response studies, conjugated to 2% BSA alone or with NAc (a precursor compound for glutathione formation), aminoguanidine, an iNOS inhibitor, or FeTPPS, a peroxynitrite inhibitor (38). NAc, aminoguanidine, and FeTPPS all prevented palmitate-induced mtDNA damage (Fig. 4AGo). To assess cell viability, a trypan blue exclusion assay was used. Briefly, cells were grown in 24-well culture plates and treated with varying concentrations of palmitate for 24 h. Additionally, at each concentration of palmitate used, selected L6 myotubes cultures were incubated with palmitate and 5 mM NAc, 1 mM aminoguanidine, or 200 µM FeTPPS. As shown in Fig. 4BGo, NAc, aminoguanidine, and FeTPPS all diminished palmitate-induced cell death.


Figure 4
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 4. NAc, aminoguanidine, and FeTPPS reduced palmitate-induced mtDNA damage and cytotoxicity in L6 myotubes. A, L6 myotubes were exposed to 2 mM palmitate (P) for 6 h alone or in combination with 5 mM NAc, 1 mM aminoguanidine (A), and 200 µM FeTPPS. Break frequency per 10.8-kb fragment of mtDNA. B, L6 myotubes were incubated with palmitate (P) alone or with palmitate (P) and 5 mM NAc, 1 mM aminoguanidine, or 200 µM FeTPPS. Control (C) cultures were treated with drug diluent alone (untreated control) or with drug diluent in combination with 5 mM NAc, 1 mM aminoguanidine, or 200 µM FeTPPS. Twenty-four hours later, trypan blue was added, and viable cells were counted using light microscopy. Viability was calculated and shown as a percentage of corresponding untreated control. The mean ± SE are shown (n = 3). *, P < 0.05 vs. untreated control; #, P < 0.05 vs. palmitate-treated condition.

 
Palmitate decreased ATP levels and mitochondrial viability in L6 myotubes
To investigate whether the observed increase in mtDNA damage correlated with a decrease of production of ATP, total cellular ATP levels were measured 12 h after exposure to different concentrations of palmitate (0.5, 1, or 2 mM). Palmitate was found to reduce ATP levels in a dose-dependent fashion (Fig. 5AGo). To ascertain whether the progressive increase in mtDNA damage and reduced ATP levels after exposure to palmitate correlated with a decrease in mitochondrial viability in the cell cultures, mitochondrial function was assessed by the MTS assay 24 h after exposure to 0.5, 1, or 2 mM palmitate (Fig. 5BGo). The results showed a dose-dependent decrease in cellular dehydrogenase activity.


Figure 5
View larger version (20K):
[in this window]
[in a new window]

  		FIG. 5. Palmitate decreased ATP levels and mitochondrial function in L6 myotubes. A, Palmitate dose-dependent reduction in ATP levels in L6 myotubes. B, Palmitate decreased mitochondrial function in L6 myotubes. The mean ± SE are shown (n = 3). *, P < 0.05.

 
Palmitate induced apoptosis, caspase-3 cleavage, and cytochrome c release from mitochondria
To determine whether exposure to palmitate was associated with an increase in apoptosis, apoptosis was evaluated using a DNA fragmentation assay and confirmed by assessing caspase 3 activation. Additionally, to establish that palmitate-mediated apoptosis is induced by a pathway initiated through mitochondria, cytochrome c release into the cytosol was determined. Myotubes were exposed to different concentrations of palmitate (0.5, 1, or 2 mM) for 24 h. After incubation, cells were rinsed, and DNA fragmentation was evaluated using the Cell Death Detection ELISA kit (Fig. 6AGo). After 24 h palmitate treatment, activated caspase-3 was found in L6 myotubes (Fig. 6BGo). Additionally, palmitate treatment for 24 h caused cytochrome c to be released from mitochondria into the cytoplasm as determined by Western blot analysis of the cytosolic fractions (Fig. 6CGo). These results indicate that palmitate-mediated apoptosis is induced by a pathway initiated through mitochondria.


Figure 6
View larger version (34K):
[in this window]
[in a new window]

  		FIG. 6. Palmitate induced apoptosis, caspase-3 activation, and cytochrome c release from mitochondria in L6 myotubes. DNA fragmentation, as measured by an ELISA using absorbance (OD 405–490 values). Culture of L6 myotubes 24 h in the presence of palmitate resulted in a dose-dependent fragmentation of DNA. A, Western blots were performed on the cytosolic protein fraction from untreated control (C) cells and L6 myotubes exposed to different concentrations of palmitate for 24 h. B, Caspase-3 antibodies were used to recognize the full-length (35 kDa) and large (17 kDa) fragments of caspase-3 resulting from its cleavage. C, Western blot of the cytochrome c release into the cytosol. Equal loading for both Western blots (B and C) was confirmed using an actin antibody. Note the increase of cleaved fragment of caspase-3 and cytochrome c in the cytosolic fraction isolated from palmitate-treated L6 myotubes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The hypothesis that mitochondrial dysfunction caused by oxidative stress resulting from FFA-induced lipotoxicity contributes to insulin resistance in skeletal muscle recently has come to the forefront in diabetes research (14). This hypothesis has been supported by reports that showed an increase in intramyocellular lipid content (39), reduced oxidative enzyme activity, which can result in mitochondrial oxidative stress, and impaired functional capacity in skeletal muscle from T2DM patients (15, 16).

The aims of the present study were to assess whether FFA caused 1) mtDNA damage, 2) mitochondrial dysfunction, and 3) apoptosis in rat L6 skeletal muscle cells. As a source of FFA, we used palmitate, which is the most abundant saturated FFA in the circulation. Moreover, palmitate and the unsaturated FFA, oleate, comprise the largest percentage of diglyceride and triglyceride fractions in rat soleus and gastrocnemius muscles (40). In C2C12 myotubes, palmitate, but not oleate, has been found to inhibit the action of insulin (41). Also, saturated fatty acids like palmitate have been found to cause marked apoptosis in different cell types (42, 43, 44). Conversely, unsaturated fatty acids, such as oleate, are less cytotoxic and may even exert some protection against the proapoptotic effects generated by saturated fatty acids (43, 44). Thus, palmitate appears to be more involved in promoting insulin resistance than unsaturated FFA (41, 45). Therefore, for the present study, we chose to focus only on the effects of palmitate.

We found a progressive increase in mtDNA damage after exposure to increasing concentrations of palmitate, with the greatest amount of damage to mtDNA resulting from exposure to 2 mM palmitate (~one break per restriction fragment). It is important to point out that although this concentration caused the greatest amount of damage, significant mtDNA damage was seen at concentrations as low as 0.5 mM. Nevertheless, the 2 mM concentration of FFA and BSA (2%) placed the unbound FFA levels in the range of 600–800 µM, which is consistent with the FFA concentrations observed in T2DM (46). Moreover, this concentration of palmitate has been found previously to cause insulin resistance at the level of glycogen synthesis in rat soleus muscle (47).

Previously, our laboratory has shown that endogenously generated peroxynitrite produced by exposure to cytokines can damage mtDNA in oligodendrocytes (33). Based on the fact that palmitate stimulated iNOS expression and the production of both superoxide and NO in L6 myotubes, we believe that peroxynitrite, the end product of the reaction of NO with superoxide (for review, see Ref. 48), is the oxidant that damaged mtDNA in these cells. The most direct evidence supporting that peroxynitrite caused the mtDNA damage and cell death in the present report comes from the studies using the peroxynitrite decomposition catalyst, FeTPPS, which significantly reduced palmitate-induced mtDNA damage even at the highest concentration studied. Additionally, aminoguanidine, which blocks NO production, and NAc, which enhances the scavenging of superoxide, also reduced palmitate-induced mtDNA damage and cytotoxicity in L6 myotubes. Therefore, when either of the precursors required for peroxynitrite formation were removed, both mtDNA damage and cell death were attenuated.

Although it is as yet uncertain whether a frank elevation in the levels of FFA that directly damage mitochondria or inherited defects in mitochondrial metabolism that decrease oxidative capacity and cause increased FFA levels ultimately leads to insulin resistance, it is apparent that elevated levels of FFA contribute to the initiation of insulin resistance. One significant problem which occurs in muscle after the onset of insulin resistance is muscle atrophy. Marked muscle atrophy is a major characteristic of many catabolic conditions that result from both increased proteolysis and the inability to repair damaged skeletal muscle through protein synthesis (49). Several proteolytic systems contribute to the degradation of muscle proteins, including the ATP-dependent ubiquitin-proteasome system (for review, see Ref. 50) and caspase-3 activation through apoptosis (51). Although little is known about the proteolytic systems and the pathways contributing to muscle loss in T2DM, increased degradation of skeletal muscle protein by the apoptotic pathway has been shown in a model of type 1 diabetes (52). Therefore, discovering methods to attenuate apoptosis in skeletal muscle could provide a means for protecting this vital tissue from atrophy. Insulin resistance has long been considered to play a central role in the derangements in metabolism that ultimately lead to muscle wasting. We have found that palmitate induced mtDNA damage and apoptosis via a mitochondrial pathway in L6 myotubes. Others have demonstrated that similar concentrations of palmitate induced insulin resistance in L6 myotubes (53, 54). In addition, insulin resistance was found to increase in conditions that have been associated with the loss of skeletal muscle due to activation of apoptotic signals (55). Therefore, in considering the complexity of the pathways governing insulin resistance in skeletal muscle, it is of importance to note that regardless of the exact mechanism, diminished skeletal muscle oxidative capacity through FFA-induced mitochondrial dysfunction is likely related to the induction of muscle insulin resistance and apoptosis and contributes to the development of T2DM. The question as to whether skeletal muscle apoptosis is the result of insulin resistance, a contributor to insulin resistance, or a contributor to T2DM still remains to be fully resolved. The findings reported here suggest that chronic damage to mtDNA, produced by the continuous exposure to FFA, is a critical contributor to skeletal muscle atrophy likely through the induction of apoptosis. Therefore, novel strategies to prevent this damage to mtDNA may be of significant therapeutic benefit for the treatment of some of the secondary complications resulting from T2DM.


   Acknowledgments
 
We thank Dr. A. B. Al-Mehdi and Darla Reed for the help with the fluorometer.


   Footnotes
 
This work was supported by National Institutes of Health Grants ES03456 (to G.L.W.), AG19602 (to G.L.W.), ES05865 (to S.P.L.), and NS 041208 (to S.P.L.).

The authors have nothing to disclose.

First Published Online October 5, 2006

Abbreviations: DHE, Dihydroethidium; FeTPPS, 5,10,15,20-tetrakis(4-sulphonatophenyl) porphyrinato iron (III); FFA, free fatty acid; iNOS, inducible NO synthase; mtDNA, mitochondrial DNA; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; NAc, N-acetyl cysteine; NO, nitric oxide; RNS, reactive nitrogen species; ROS, reactive oxygen species; T2DM, type 2 diabetes mellitus.

Received July 25, 2006.

Accepted for publication September 28, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. DeFronzo RA, Gunnarsson R, Bjorkman O, Olsson M, Wahren J 1985 Effects of insulin on peripheral and splanchnic glucose metabolism in noninsulin-dependent (type II) diabetes mellitus. J Clin Invest 76:149–155[Medline]
  2. McGarry JD 1992 What if Minkowski had been ageusic? An alternative angle on diabetes. Science 258:766–770[Abstract/Free Full Text]
  3. Randle PJ, Kerbey AL, Espinal J 1988 Mechanisms decreasing glucose oxidation in diabetes and starvation: role of lipid fuels and hormones. Diabetes Metab Rev 4:623–638[Medline]
  4. Shulman GI 2000 Cellular mechanisms of insulin resistance. J Clin Invest 106:171–176[Medline]
  5. Evans JL, Goldfine ID, Maddux BA, Grodsky GM 2003 Are oxidative stress-activated signaling pathways mediators of insulin resistance and ß-cell dysfunction? Diabetes 52:1–8[Abstract/Free Full Text]
  6. Rosen P, Nawroth P, King G, Moller W, Tritschler HJ, Packer L 2001 The role of oxidative stress in the onset and progression of diabetes and its complications: a summary of a Congress Series sponsored by UNESCO-MCBN, the American Diabetes Association and the German Diabetes Society. Diabetes Metab Res Rev 17:189–212[CrossRef][Medline]
  7. Houstis N, Rosen ED, Lander ES 2006 Reactive oxygen species have a causal role in multiple forms of insulin resistance. Nature 440:944–948[CrossRef][Medline]
  8. Chance B, Sies H, Boveris A 1979 Hydroperoxide metabolism in mammalian organs. Physiol Rev 59:527–605[Free Full Text]
  9. Wojtczak L, Schonfeld P 1993 Effect of fatty acids on energy coupling processes in mitochondria. Biochim Biophys Acta 1183:41–57[Medline]
  10. Bakker SJ, Ijzerman RG, Teerlink T, Westerhoff HV, Gans RO, Heine RJ 2000 Cytosolic triglycerides and oxidative stress in central obesity: the missing link between excessive atherosclerosis, endothelial dysfunction, and ß-cell failure? Atherosclerosis 148:17–21[CrossRef][Medline]
  11. Toborek M, Hennig B 1994 Fatty acid-mediated effects on the glutathione redox cycle in cultured endothelial cells. Am J Clin Nutr 59:60–65[Abstract/Free Full Text]
  12. Hennig B, Meerarani P, Ramadass P, Watkins BA, Toborek M 2000 Fatty acid-mediated activation of vascular endothelial cells. Metabolism 49:1006–1013[CrossRef][Medline]
  13. Green K, Brand MD, Murphy MP 2004 Prevention of mitochondrial oxidative damage as a therapeutic strategy in diabetes. Diabetes 53(Suppl 1):S110–S118
  14. Fridlyand LE, Philipson LH 2006 Reactive species and early manifestation of insulin resistance in type 2 diabetes. Diabetes Obes Metab 8:136–145[CrossRef][Medline]
  15. Ritov VB, Menshikova EV, He J, Ferrell RE, Goodpaster BH, Kelley DE 2005 Deficiency of subsarcolemmal mitochondria in obesity and type 2 diabetes. Diabetes 54:8–14[Abstract/Free Full Text]
  16. Kelley DE, He J, Menshikova EV, Ritov VB 2002 Dysfunction of mitochondria in human skeletal muscle in type 2 diabetes. Diabetes 51:2944–2950[Abstract/Free Full Text]
  17. Liang P, Hughes V, Fukagawa NK 1997 Increased prevalence of mitochondrial DNA deletions in skeletal muscle of older individuals with impaired glucose tolerance: possible marker of glycemic stress. Diabetes 46:920–923[Abstract]
  18. Park SY, Choi GH, Choi HI, Ryu, J, Jung CY, Lee W 2005 Depletion of mitochondrial DNA causes impaired glucose utilization and insulin resistance in L6 GLUT4myc myocytes. J Biol Chem 280:9855–9864[Abstract/Free Full Text]
  19. Vartanian V, Lowell B, Minko IG, Wood TG, Ceci JD, George S, Ballinger SW, Corless CL, McCullough AK, Lloyd RS 2006 The metabolic syndrome resulting from a knockout of the NEIL1 DNA glycosylase. Proc Natl Acad Sci USA 103:1864–1869[Abstract/Free Full Text]
  20. Hu J, de Souza-Pinto NC, Haraguchi K, Hogue BA, Jaruga P, Greenberg MM, Dizdaroglu M, Bohr VA 2005 Repair of formamidopyrimidines in DNA involves different glycosylases: role of the OGG1, NTH1, and NEIL1 enzymes. J Biol Chem 280:40544–40551[Abstract/Free Full Text]
  21. Petros JA, Baumann AK, Ruiz-Pesini E, Amin MB, Sun CQ, Hall J, Lim S, Issa MM, Flanders Wd, Hosseini SH, Marshall FF, Wallace DC 2005 mtDNA mutations increase tumorigenicity in prostate cancer. Proc Natl Acad Sci USA 102:19–724
  22. Schrauwen P, Hesselink MK 2004 Oxidative capacity, lipotoxicity, and mitochondrial damage in type 2 diabetes. Diabetes 53:1412–1417[Abstract/Free Full Text]
  23. Grishko V, Rachek L, Musiyenko S, LeDoux SP, Wilson GL 2005 Involvement of mtDNA damage in free fatty acid-induced apoptosis. Free Radic Biol Med 38:755–762[CrossRef][Medline]
  24. Torres SH, De Sanctis JB, de L Briceno M, Hernandez N, Finol HJ 2004 Inflammation and nitric oxide production in skeletal muscle of type 2 diabetic patients. J Endocrinol 181:419–427[Abstract]
  25. Green LC, Wagner DA, Goglowski J, Skipper PL, Wishnok JS, Tannenbaum SR 1982 Analysis of nitrate, nitrite, and [15 N]nitrate in biological fluids. Anal Biochem 126:131–138[CrossRef][Medline]
  26. Cnop M, Hannaert JC, Hoorens A, Eizirik DL, Pipeleers DG 2001 Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation. Diabetes 50:1771–17772[Abstract/Free Full Text]
  27. Rachek LI, Thornley NP, Grishko VI, LeDoux SP, Wilson GL 2006 Protection of INS-1 cells from free fatty acid-induced apoptosis by targeting hOGG1 to mitochondria. Diabetes 55:1022–1028[Abstract/Free Full Text]
  28. Pettepher CC, LeDoux SP, Bohr VA, Wilson GL 1991 Repair of alkali-labile sites within the mitochondrial DNA of RINr 38 cells after exposure to the nitrosourea streptozotocin. J Biol Chem 266:3113–3117[Abstract/Free Full Text]
  29. Driggers WJ, LeDoux SP, Wilson GL 1993 Repair of oxidative damage within the mitochondrial DNA of RINr 38 cells. J Biol Chem 268:22042–22045[Abstract/Free Full Text]
  30. Dobson AW, Xu Y, Kelley MR, LeDoux SP, Wilson GL 2000 Enhanced mitochondrial DNA repair and cellular survival after oxidative stress by targeting the human 8-oxoguanine glycosylase repair enzyme to mitochondria. J Biol Chem 275:37518–37523[Abstract/Free Full Text]
  31. Rachek LI, Grishko VI, Musiyenko SI, Kelley MR, LeDoux SP, Wilson GL 2002 Conditional targeting of the DNA repair enzyme hOGG1 into mitochondria. J Biol Chem 277:44932–44937[Abstract/Free Full Text]
  32. Grishko VI, Rachek LI, Spitz DR, Wilson GL, LeDoux SP 2005 Contribution of mitochondrial DNA repair to cell resistance from oxidative stress. J Biol Chem 280:8901–8905[Abstract/Free Full Text]
  33. Druzhyna NM, Musiyenko SI, Wilson GL, LeDoux SP 2005 Cytokines induce nitric oxide-mediated mtDNA damage and apoptosis in oligodendrocytes. Protective role of targeting 8-oxoguanine glycosylase to mitochondria. J Biol Chem 280:21673–21679[Abstract/Free Full Text]
  34. Wassermann K, Kohn KW, Bohr VA 1990 Heterogeneity of nitrogen mustard-induced DNA damage and repair at the level of the gene in Chinese hamster ovary cells. J Biol Chem 265:13906–13913[Abstract/Free Full Text]
  35. Crouch SP, Kozlowski R, Slater K J, Fletcher J 1993 The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. J Immunol Methods 160:81–88[CrossRef][Medline]
  36. Inoguchi T, Li P, Umeda F, Yu HY, Kakimoto M, Imamura M, Aoki T, Etoh T, Hashimoto T, Naruse M, Sano H, Usumi H, Nawata H 2000 High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C-dependent activation of NAD(P)H oxidase in cultured vascular cells. Diabetes 49:1939–1945[Abstract]
  37. Rothe G, Valet G 1990 Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2',7'-dichlorofluorescein. J Leukoc Biol 47:440–448[Abstract]
  38. Misko TP, Highkin MK, Veenhuizen AW, Manning PT, Stern MK, Currie MG, Salvemini D 1998 Characterization of the cytoprotective action of peroxynitrite decomposition catalysts. J Biol Chem 273:15646–15653[Abstract/Free Full Text]
  39. Petersen KF, Dufour S, Befroy D, Garcia R, Shulman GI 2004 Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes. N Engl J Med 350:664–671[Abstract/Free Full Text]
  40. Gorski J, Nawrocki A, Murthy M 1998 Characterization of free and glyceride-esterified long chain fatty acids in different skeletal muscle types of the rat. Mol Cell Biochem 178:113–118[CrossRef][Medline]
  41. Chavez JA, Summers SA 2003 Characterizing the effects of saturated fatty acids on insulin signaling and ceramide and diacylglycerol accumulation in 3T3–L1 adipocytes and C2C12 myotubes. Arch Biochem Biophys 419:101–109[CrossRef][Medline]
  42. de Vries JE, Vork MM, Roeman TH, de Jong YF, Cleutjens JP, van der Vusse GJ, van Bilsen M 1997 Saturated but not mono-unsaturated fatty acids induce apoptotic cell death in neonatal rat ventricular myocytes. J Lipid Res 38:1384–1394[Abstract]
  43. Maedler K, Oberholzer J, Bucher P, Spinas GA, Donath MY 2003 Monosaturated fatty acids prevent the deleterious effects of palmitate and high glucose on human pancreatic ß-cell turnover and function. Diabetes 52:726–733[Abstract/Free Full Text]
  44. Hardy S, El-Assaad W, Przybytkowski E, Joly E, Prentki M, Langelier Y 2003 Saturated fatty acid-induced apoptosis in MDA-MB-231 breast cancer cells: a role for cardiolipin. J Biol Chem 278:31861–31870[Abstract/Free Full Text]
  45. Schmitz-Peiffer C, Craig DL, Biden TJ 1999 Ceramide generation is sufficient to account for the inhibition of the insulin-stimulated PKB pathway in C2C12 skeletal muscle cells pretreated with palmitate. J Biol Chem 274:24202–24210[Abstract/Free Full Text]
  46. Kashyap SR, Belfort R, Berria R, Suraamornkul S, Pratipranawtr T, Finlayson J, Barrentine A, Bajaj M, Mandariono L, DeFronzo R, Cusi K 2004 Discordant effects of a chronic physiological increase in plasma FFA on insulin signaling in healthy subjects with or without a family history of type 2 diabetes. Am J Physiol Endocrinol Metab 287:E537–E546
  47. Thompson AL, Lim-Fraser MY, Kraegen EW, Cooney GJ 2000 Effects of individual fatty acids on glucose uptake and glycogen synthesis in soleus muscle in vitro. Am J Physiol Endocrinol Metab 279:E577–E584
  48. Brown GC, Borutaite V 2002 Nitric oxide inhibition of mitochondrial respiration and its role in cell death. Free Radic Biol Med 33:1440–1450[CrossRef][Medline]
  49. Smith OL, Wong CY, Gelfand RA 1989 Skeletal muscle proteolysis in rats with acute streptozocin-induced diabetes. Diabetes 38:1117–1122[Abstract]
  50. Powers SK, Kavazis AN, DeRuisseau KC 2005 Mechanisms of disuse muscle atrophy: role of oxidative stress. Am J Physiol Regul Integr Comp Physiol 288:R337–R344
  51. Du J, Wang X, Miereles C, Bailey JL, Debigare R, Zheng B, Price SR, Mitch WE 2004 Activation of caspase-3 is an initial step triggering accelerated muscle proteolysis in catabolic conditions. J Clin Invest 113:115–123[CrossRef][Medline]
  52. Lee SW, Dai G, Hu Z, Wang X, Du J, Mitch WE 2004 Regulation of muscle protein degradation: coordinated control of apoptotic and ubiquitin-proteasome systems by phosphatidylinositol 3 kinase. J Am Soc Nephrol 15:1537–1545[Abstract/Free Full Text]
  53. Powell DJ, Turban S, Gray A, Hajduch E, Hundal HS 2004 Intracellular ceramide synthesis and protein kinase C{zeta} activation play an essential role in palmitate-induced insulin resistance in rat L6 skeletal muscle cells. Biochem J 382:619–629[CrossRef][Medline]
  54. Sihna S, Perdomo G, Brown NF, O’Doherty RM 2004 Fatty acid-induced insulin resistance in L6 myotubes is prevented by inhibition of activation and nuclear localization of nuclear factor {kappa}B. J Biol Chem 279:41294–41301[Abstract/Free Full Text]
  55. Sugita H, Kaneki M, Sugita M, Yasukawa T, Yasuhara S, Martyn JA 2005 Burn injury impairs insulin-stimulated Akt/PKB activation in skeletal muscle. Am J Physiol Endocrinol Metab 288:E585–E591



This article has been cited by other articles:


Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
H. Sell, K. Eckardt, A. Taube, D. Tews, M. Gurgui, G. Van Echten-Deckert, and J. Eckel
Skeletal muscle insulin resistance induced by adipocyte-conditioned medium: underlying mechanisms and reversibility
Am J Physiol Endocrinol Metab, June 1, 2008; 294(6): E1070 - E1077.
[Abstract] [Full Text] [PDF]

Home page
 J. Physiol.Home page
P. Newsholme, E. P. Haber, S. M. Hirabara, E. L. O. Rebelato, J. Procopio, D. Morgan, H. C. Oliveira-Emilio, A. R. Carpinelli, and R. Curi
Diabetes associated cell stress and dysfunction: role of mitochondrial and non-mitochondrial ROS production and activity
J. Physiol., August 15, 2007; 583(1): 9 - 24.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Crunkhorn, F. Dearie, C. Mantzoros, H. Gami, W. S. da Silva, D. Espinoza, R. Faucette, K. Barry, A. C. Bianco, and M. E. Patti
Peroxisome Proliferator Activator Receptor {gamma} Coactivator-1 Expression Is Reduced in Obesity: POTENTIAL PATHOGENIC ROLE OF SATURATED FATTY ACIDS AND p38 MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION
J. Biol. Chem., May 25, 2007; 282(21): 15439 - 15450.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
148/1/293    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rachek, L. I.
Right arrow Articles by Wilson, G. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rachek, L. I.
Right arrow Articles by Wilson, G. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals