From the Diabetes Research Laboratory (B.A.M., W.S., A.L.G., I.D.G.), Mount Zion Hospital, San Francisco, California; the Department of Pharmacology (J.C.L.), University of Virginia, Charlottesville, Virginia; and the Medical Research Institute (J.L.E.), San Bruno, California.
Address correspondence and reprint requests to Dr. Betty A. Maddux, Diabetes Research Laboratory, Mount Zion Hospital, University of California at San Francisco, 2200 Post St., San Francisco, CA 94143-1616. E-mail: bmaddux{at}itsa.ucsf.edu .
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ABSTRACT |
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INTRODUCTION |
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LA levels are decreased in diabetic patients (5). In Germany, LA has been used for 30 years to treat diabetic neuropathy and liver cirrhosis (2,3,6). It was believed that both the oxidative stress and abnormal 2-oxo-acid oxidation that occur in these conditions are corrected by LA administration. LA has also been used to treat heavy metal poisoning via its metal chelating activity (1).
Evidence suggests that improvements in glucose metabolism occur in diabetic animals and diabetic humans treated with LA (7). It was first reported that LA stimulates glucose utilization in rat hemidiaphragm studied in vitro (8). More recent studies have indicated that LA administration to obese Zucker (fa/fa) rats improves insulin-stimulated glucose uptake in muscle (9,10,11). In addition, in diabetic and nondiabetic fasted rats, LA was reported to cause acute hypoglycemia by decreasing hepatic glucose output (12). This effect on hepatic glucose output could have been attributable to an effect of LA on the liver directly or on adipose tissue, where free fatty acid release regulates hepatic glucose output (13). In spontaneously hypertensive rats, dietary supplementation with LA lowered systolic blood pressure, glucose and insulin levels, and tissue aldehyde conjugates, and attenuated adverse renal vascular changes (14).
In patients with insulin-resistant type 2 diabetes, chronic and acute parenteral administration of LA improves insulinmediated glucose disposal by 30 and 55%, respectively (15,16). More recently, it has been observed that chronic oral administration of LA exerts a small but significant effect on insulin sensitivity in patients with type 2 diabetes (17,18). Because the major action of insulin in vivo is to enhance glucose disposal via skeletal muscle glucose transport, it is likely that skeletal muscle is a primary target for LA action.
LA has been studied in cultured cells in order to understand and characterize its effects on glucose metabolism. In 3T3-L1 adipocytes, one group reported that oxidative stress induced by the generation of low amounts of H2O2 decreased insulin-stimulated glucose transport and GLUT4 translocation (19,20). Pretreatment with LA, at micromolar concentrations, protected the effects of insulin (21). Because the therapeutic effects of LA occur at plasma concentrations in the micromolar range (17,22), it is possible that protection against oxidative stress is one mechanism by which LA improves insulin action. However, the potential effect of LA on oxidative stress in muscle cells has not been evaluated.
In the present report, we investigated the effects of LA on cultured rat L6 muscle cells that had undergone oxidative stress. To improve both the responsiveness and sensitivity to insulin, we studied cells that were engineered to overexpress GLUT4 (23). In these cells, we find that oxidative stress inhibits insulin action on glucose transport. We now report that LA has little effect on nonstressed cells; however, in stressed cells, LA at micromolar concentrations restores responsiveness to insulin.
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RESEARCH DESIGN AND METHODS |
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Cell culture. L6 wild-type (L6 WT) cells and L6 GLUT4 cells (clone
SG4-811) (23) were cultured
(37°C, 5% CO2) in DMEM, supplemented with 10% FCS, 100 units/ml
penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. For
glucose transport assays, cells were subcultured into 24-well cluster plates.
L6 WT cells were plated at 10,000 cells/ml and allowed to differentiate
spontaneously (7-10 days after plating), as described previously
(24). L6 GLUT4 cells were
plated at 40,000 cells/ml, and transport assays were performed at 80%
confluence. These cells did not require differentiation.
Glucose transport. L6 GLUT4 and L6 WT cells were incubated (in triplicate) in DMEM with 0.5% bovine serum albumin (BSA) for various times, and then placed in transport buffer consisting of 20 mmol/l HEPES (pH 7.4), 140 mmol/l NaCl, 5 mmol/l KCl, 2.5 mmol/l MgCl2, 1 mmol/l CaCl2, and 0.1% (wt/vol) BSA. Next, insulin (1-1000 nmol/l) was added for 30 min, followed by 10 µmol/l 2-DG (1.0 µCi/ml) for 30 min at 37°C. Reactions were stopped by aspirating the media and thoroughly washing the monolayers with phosphate-buffered saline (PBS) containing 20 mmol/l D-glucose (at 4°C). Cells were solubilized in 0.03% (wt/vol) SDS, and radioactivity was determined by liquid scintillation counting. Data were expressed per milligram of lysate protein, which was determined using the bicinchoninic acid method (Pierce Chemical, Rockford, IL). Preliminary studies indicated that insulin stimulation of glucose transport in L6 WT cells was maximum when cells were serum starved for 4 h, whereas in L6 GLUT4 cells the maximum response occurred when cells were serum starved for 18 h.
Treatment with antioxidants. L6 GLUT4 cells were washed in DMEM supplemented with 0.5% BSA. Next, LA or other antioxidants were added, and cells were incubated for 18 h. Cells were then washed and incubated in 0.5 ml DMEM (phenol red-free) supplemented with 0.5% BSA, 100 mU/ml glucose oxidase, and 5 mmol/l D-glucose for 2 h. To measure the amount of H2O2 generated, media were collected from triplicate wells, transferred to tubes containing 0.25 ml 50% (wt/vol) trichloroacetic acid, chilled on ice, and centrifuged (5000g for 10 min). Aliquots (1 ml) of the supernatant were added to 0.2 ml of 10 mmol/l ferrous ammonium sulfate and 0.1 ml of 2.5 mol/l potassium thiocyanate. Absorbance was measured spectrophotometrically at 491 nm using t-butyl hydroperoxide as a standard. Lactate dehydrogenase was measured in the culture medium using a colorimetric kit purchased from Sigma (Product No. 500C).
Reduced glutathione determination. Cells were treated to produce oxidative stress as described above. Next, cells were washed 3 times with PBS, scraped, and sonicated for 20 s. Then an equal volume of 10% (vol/vol) metaphosphoric acid was added to samples, incubated at room temperature for 5 min, and centrifuged for 5 min. Samples were assayed for glutathione (GSH) using a glutathione assay kit (Cayman Chemical, #703002).
p38 Mitogen-activated protein kinase. Cells were grown until 80% confluent, then treated to produce oxidative stress as described above. Next, cells were washed and solubilized, and 20 µg protein was loaded onto 8-16% Trisglycine gel (Novex). Proteins were transferred to nitrocellulose, then incubated overnight with antiphospho p38 mitogen-activated protein kinase (MAPK; 1:1000). Signal was detected using a Phototope-HRP Western Detection Kit (New England Biolabs).
Statistical analyses. Data are expressed as means ± SE. Differences between means were assessed by Student's t test or one-way analysis of variance (ANOVA). Post hoc comparisons were performed using either a Dunnett's or Newman-Keuls test for multiple comparisons. Statistical significance was accepted at P < 0.05. All analyses (and graphics) were performed using Graph-Pad Prism (MS Windows version 3.02; GraphPad Software, San Diego, CA; www.graphpad.com ).
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RESULTS |
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Direct effects of LA on glucose transport in L6 GLUT4 cells. We next
investigated whether LA exerted a direct effect on glucose transport. L6 GLUT4
cells were exposed to LA for up to 18 h, followed by the addition of 100
nmol/l insulin for 30 min. Over the concentration range of 1-1,000 µmol/l,
LA produced only a small effect on both basal and insulin-stimulated glucose
transport (Fig. 2). In the
absence of insulin, 1000 µmol/l LA stimulated 2-DG uptake by 65%
(P < 0.05). In the presence of insulin (1 µmol/l), 1,000
µmol/l LA stimulated 2-DG uptake by 22% (NS). At concentrations >
1,000 µmol/l, cell death occurred.
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Effects of LA on glucose transport in L6 GLUT4 cells exposed to oxidative stress. We next used glucose and glucose oxidase to generate H2O2, causing oxidative stress as previously described (21). Increasing the glucose oxidase concentration from 10 to 100 mU/ml caused a linear increase in H2O2 production (Fig. 3). Pretreatment of cells with 100 mU/ml of glucose oxidase produced an H2O2 concentration of 40-50 µmol/l. When cells were treated with this concentration of glucose oxidase, basal glucose transport was decreased slightly (but not significantly), whereas insulin-stimulated transport was nearly abolished (P < 0.05) (Fig. 4). Pretreatment with 300 µmol/l LA had a small, but not significant, stimulatory effect on basal glucose transport, but completely restored the responsiveness to insulin (P < 0.05).
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To evaluate whether the protective effect of racemic LA on insulin-stimulated glucose transport could be attributed to the activity of a particular isomer, we studied the effects of the two individual isomers of LA, R and S, on oxidative stressinduced insulin resistance. For each isomer, a protective effect was observed at 30 µmol/l (P < 0.05), and a maximal effect was achieved at 1,000 µmol/l (P < 0.001) (Fig. 5). The EC50 values for the R and S isomers were 96 and 131 µmol/l, respectively, but these values were not statistically different.
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In addition to LA, exposure to another antioxidant, vitamin E, also afforded significant protection against oxidative stressinduced insulin resistance (Fig. 6). In cells treated with vitamin E (5 µmol/l), the response to insulin was nearly normalized. In contrast, vitamin C (300 µmol/l) and troglitazone (5 µmol/l), a thiazolidinedione possessing the vitamin E moiety, were ineffective at protecting against oxidative stress at the concentrations tested. There was a trend toward increased basal rates of 2-DG uptake in cells treated with LA and vitamins C and E, although this difference was not statistically significant.
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Cell toxicity and intracellular GSH content. To determine whether H2O2 generation was causing cell toxicity or death, we measured the release of the cytosolic marker enzyme, lactate dehydrogenase (LDH). At 100 µmol/l H2O2, there was no significant increase in LDH activity released into the incubation medium (data not shown). The release of LDH activity was detected only at concentrations of H2O2 >1 mmol/l. Thus, under the experimental conditions of the present study, it is unlikely that the effects of LA could be attributed to simply protecting the cells from cell death.
Treatment of cells with 100 mU/ml of glucose oxidase and 5 mmol/l glucose for 2 h resulted in a reduction in the intracellular GSH content from 56.7 ± 10.7 to 32 ± 11.3 nmol/mg protein (P < 0.05) (Fig. 7), reflecting a condition of oxidative stress resulting from an alteration in intracellular redox state (21). To determine if LA could protect against the reduction in GSH content brought about by increased H2O2, cells were incubated for 18 h with racemic LA in the absence or presence of glucose oxidase. In cells pretreated with LA, GSH content was 68.3 ± 11.9 nmol/mg protein (NS vs. control) in cells not subsequently exposed to glucose oxidase and 52.0 ± 14.5 nmol/mg protein in cells that were treated with glucose oxidase. These results suggest that LA treatment provides protection against the oxidative stressinduced decrease in GSH content.
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Protection against p38 MAPK activation. In L6 cells, it was reported recently that acute exposure to H2O2 activates p38 MAPK coincident with the inhibition of insulin action (25). The H2O2-induced effects could be effectively antagonized by two synthetic inhibitors of the p38 MAPK. To assess whether LA would also protect against H2O2-stimulated p38 MAPK activation, cells were incubated in the presence or absence of 300 µmol/l LA, followed by the addition of the H2O2-generating system. As reported by Blair et al. (25), H2O2 caused a marked activation of p38 MAPK, as judged by the increase in p38 MAPK phosphorylation (Fig. 8; compare lanes 1 and 2). This effect was substantially blocked when cells were preincubated with LA (Fig. 8; compare lanes 2 and 4).
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DISCUSSION |
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In L6 GLUT4 cells, we found that LA had only small effects on cells that were not subjected to oxidative stress. When we exposed cells to oxidative stress using an H2O2-generating system (glucose and glucose oxidase), we found that insulin stimulation of glucose transport was nearly abolished. Pretreatment with LA for 18 h prevented this loss of insulin action. The beneficial effect of LA was detected at a concentration of 30 µmol/l, and a maximal effect was observed at 1,000 µmol/l. In 3T3-L1 adipocytes, similar results were reported with respect to the ability of LA to protect against the H2O2-induced loss of insulin-stimulated glucose uptake (21). Because therapeutic concentrations of LA fall within this micromolar range (17,18,22), it is possible that the protective effect of LA on insulin action in vitro is linked to its therapeutic effect in vivo.
One group has reported a direct effect of LA on glucose transport in cultured L6 rat muscle cells and mouse 3T3-L1 adipocytes (24). This effect was blocked by inhibitors of phosphatidylinositol 3-kinase, but was additive to the effect of insulin, suggesting that it was using some but not all of the insulin-signaling system. However, in that study, the LA effect on glucose transport was observed at 10-fold higher concentrations of LA (i.e., millimolar or greater) than the effective concentrations reported here and in a previous study (21). Similarly, a direct stimulatory effect on glucose transport in response to a millimolar concentration of LA was recently reported in isolated cardiac myocytes (32). In contrast, in both the present study with L6 cells and the earlier study with 3T3-L1 adipocytes (21), only a small direct effect of LA on glucose transport could be detected, whereas major effects were observed in cells that had been subjected to oxidative stress. Thus the relationship and significance of the direct effect exerted by millimolar concentrations of LA on cellular glucose transport to LA's therapeutic effects in patients remains to be defined. Our results are consistent with the observations of Jacob et al. (9) that administration of LA in vivo improved insulin-stimulated glucose transport in skeletal muscle only in the insulin-resistant obese Zucker (fa/fa) rats (which are under increased oxidative stress at the tissue level) (33) and not in the insulin-sensitive lean Zucker rats.
Synthetic LA exists as an 50:50 mixture of two different isomers: the
naturally occurring R isomer and the synthetic S isomer. In one study
comparing the ability of the two isomers to directly stimulate glucose
transport in vitro, R-LA was reported to be more potent than S-LA
(24). In obese
insulin-resistant Zucker rats, parenteral administration of R-LA improved both
oxidative and nonoxidative glucose metabolism to a greater degree than did
S-LA (11). However, in other
studies, S-LA was reported to be equipotent or more potent than R-LA. For
example, in studies of 3T3-L1 adipocytes, S-LA was more potent than R-LA in
protecting against the inhibition of insulin action induced by oxidative
stress (21). In the present
study using muscle cells, we also compared the two isomers and found that both
the R and S isomers were biologically active; each was able to protect against
oxidative stress-induced insulin resistance. The potency of the two isomers
was similar and not statistically different (96 vs. 131 µmol/l for R-LA
EC50 and S-LA EC50, respectively)
(Fig. 5). Thus the biological
activities of the individual enantiomers merit further study.
The mechanisms by which H2O2 and other mediators of oxidative stress cause insulin resistance are unknown. Similarly, the mechanisms by which LA offers protection against the H2O2-induced attenuation of insulin action are also unknown. A possible explanation for the inhibitory effect of H2O2 on insulin action is that it triggers an alteration of the cellular redox balance because of prolonged exposure to reactive oxygen molecules. The inhibitory effects of H2O2 have been reported to target the proximal steps in the insulin-signaling cascade, including the suppression of insulin-stimulated insulin receptor and insulin receptor substrate-1 tyrosine phosphorylation (20,34). Stress inducers, including H2O2, activate a variety of serine/threonine kinase cascades (35,36,37). Increased phosphorylation of insulin receptor substrates on discrete serine or threonine sites decreases the extent of their tyrosine phosphorylation and is consistent with impaired insulin action (38,39,40,41,42,43,44). Therefore, it is possible that one component of the inhibitory effect of H2O2 on insulin action might be mediated via the activation of inhibitory serine/threonine kinase activity.
Evidence to support this possibility has been provided in a recent study by Blair et al. (25). In L6 cells, it was reported that acute exposure to H2O2 activates p38 MAPK coincident with the inhibition of insulin action (25). The H2O2-induced effects were antagonized by two synthetic inhibitors of the p38 MAPK. Results from the present study in L6 GLUT4 cells confirmed that oxidative stress via H2O2 generation acutely stimulates p38 MAPK activity. This report, therefore, is the first to identify LA as an additional pharmacological agent capable of blocking the activation of this inhibitory kinase coincident with its ability to provide protection against oxidative stress-induced insulin resistance. It cannot be determined from this study if LA exerts its inhibitory effect on p38 MAPK directly via inhibition of kinase activity or indirectly via modulation of the cellular redox state (see below). Nonetheless, taken together, these results provide additional support for a link between the p38 MAPKsignaling pathway and the insulin-signaling pathway to regulate glucose transport in skeletal muscle cells.
In muscle cells undergoing oxidative stress, the specific sites in the insulin-signaling pathway that are protected by LA are unknown. However, under equivalent experimental conditions, Tirosh et al. (20) reported that the protection afforded by LA against oxidative stressinduced insulin resistance in 3T3-L1 adipocytes involved the preservation of insulin-induced cellular redistribution of IRS-1 and PI 3-kinase. It is likely, therefore, that a similar mechanism occurs in L6 cells.
A potential explanation for the protective effects of LA on H2O2-induced insulin resistance may be related to its ability to preserve the intracellular redox balance, acting either directly or through other endogenous antioxidants, such as glutathione. The limiting factor in glutathione synthesis is the bioavailability of intracellular cysteine. LA and other antioxidants generate intracellular cysteine from extracellular cystine and thus maintain reduced glutathione levels (45). Rudich et al. (21) and other researchers (45) have reported that in 3T3-L1, human erythrocytes, human Jurkat cells, and others, LA pretreatment maintains the cellular reduced glutathione concentration in response to subsequent oxidative stress and have suggested that preservation of the normal reduced glutathione concentration is the major mode of action of LA. We found that pretreatment with LA protects against the fall in reduced glutathione caused by oxidative stress. Thus this protective effect of LA occurs in multiple cell types.
When we studied other antioxidants and related compounds, vitamin E also protected cells against oxidative stress, whereas vitamin C and troglitazone were ineffective. Whether vitamin E and LA possess similar modes of action is unknown. It is of interest that troglitazone, a clinically effective insulin sensitizer that contains the vitamin E moiety, was ineffective in protecting cells (46).
One possible explanation for the effects of oxidative stress on insulin signaling is via a lowering of ATP levels. For several reasons, we believe that changes in this parameter did not occur under our experimental conditions. First, several other groups studying H2O2-mediated oxidative stress have not observed significant falls in intracellular ATP levels (47,48). Second, if a decrease in ATP levels did occur, basal glucose transport would have increased because of the activation of the 5' AMP-kinase (49). This enzyme is activated by increases in the AMP:ATP and creatine:phosphocreatine ratios (50). Third, the study of Blair et al. (25) demonstrated that under conditions of oxidative stress, inhibition of p38 MAPK restored the insulin effect on glucose transport. This result would have been extremely unlikely if ATP was substantially decreased.
In summary, we found that in L6 muscle cells, micromolar concentrations of LA protect the insulin-signaling system from oxidative stress. These findings are in agreement with similar studies using 3T3-L1 adipocytes. Thus, in two of the major insulin-sensitive target tissues, LA action in vitro possesses potent protective effects that are in concert with its reported therapeutic effects in vivo. Taken together, these studies support the concept that the antioxidant actions of LA are an important feature of its clinical efficacy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2-DG, 2-deoxy-D-[H3]glucose; ANOVA, analysis of variance; BSA,
bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal
calf serum; GSH, glutathione; LA, -lipoic acid; LDH, lactate
dehydrogenase; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered
saline.
Received for publication May 30, 2000 and accepted in revised form October 4, 2000
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REFERENCES |
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