Metallothionein-Mediated Antioxidant Defense System and Its Response to Exercise Training Are Impaired in Human Type 2 Diabetes
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《糖尿病学杂志》
1 Copenhagen Muscle Research Center, The Panum Institute, Department of Medical Physiology, University of Copenhagen, Copenhagen, Denmark
2 Centre of Inflammation and Metabolism, Department of Medical Anatomy at the Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
3 Institute of Neurosciences and Department of Cellular Biology, Physiology and Immunology, Animal Physiology Unit, Faculty of Sciences, Autonomous University of Barcelona, Barcelona, Spain
4 Copenhagen Muscle Research Centre, Department of Molecular Muscle Biology, Rigshospitalet, Copenhagen, Denmark
5 Department of Exercise Science, Concordia University, Montreeal, Canada
GSH, glutathione; MDA, malondialdehyde; MT-I+II, metallothioneins I and II; NITT, nitrotyrosine; ROS, reactive oxygen species; SOD, superoxide dismutase
ABSTRACT
Oxidative stress is implicated in diabetes complications, during which endogenous antioxidant defenses have important pathophysiological consequences. To date, the significance of endogenous antioxidants such as metallothioneins I and II (MT-I+II) in type 2 diabetes remains unclear. To examine the MT-I+IIeCmediated antioxidant capacity and its response to exercise training in the skeletal muscle of patients with type 2 diabetes, biopsies and blood samples were taken from 13 matched subjects (type 2 diabetes n = 8, control subjects n = 5) both before and after 8 weeks of exercise training. Immunohistochemical analysis revealed reduced MT-I+II levels in the skeletal muscle of type 2 diabetic subjects compared with control subjects. Control subjects produced a robust increase of MT-I+II in response to training; however, in type 2 diabetes, MT-I+II levels remained essentially unchanged. Significantly lower levels of MT-I+II were also detected in the plasma of type 2 diabetic subjects compared with control subjects. These results suggest that, in control subjects, the MT-I+II defense system is active and inducible within skeletal muscle tissue and plasma. In type 2 diabetes, reduced levels of MT-I+II in muscle and plasma, as well as the deficient MT-I+II response to exercise, indicate that this antioxidant defense is impaired. This study presents a novel candidate in the pathogenesis of complications related to oxidative stress in type 2 diabetes.
Although the mechanisms involved in the pathogenesis of complications associated with type 2 diabetes have yet to be fully elucidated, there is growing evidence that oxidative stress contributes to the development and acceleration of related conditions such as nephropathy, neuropathy, retinopathy, and macro- and microvascular damage (1). In a healthy state, reactive oxygen species (ROS) formation is countered by the presence of an effective antioxidant defense system. When ROS formation overwhelms endogenous (and exogenous) antioxidant capacity, the cellular redox balance becomes altered and oxidative stress ensues. Components of this antioxidant defense system appear to be compromised in type 2 diabetes (2eC5), thus likely resulting in occurrence of associated complications.
Metallothioneins are cysteine-rich, low molecular weight intracellular proteins that were initially shown to regulate the metabolism of metals (such as zinc, copper, cadmium, and mercury) and to be efficient scavengers of ROS (6eC8). In mammals, four metallothionein isoforms (I-IV) have been identified, of which metallothionein I and II (MT-I+II) are expressed ubiquitously in most mammalian tissues and cell types (9eC11). Due to their structural similarities and analogous functions, the MT-I and MT-II isoforms are nearly indistinguishable and most often examined in unison. MT-I+II are induced by almost any inflammatory or pathological stimulus like proinflammatory cytokines (interleukin 3 and 6, tumor necrosis factor-, macrophage-specific colony-stimulating factor, and interferon and ), oxidative stress, glucocorticoids, and catecholamines (10,12eC14). During oxidative stress, MT-I+II inhibit ROS-induced nuclear toxicity and cytotoxicity more effectively than proteins 10eC50 times their molecular weight (15), and MT-I+II protect against ROS-induced DNA degradation with much higher molar efficiency than glutathione (16eC19). In addition to their antioxidant properties, MT-I+II also possess important anti-inflammatory and antiapoptotic functions, providing protection to cells and tissue during various pathological conditions (11,20,21). The metal thiolate clusters of metallothionein have also been implicated in nitric oxide signaling in the vascular wall (22).
In addition to the importance of MT-I+II as an antioxidant defense system in its own right, MT-I+II interacts with glutathione (GSH) as a coprotein in the preservations of intracellular zinc concentractions (23) and serves as a protective mechanism in maintenance of GSH levels (24). MT-I+II were also suggested to functionally substitute for Cu/Zn superoxide dismutase (SOD) deficiency in yeast (25) and antagonize the deleterious effects of oxidative stress on catalase (24).
animal studies, it has been shown that MT-I+II protect cardiomyocytes (26,27) and pancreatic -cells (28,29) against hyperglycemia-induced oxidative stress. Despite the known potency of MT-I+II as an antioxidant defense system, very little is known about the effects of type 2 diabetes on MT-I+II in skeletal muscle. Considering that oxidative stress has been associated with decreased insulin sensitivity by means of the activation of stress-sensitive pathways (3) and impaired insulin-induced GLUT4 translocation (30) and expression (31), the status of antioxidants in skeletal muscle is particularly relevant due to its importance in glucose homeostasis.
Although the findings are equivocal, a trend in the literature demonstrates that exercise training increases SOD and GSH but not catalase activity (rev. in 32,33). In comparison, very little is known about the response of MT-I+II to exercise-generated ROS production. Previously, it has been shown that acute exercise increases MT-I+II levels in the liver of the rat (34,35). Acute exercise has also been shown to induce MT-I+II levels in healthy human skeletal muscle (36,37). Studies investigating the effects of physical training have been very limited: chronic exercise has been associated with a decrease in hepatic and cardiac metallothionein, an increase in aortic metallothionein, and no effects of physical activity on quadriceps or kidney levels of metallothionein in rats (38). At present, no study has investigated the effects of exercise training on MT-I+II levels in human skeletal muscle and, in particular, not in human type 2 diabetes. This study examined whether the MT-I+IIeCmediated antioxidant defense system is altered in skeletal muscle of patients with type 2 diabetes and whether exercise training, known for both its therapeutic benefits and acute augmentation of oxidative stress, can induce MT-I+II in the skeletal muscle of these subjects.
RESEARCH DESIGN AND METHODS
Thirteen matched male subjects (type 2 diabetes n = 8, control subjects n = 5) were accepted for participation in this investigation after informed written consent as approved by the ethical committee of Copenhagen and Frederiksberg (01eC069/02) and in accordance with the Declaration of Helsinki. Physical characteristics of the subjects are presented in Table 1. It was established that none of the subjects regularly performed exercise before the commencement of the study. Normal glucose tolerance was confirmed in all healthy control subjects through a standard 75-g oral glucose tolerance test. None of the control subjects were taking any medications, while type 2 diabetes was treated with antidiabetes drugs (metformin [n = 7], glipizide [n = 2], glimepiride [n = 1], or tolbutamid [n = 1]), antihypertensives (amlodipin [n = 1], enalapril [n = 2], thiazid [n = 3], or angiotensin II receptor antagonist [n = 1]), or cholesterol-lowering drugs (simvastatin [n = 4]).
All participants undertook 8 weeks of exercise training (65% of maximal heart rate, 3eC4 sessions per week, 30 min per session) on a rowing ergometer. In order to minimize effects of acute exercise, subjects were instructed to not exercise on the day immediately before coming into the laboratory for testing. Muscle biopsies were obtained from the vastus lateralis (Bergstrm technique) under local anesthetic at baseline and after completion of the training period (same leg for both biopsies). Tissue was immediately set in TissueTek and flash frozen in liquid nitrogeneCcooled isopentane. Samples were stored at eC80°C. Blood was sampled from a brachial artery catheter.
Blood sample processing.
Blood samples were immediately mixed with 10% EDTA and centrifuged at 4,000 rpm for 10 min at 4°C. Plasma was then maintained at eC80°C until analysis.
Tissue processing.
Muscle tissue was cut in 6-e蘭 consecutive sections on a cryostat, and the sections were immediately collected on glass slides to be used for immunohistochemistry. For some immunohistochemical stainings, sections were preincubated in 20 mg/ml Proteinase K (Sigma-Aldrich, St. Louis, MO) for 5 min or sections were incubated overnight in tris-EGTA buffer (1.211 g Tris, 0.95 g EGTA, one l dest. H2O, pH 10) for epitope retrieval. Afterward, sections were incubated in H2O2 to block endogenous peroxidase, followed by incubation in 10% goat serum to block unspecific background staining.
For epitope retrieval, some sections were preincubated in 20 mg/ml Proteinase K (Sigma-Aldrich) for 5 min or sections were incubated overnight in tris-EGTA buffer (1.211 g Tris, 0.95 g EGTA, one l dest. H2O, pH 10) followed by blocking of endogenous peroxidase by incubation in H2O2, and, afterward, sections were incubated in 10% goat serum to block unspecific background staining.
Immunohistochemistry.
Sections were incubated overnight at 4°C with primary antibody: rabbit antieCMT-I+II diluted 1:500 (39), rabbit anti-nitrotyrosine (NITT) diluted 1:100 (marks peroxynitrite-induced nitration of tyrosine residues/oxidative stress), rabbit anti-malondialdehyde (MDA) diluted 1:100 (marks MDA, a byproduct of fatty acid peroxidation/oxidative stress), and mouse antieC8-oxoguanine 1:100 (Chemicon, Hampshire, U.K.) (marking a free radicaleCinduced base modification in the genome/oxidative stress).
The primary antibodies were detected using biotinylated mouse anti-rabbit IgG diluted 1:400 (Sigma-Aldrich), goat anti-mouse IgG diluted 1:200 (Sigma-Aldrich), or goat anti-mouse IgM diluted 1:40 (Jackson ImmunoResearch, West Grove, PA) for 30 min at room temperature followed by streptavidin-biotineCperoxidase complex (StreptABComplex/HRP; Dakocytomation, Glostrup, Denmark) prepared at manufacturer’s recommended dilutions for 30 min at room temperature. Afterward, sections were incubated with biotinylated tyramide and streptavidin-peroxidase complex (New England Nuclear Life Science Products) prepared following manufacturer recommendations. The immunoreaction was visualized using 0.015% H2O2 in 3,3-diaminobenzidine-tetrahydrochloride/tris-buffered solution for 10 min at room temperature.
In order to evaluate the extent of nonspecific binding in the immunohistochemical analysis, control sections were incubated in the absence of primary antibody or in the blocking serum. Results were considered only if controls were negative. For the simultaneous examination and recording of the stainings, a Zeiss Axioplan two-light microscope was used.
MT-I+II mRNA.
Total RNA was isolated from muscle biopsies by phenol extraction (TriReagent; Molecular Research Center, Cincinatti, OH) as previously described (40). Intact RNA was confirmed by denaturing agarose gel electrophoresis. Five hundred nanograms total RNA was converted into cDNA in 20 ml using the OmniScript reverse transcriptase (Qiagen, Valencia, CA) according to the manufacture’s protocol. For each target mRNA, 0.25 e蘬 cDNA was amplified in a 25 e蘬 SYBR Green PCR containing 1 x Quantitect SYBR Green Master Mix (Qiagen) and 100 nmol/l of each primer. The amplification was monitored real time using the MX3000P real-time PCR machine (Stratagene, La Jolla, CA). The primers for metallothionein were designed to target mRNA for all the different MT-I+II genes (TCC TGC TGC CCT GTG GGC TGT G and CAT CAGGCG CAG CAG CTG CAC TT). The Ct values were related to a standard curve made with pooled cDNA. The quantities were normalized to mRNA for the large ribosomal protein P0 (RPLP0) as internal control (41) using the primers GGA AAC TCT GCA TTC TCG CTT CCT and CCA GGA CTC GTT TGT ACC CGT TG.
Plasma MT-I+II.
Circulating MT-I+II levels were measured in arterial plasma as aputative index of the organism integrated response to training and/or diabetes. MT-I+II levels were measured by a radioimmunoassay (39). The antibody was raised against rat MT-II and showed full cross-reactivity with human MT-II (the latter provided by Dr. Milan Vasak). Undetermined factors present in human plasma eventually bind labeled metallothionein, and thus in the radioimmunoassay the unspecific binding (i.e., binding of 125I-metallothionein in the absence of primary antibody) was determined for each sample, which was then substracted from total binding (i.e., binding in the presence of primary antibody) to get the specific binding.
Statistics.
All data are presented as means ± SE. Two-way repeated- measures ANOVA testing was utilized to determine statistical significance in results. Differences between type 2 diabetic and control groups (Table 1) were established with Student’s t tests.
RESULTS
Immunohistochemistry
MT-I+II.
Immunohistochemical analysis revealed that MT-I+II expression is noticeably reduced in type 2 diabetic compared with control subjects at both baseline and, in particular, posttraining time points. Control subjects had a robust increase in MT-I+II expression levels in response to exercise training, where MT-I+II immunohistochemical staining was pronounced in cytoplasm, nuclei, and membranes of all muscle fibers. However, this was not the case with type 2 diabetes, where MT-I+II levels remained essentially unchanged after the 8-week training period (Fig. 1). Blinded, nonbiased evaluations of these stains were performed and are presented as semiquantitative data in Table 2.
Oxidative stress.
Simultaneously, tissue oxidative stress was assessed by immunoreactivity to MDA, 8-oxoguanine, and NITT. At baseline, type 2 diabetic patients had immunohistochemical stains corresponding to a higher level of tissue oxidative stress than control subjects. While both study groups displayed evidence of increased oxidative stress in response to exercise training, type 2 diabetes tissue appeared to be more susceptible to damage and showed higher levels of MDA, 8-oxoguanine, and NITT (Fig. 2). MDA and 8-oxoguanine immunohistochemical stainings reveal a similar pattern to NITT; however, these data are not shown.
mRNA.
Eight weeks of exercise training had no significant effect on MT-I+II mRNA in either control (pretraining 1.08 ± 0.06, posttraining 1.10 ± 0.05) or type 2 diabetic (pretraining 1.13 ± 0.10, posttraining 1.19 ± 0.17) experimental groups. Also, no differences were found in MT-I+II mRNA levels when comparing type 2 diabetic groups (1.13 ± 0.1) to control groups (1.08 ± 0.06) at pretraining values. All measurements are given in arbitrary units. Data shown in Fig. 3.
Plasma radioimmunoassay.
Significantly lower levels of MT-I+II (P = 0.037) were detected in the plasma (arterial) of type 2 diabetic (pretraining 16.13 ± 1.06 ng/ml, posttraining 16.05 ± 1.15 ng/ml) compared with control subjects (pretraining 22.45 ± 4.11 ng/ml, posttraining 23.29 ± 3.69 ng/ml). Contrary to the immunohistochemical results, 8 weeks of exercise training had no statistical effect on plasma concentration of MT-I+II (Fig. 4).
DISCUSSION
The major findings of this study are that 1) endogenous MT-I+II is apparent both within plasma and skeletal muscle of healthy middle-aged subjects and, in tissue, is inducible, upregulated by the increased levels of oxidative stress associated with 8 weeks of exercise training and 2) in type 2 diabetes, lower levels of MT-I+II in both plasma and skeletal muscle at both pre- and posttraining time points and the absence of an inducible response of tissue MT-I+II to exercise training indicate that this antioxidant defense system is impaired in human type 2 diabetes.
Our results show that 8 weeks of exercise training were able to induce MT-I+II in the skeletal muscle of healthy, middle-aged subjects but not in patients with type 2 diabetes. This impaired induction of MT-I+II in subjects with type 2 diabetes also corresponded with a higher intensity of tissue staining for oxidative stress markers (MDA, 8-oxoguanine, and NITT) compared with control subjects posttraining. From these concurrent immunohistochemical stains, it can be interpreted that MT-I+II is one of many candidate antioxidants that are dysregulated by chronic hyperglycemia and that skeletal muscle tissue in type 2 diabetes appears to be more susceptible to the oxidative stress associated with exercise training as a result of these impairments.
Past work investigating the effects of chronic exercise (swimming) on tissue metallothionein levels in both spontaneously hypertensive and control rats resulted in no change in the quadriceps levels of metallothioneinin in either study group (37). Contrary to this last report, the data presented in this study show that exercise training produced a robust increase in quadriceps (vastus lateralis) tissue MT-I+II in human control subjects as determined by immunohistochemistry. The difference in results may be species related (rats versus humans) or due to the difference in exercise mode (swimming versus rowing ergometry), the intensity of exercise, or the method of detection of MT-I+II (indirect quantification by cadmium binding versus immunohistochemistry).
Acute exercise, known to elicit increased ROS production, has been previously shown to induce MT-I+II, both on the protein and mRNA level, in young healthy subjects (36). Similar increases in skeletal muscle metallothionein mRNA were also reported in recent work that employed high-intensity endurance cycling in healthy young subjects (37). The goal of the present study was to examine the effects of both type 2 diabetes and chronic exercise training on MT-I+II in both skeletal muscle and plasma. For this reason, subjects were specifically instructed not to exercise on the day before the collection of samples, allowing for a period of at least 36eC48 h to avoid the effects of acute exercise. This time frame was chosen in light of previous work that demonstrated a peak of MT-I+II mRNA at 24 h postexercise (36). The study by Mahoney et al. (37), published after our experiments were conducted, shows that skeletal muscle metallothionein mRNA remains elevated 48 h after a single bout of exhaustive endurance exercise. In contrast to the former study, our results show no difference between pre- and posttraining in either control (1.02 ± 0.04) or type 2 diabetes (1.06 ± 0.13) biopsies, which were taken at least 36 h after the last bout of exercise of an 8-week training program. This most likely implies that, in our case, MT-I+II mRNA levels have returned to a baseline level for a given training status by 36eC48 h and the effects of acute exercise have been eliminated. The different time courses of metallothionein mRNA are possibly related to the intensity of the previous bout of exercise (degree of oxidative stress produced) or, in the case of exercise training, may be associated with the adaptation of the organism to the repetitive exposure to oxidative stress. The induction of metallothionein in response to varying degrees of exposure to oxidative stress has yet to be studied.
To date, very few studies have examined MT-I+II in skeletal muscle and, in particular, the response and effects in type 2 diabetes. Microarray data has shown that skeletal muscle MT-I+II mRNA is increased in streptozotocin-induced diabetes, but no assessment of tissue protein was performed (42). Interestingly, our results support the notion that chronic exposure to a hyperglycemic condition in a human model results in a decrease of metallothionein protein in both skeletal muscle tissue and plasma and suppresses the ability for exercise training to stimulate a MT-I+II response in skeletal muscle. Our mRNA data showed no differences in pretraining values between control and type 2 diabetic subjects. Whether this is a result of an impairment in the translation of mRNA into protein is not currently known. The contrasting results between the microarray data in the study conducted by Lecker et al. (42) and our own may shed insight into the variation of the MT-I+II response in type 1 versus type 2 diabetes or a time-course reaction to a hyperglycemic milleu (increase of metallothionein in short-term exposure versus decrease in long-term exposure).
Our data also indicate that, in both type 2 diabetic and healthy control subjects, exercise training did not have a significant effect on plasma MT-I+II. A similar lack of response has been found with acute exercise (M.P. et al., unpublished observations). It is not known whether the lack of responsiveness of plasma MT-I+II in both experiments is due to the time course of the antioxidant. For example, more stress than a single bout of exercise may be required to elevate plasma MT-I+II to detectable levels. It is also possible that, in this study, circulating MT-I+II peaks and drops off before the completion of 8 weeks of chronic physical training or that this system is primarily responsive within organ compartments other than blood with exercise training. The time course of MT-I+II in the plasma has yet to be elucidated.
Despite the apparent nonresponsiveness of plasma MT-I+II to exercise (both acute and chronic), we did find an effect of type 2 diabetes on plasma MT-I+II. Type 2 diabetic patients had a significantly lower (P = 0.037) level of MT-I+II in the plasma than control subjects at both pre- and postexercise time points. This observation implies that circulating MT-I+II is restricted in type 2 diabetes, possibly due to an impairment in either the formation of MT-I+II in the adjacent cells (for example: endothelial, erythrocyte, smooth muscle, and skeletal muscle) or release from these sites into the blood stream. Another hypothesis may be that MT-I+II is removed from the circulation due to the formation of disease-related antibodies, contributing to a dysfunction or depletion of plasma MT-I+II. This is consistent with previous work that shows the presence of MT-I+II antibodies in the serum of subjects with atopic dermatitis with concurrent metal allergy (43). The presence of similar antibodies has not yet been studied in patients with type 2 diabetes.
We were also interested in determining whether differences exist between plasma MT-I+II levels obtained from both femoral arterial and venous samples; however, no such differences were observed. Due to the lack of detectable trends between arterial and venous plasma, we report only arterial values in this article.
In summary, this work provides novel evidence that the MT-I+II antioxidant defense system is impaired in human type 2 diabetes. This finding is of importance as oxidative stress plays a major role in the development and exacerbation of diabetes-associated complications. In light of the associations between oxidative stress and type 2 diabetes, it remains imperative to further explore candidate antioxidants and the possible interactions that exist in maintaining an adequate defense system. For this reason, this study examines and demonstrates MT-I+II antioxidant defense dysfunction in the skeletal muscle of patients with type 2 diabetes. As there is very little information in the literature regarding metallothionein in skeletal muscle, the data presented in this article lays groundwork for further avenues of research. Further studies addressing MT-I+II in type 2 diabetes will provide valuable insight into potential pharmacological or therapeutic interventions and contribute to the understanding of the metallothionein antioxidant defense system.
ACKNOWLEDGMENTS
This work was funded by Novo Nordisk Fonds Komitee, Novo Nordisk Fonden, Diabetesforeningen, jenforeningen, Ministerio de Ciencia y Tecnologe猘, Feder SAF 2002-01268, Direccie?General de Recerca 2001SGR 00203, Fonds de Recherche en Santee du Queebec, and Concordia University.
The authors acknowledge the excellent technical assistance of Regitze Kraunse, Jeppe Bach, Thomas Beck (F.D.’s lab), Hanne Hadberg, Pernille Froh, Ha Nguyen (M.P.’s lab), and Dr. Mercedes Giralt (J.H.’s lab). Invaluable administrative assistance was provided by Vibeke Hvass.
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2 Centre of Inflammation and Metabolism, Department of Medical Anatomy at the Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
3 Institute of Neurosciences and Department of Cellular Biology, Physiology and Immunology, Animal Physiology Unit, Faculty of Sciences, Autonomous University of Barcelona, Barcelona, Spain
4 Copenhagen Muscle Research Centre, Department of Molecular Muscle Biology, Rigshospitalet, Copenhagen, Denmark
5 Department of Exercise Science, Concordia University, Montreeal, Canada
GSH, glutathione; MDA, malondialdehyde; MT-I+II, metallothioneins I and II; NITT, nitrotyrosine; ROS, reactive oxygen species; SOD, superoxide dismutase
ABSTRACT
Oxidative stress is implicated in diabetes complications, during which endogenous antioxidant defenses have important pathophysiological consequences. To date, the significance of endogenous antioxidants such as metallothioneins I and II (MT-I+II) in type 2 diabetes remains unclear. To examine the MT-I+IIeCmediated antioxidant capacity and its response to exercise training in the skeletal muscle of patients with type 2 diabetes, biopsies and blood samples were taken from 13 matched subjects (type 2 diabetes n = 8, control subjects n = 5) both before and after 8 weeks of exercise training. Immunohistochemical analysis revealed reduced MT-I+II levels in the skeletal muscle of type 2 diabetic subjects compared with control subjects. Control subjects produced a robust increase of MT-I+II in response to training; however, in type 2 diabetes, MT-I+II levels remained essentially unchanged. Significantly lower levels of MT-I+II were also detected in the plasma of type 2 diabetic subjects compared with control subjects. These results suggest that, in control subjects, the MT-I+II defense system is active and inducible within skeletal muscle tissue and plasma. In type 2 diabetes, reduced levels of MT-I+II in muscle and plasma, as well as the deficient MT-I+II response to exercise, indicate that this antioxidant defense is impaired. This study presents a novel candidate in the pathogenesis of complications related to oxidative stress in type 2 diabetes.
Although the mechanisms involved in the pathogenesis of complications associated with type 2 diabetes have yet to be fully elucidated, there is growing evidence that oxidative stress contributes to the development and acceleration of related conditions such as nephropathy, neuropathy, retinopathy, and macro- and microvascular damage (1). In a healthy state, reactive oxygen species (ROS) formation is countered by the presence of an effective antioxidant defense system. When ROS formation overwhelms endogenous (and exogenous) antioxidant capacity, the cellular redox balance becomes altered and oxidative stress ensues. Components of this antioxidant defense system appear to be compromised in type 2 diabetes (2eC5), thus likely resulting in occurrence of associated complications.
Metallothioneins are cysteine-rich, low molecular weight intracellular proteins that were initially shown to regulate the metabolism of metals (such as zinc, copper, cadmium, and mercury) and to be efficient scavengers of ROS (6eC8). In mammals, four metallothionein isoforms (I-IV) have been identified, of which metallothionein I and II (MT-I+II) are expressed ubiquitously in most mammalian tissues and cell types (9eC11). Due to their structural similarities and analogous functions, the MT-I and MT-II isoforms are nearly indistinguishable and most often examined in unison. MT-I+II are induced by almost any inflammatory or pathological stimulus like proinflammatory cytokines (interleukin 3 and 6, tumor necrosis factor-, macrophage-specific colony-stimulating factor, and interferon and ), oxidative stress, glucocorticoids, and catecholamines (10,12eC14). During oxidative stress, MT-I+II inhibit ROS-induced nuclear toxicity and cytotoxicity more effectively than proteins 10eC50 times their molecular weight (15), and MT-I+II protect against ROS-induced DNA degradation with much higher molar efficiency than glutathione (16eC19). In addition to their antioxidant properties, MT-I+II also possess important anti-inflammatory and antiapoptotic functions, providing protection to cells and tissue during various pathological conditions (11,20,21). The metal thiolate clusters of metallothionein have also been implicated in nitric oxide signaling in the vascular wall (22).
In addition to the importance of MT-I+II as an antioxidant defense system in its own right, MT-I+II interacts with glutathione (GSH) as a coprotein in the preservations of intracellular zinc concentractions (23) and serves as a protective mechanism in maintenance of GSH levels (24). MT-I+II were also suggested to functionally substitute for Cu/Zn superoxide dismutase (SOD) deficiency in yeast (25) and antagonize the deleterious effects of oxidative stress on catalase (24).
animal studies, it has been shown that MT-I+II protect cardiomyocytes (26,27) and pancreatic -cells (28,29) against hyperglycemia-induced oxidative stress. Despite the known potency of MT-I+II as an antioxidant defense system, very little is known about the effects of type 2 diabetes on MT-I+II in skeletal muscle. Considering that oxidative stress has been associated with decreased insulin sensitivity by means of the activation of stress-sensitive pathways (3) and impaired insulin-induced GLUT4 translocation (30) and expression (31), the status of antioxidants in skeletal muscle is particularly relevant due to its importance in glucose homeostasis.
Although the findings are equivocal, a trend in the literature demonstrates that exercise training increases SOD and GSH but not catalase activity (rev. in 32,33). In comparison, very little is known about the response of MT-I+II to exercise-generated ROS production. Previously, it has been shown that acute exercise increases MT-I+II levels in the liver of the rat (34,35). Acute exercise has also been shown to induce MT-I+II levels in healthy human skeletal muscle (36,37). Studies investigating the effects of physical training have been very limited: chronic exercise has been associated with a decrease in hepatic and cardiac metallothionein, an increase in aortic metallothionein, and no effects of physical activity on quadriceps or kidney levels of metallothionein in rats (38). At present, no study has investigated the effects of exercise training on MT-I+II levels in human skeletal muscle and, in particular, not in human type 2 diabetes. This study examined whether the MT-I+IIeCmediated antioxidant defense system is altered in skeletal muscle of patients with type 2 diabetes and whether exercise training, known for both its therapeutic benefits and acute augmentation of oxidative stress, can induce MT-I+II in the skeletal muscle of these subjects.
RESEARCH DESIGN AND METHODS
Thirteen matched male subjects (type 2 diabetes n = 8, control subjects n = 5) were accepted for participation in this investigation after informed written consent as approved by the ethical committee of Copenhagen and Frederiksberg (01eC069/02) and in accordance with the Declaration of Helsinki. Physical characteristics of the subjects are presented in Table 1. It was established that none of the subjects regularly performed exercise before the commencement of the study. Normal glucose tolerance was confirmed in all healthy control subjects through a standard 75-g oral glucose tolerance test. None of the control subjects were taking any medications, while type 2 diabetes was treated with antidiabetes drugs (metformin [n = 7], glipizide [n = 2], glimepiride [n = 1], or tolbutamid [n = 1]), antihypertensives (amlodipin [n = 1], enalapril [n = 2], thiazid [n = 3], or angiotensin II receptor antagonist [n = 1]), or cholesterol-lowering drugs (simvastatin [n = 4]).
All participants undertook 8 weeks of exercise training (65% of maximal heart rate, 3eC4 sessions per week, 30 min per session) on a rowing ergometer. In order to minimize effects of acute exercise, subjects were instructed to not exercise on the day immediately before coming into the laboratory for testing. Muscle biopsies were obtained from the vastus lateralis (Bergstrm technique) under local anesthetic at baseline and after completion of the training period (same leg for both biopsies). Tissue was immediately set in TissueTek and flash frozen in liquid nitrogeneCcooled isopentane. Samples were stored at eC80°C. Blood was sampled from a brachial artery catheter.
Blood sample processing.
Blood samples were immediately mixed with 10% EDTA and centrifuged at 4,000 rpm for 10 min at 4°C. Plasma was then maintained at eC80°C until analysis.
Tissue processing.
Muscle tissue was cut in 6-e蘭 consecutive sections on a cryostat, and the sections were immediately collected on glass slides to be used for immunohistochemistry. For some immunohistochemical stainings, sections were preincubated in 20 mg/ml Proteinase K (Sigma-Aldrich, St. Louis, MO) for 5 min or sections were incubated overnight in tris-EGTA buffer (1.211 g Tris, 0.95 g EGTA, one l dest. H2O, pH 10) for epitope retrieval. Afterward, sections were incubated in H2O2 to block endogenous peroxidase, followed by incubation in 10% goat serum to block unspecific background staining.
For epitope retrieval, some sections were preincubated in 20 mg/ml Proteinase K (Sigma-Aldrich) for 5 min or sections were incubated overnight in tris-EGTA buffer (1.211 g Tris, 0.95 g EGTA, one l dest. H2O, pH 10) followed by blocking of endogenous peroxidase by incubation in H2O2, and, afterward, sections were incubated in 10% goat serum to block unspecific background staining.
Immunohistochemistry.
Sections were incubated overnight at 4°C with primary antibody: rabbit antieCMT-I+II diluted 1:500 (39), rabbit anti-nitrotyrosine (NITT) diluted 1:100 (marks peroxynitrite-induced nitration of tyrosine residues/oxidative stress), rabbit anti-malondialdehyde (MDA) diluted 1:100 (marks MDA, a byproduct of fatty acid peroxidation/oxidative stress), and mouse antieC8-oxoguanine 1:100 (Chemicon, Hampshire, U.K.) (marking a free radicaleCinduced base modification in the genome/oxidative stress).
The primary antibodies were detected using biotinylated mouse anti-rabbit IgG diluted 1:400 (Sigma-Aldrich), goat anti-mouse IgG diluted 1:200 (Sigma-Aldrich), or goat anti-mouse IgM diluted 1:40 (Jackson ImmunoResearch, West Grove, PA) for 30 min at room temperature followed by streptavidin-biotineCperoxidase complex (StreptABComplex/HRP; Dakocytomation, Glostrup, Denmark) prepared at manufacturer’s recommended dilutions for 30 min at room temperature. Afterward, sections were incubated with biotinylated tyramide and streptavidin-peroxidase complex (New England Nuclear Life Science Products) prepared following manufacturer recommendations. The immunoreaction was visualized using 0.015% H2O2 in 3,3-diaminobenzidine-tetrahydrochloride/tris-buffered solution for 10 min at room temperature.
In order to evaluate the extent of nonspecific binding in the immunohistochemical analysis, control sections were incubated in the absence of primary antibody or in the blocking serum. Results were considered only if controls were negative. For the simultaneous examination and recording of the stainings, a Zeiss Axioplan two-light microscope was used.
MT-I+II mRNA.
Total RNA was isolated from muscle biopsies by phenol extraction (TriReagent; Molecular Research Center, Cincinatti, OH) as previously described (40). Intact RNA was confirmed by denaturing agarose gel electrophoresis. Five hundred nanograms total RNA was converted into cDNA in 20 ml using the OmniScript reverse transcriptase (Qiagen, Valencia, CA) according to the manufacture’s protocol. For each target mRNA, 0.25 e蘬 cDNA was amplified in a 25 e蘬 SYBR Green PCR containing 1 x Quantitect SYBR Green Master Mix (Qiagen) and 100 nmol/l of each primer. The amplification was monitored real time using the MX3000P real-time PCR machine (Stratagene, La Jolla, CA). The primers for metallothionein were designed to target mRNA for all the different MT-I+II genes (TCC TGC TGC CCT GTG GGC TGT G and CAT CAGGCG CAG CAG CTG CAC TT). The Ct values were related to a standard curve made with pooled cDNA. The quantities were normalized to mRNA for the large ribosomal protein P0 (RPLP0) as internal control (41) using the primers GGA AAC TCT GCA TTC TCG CTT CCT and CCA GGA CTC GTT TGT ACC CGT TG.
Plasma MT-I+II.
Circulating MT-I+II levels were measured in arterial plasma as aputative index of the organism integrated response to training and/or diabetes. MT-I+II levels were measured by a radioimmunoassay (39). The antibody was raised against rat MT-II and showed full cross-reactivity with human MT-II (the latter provided by Dr. Milan Vasak). Undetermined factors present in human plasma eventually bind labeled metallothionein, and thus in the radioimmunoassay the unspecific binding (i.e., binding of 125I-metallothionein in the absence of primary antibody) was determined for each sample, which was then substracted from total binding (i.e., binding in the presence of primary antibody) to get the specific binding.
Statistics.
All data are presented as means ± SE. Two-way repeated- measures ANOVA testing was utilized to determine statistical significance in results. Differences between type 2 diabetic and control groups (Table 1) were established with Student’s t tests.
RESULTS
Immunohistochemistry
MT-I+II.
Immunohistochemical analysis revealed that MT-I+II expression is noticeably reduced in type 2 diabetic compared with control subjects at both baseline and, in particular, posttraining time points. Control subjects had a robust increase in MT-I+II expression levels in response to exercise training, where MT-I+II immunohistochemical staining was pronounced in cytoplasm, nuclei, and membranes of all muscle fibers. However, this was not the case with type 2 diabetes, where MT-I+II levels remained essentially unchanged after the 8-week training period (Fig. 1). Blinded, nonbiased evaluations of these stains were performed and are presented as semiquantitative data in Table 2.
Oxidative stress.
Simultaneously, tissue oxidative stress was assessed by immunoreactivity to MDA, 8-oxoguanine, and NITT. At baseline, type 2 diabetic patients had immunohistochemical stains corresponding to a higher level of tissue oxidative stress than control subjects. While both study groups displayed evidence of increased oxidative stress in response to exercise training, type 2 diabetes tissue appeared to be more susceptible to damage and showed higher levels of MDA, 8-oxoguanine, and NITT (Fig. 2). MDA and 8-oxoguanine immunohistochemical stainings reveal a similar pattern to NITT; however, these data are not shown.
mRNA.
Eight weeks of exercise training had no significant effect on MT-I+II mRNA in either control (pretraining 1.08 ± 0.06, posttraining 1.10 ± 0.05) or type 2 diabetic (pretraining 1.13 ± 0.10, posttraining 1.19 ± 0.17) experimental groups. Also, no differences were found in MT-I+II mRNA levels when comparing type 2 diabetic groups (1.13 ± 0.1) to control groups (1.08 ± 0.06) at pretraining values. All measurements are given in arbitrary units. Data shown in Fig. 3.
Plasma radioimmunoassay.
Significantly lower levels of MT-I+II (P = 0.037) were detected in the plasma (arterial) of type 2 diabetic (pretraining 16.13 ± 1.06 ng/ml, posttraining 16.05 ± 1.15 ng/ml) compared with control subjects (pretraining 22.45 ± 4.11 ng/ml, posttraining 23.29 ± 3.69 ng/ml). Contrary to the immunohistochemical results, 8 weeks of exercise training had no statistical effect on plasma concentration of MT-I+II (Fig. 4).
DISCUSSION
The major findings of this study are that 1) endogenous MT-I+II is apparent both within plasma and skeletal muscle of healthy middle-aged subjects and, in tissue, is inducible, upregulated by the increased levels of oxidative stress associated with 8 weeks of exercise training and 2) in type 2 diabetes, lower levels of MT-I+II in both plasma and skeletal muscle at both pre- and posttraining time points and the absence of an inducible response of tissue MT-I+II to exercise training indicate that this antioxidant defense system is impaired in human type 2 diabetes.
Our results show that 8 weeks of exercise training were able to induce MT-I+II in the skeletal muscle of healthy, middle-aged subjects but not in patients with type 2 diabetes. This impaired induction of MT-I+II in subjects with type 2 diabetes also corresponded with a higher intensity of tissue staining for oxidative stress markers (MDA, 8-oxoguanine, and NITT) compared with control subjects posttraining. From these concurrent immunohistochemical stains, it can be interpreted that MT-I+II is one of many candidate antioxidants that are dysregulated by chronic hyperglycemia and that skeletal muscle tissue in type 2 diabetes appears to be more susceptible to the oxidative stress associated with exercise training as a result of these impairments.
Past work investigating the effects of chronic exercise (swimming) on tissue metallothionein levels in both spontaneously hypertensive and control rats resulted in no change in the quadriceps levels of metallothioneinin in either study group (37). Contrary to this last report, the data presented in this study show that exercise training produced a robust increase in quadriceps (vastus lateralis) tissue MT-I+II in human control subjects as determined by immunohistochemistry. The difference in results may be species related (rats versus humans) or due to the difference in exercise mode (swimming versus rowing ergometry), the intensity of exercise, or the method of detection of MT-I+II (indirect quantification by cadmium binding versus immunohistochemistry).
Acute exercise, known to elicit increased ROS production, has been previously shown to induce MT-I+II, both on the protein and mRNA level, in young healthy subjects (36). Similar increases in skeletal muscle metallothionein mRNA were also reported in recent work that employed high-intensity endurance cycling in healthy young subjects (37). The goal of the present study was to examine the effects of both type 2 diabetes and chronic exercise training on MT-I+II in both skeletal muscle and plasma. For this reason, subjects were specifically instructed not to exercise on the day before the collection of samples, allowing for a period of at least 36eC48 h to avoid the effects of acute exercise. This time frame was chosen in light of previous work that demonstrated a peak of MT-I+II mRNA at 24 h postexercise (36). The study by Mahoney et al. (37), published after our experiments were conducted, shows that skeletal muscle metallothionein mRNA remains elevated 48 h after a single bout of exhaustive endurance exercise. In contrast to the former study, our results show no difference between pre- and posttraining in either control (1.02 ± 0.04) or type 2 diabetes (1.06 ± 0.13) biopsies, which were taken at least 36 h after the last bout of exercise of an 8-week training program. This most likely implies that, in our case, MT-I+II mRNA levels have returned to a baseline level for a given training status by 36eC48 h and the effects of acute exercise have been eliminated. The different time courses of metallothionein mRNA are possibly related to the intensity of the previous bout of exercise (degree of oxidative stress produced) or, in the case of exercise training, may be associated with the adaptation of the organism to the repetitive exposure to oxidative stress. The induction of metallothionein in response to varying degrees of exposure to oxidative stress has yet to be studied.
To date, very few studies have examined MT-I+II in skeletal muscle and, in particular, the response and effects in type 2 diabetes. Microarray data has shown that skeletal muscle MT-I+II mRNA is increased in streptozotocin-induced diabetes, but no assessment of tissue protein was performed (42). Interestingly, our results support the notion that chronic exposure to a hyperglycemic condition in a human model results in a decrease of metallothionein protein in both skeletal muscle tissue and plasma and suppresses the ability for exercise training to stimulate a MT-I+II response in skeletal muscle. Our mRNA data showed no differences in pretraining values between control and type 2 diabetic subjects. Whether this is a result of an impairment in the translation of mRNA into protein is not currently known. The contrasting results between the microarray data in the study conducted by Lecker et al. (42) and our own may shed insight into the variation of the MT-I+II response in type 1 versus type 2 diabetes or a time-course reaction to a hyperglycemic milleu (increase of metallothionein in short-term exposure versus decrease in long-term exposure).
Our data also indicate that, in both type 2 diabetic and healthy control subjects, exercise training did not have a significant effect on plasma MT-I+II. A similar lack of response has been found with acute exercise (M.P. et al., unpublished observations). It is not known whether the lack of responsiveness of plasma MT-I+II in both experiments is due to the time course of the antioxidant. For example, more stress than a single bout of exercise may be required to elevate plasma MT-I+II to detectable levels. It is also possible that, in this study, circulating MT-I+II peaks and drops off before the completion of 8 weeks of chronic physical training or that this system is primarily responsive within organ compartments other than blood with exercise training. The time course of MT-I+II in the plasma has yet to be elucidated.
Despite the apparent nonresponsiveness of plasma MT-I+II to exercise (both acute and chronic), we did find an effect of type 2 diabetes on plasma MT-I+II. Type 2 diabetic patients had a significantly lower (P = 0.037) level of MT-I+II in the plasma than control subjects at both pre- and postexercise time points. This observation implies that circulating MT-I+II is restricted in type 2 diabetes, possibly due to an impairment in either the formation of MT-I+II in the adjacent cells (for example: endothelial, erythrocyte, smooth muscle, and skeletal muscle) or release from these sites into the blood stream. Another hypothesis may be that MT-I+II is removed from the circulation due to the formation of disease-related antibodies, contributing to a dysfunction or depletion of plasma MT-I+II. This is consistent with previous work that shows the presence of MT-I+II antibodies in the serum of subjects with atopic dermatitis with concurrent metal allergy (43). The presence of similar antibodies has not yet been studied in patients with type 2 diabetes.
We were also interested in determining whether differences exist between plasma MT-I+II levels obtained from both femoral arterial and venous samples; however, no such differences were observed. Due to the lack of detectable trends between arterial and venous plasma, we report only arterial values in this article.
In summary, this work provides novel evidence that the MT-I+II antioxidant defense system is impaired in human type 2 diabetes. This finding is of importance as oxidative stress plays a major role in the development and exacerbation of diabetes-associated complications. In light of the associations between oxidative stress and type 2 diabetes, it remains imperative to further explore candidate antioxidants and the possible interactions that exist in maintaining an adequate defense system. For this reason, this study examines and demonstrates MT-I+II antioxidant defense dysfunction in the skeletal muscle of patients with type 2 diabetes. As there is very little information in the literature regarding metallothionein in skeletal muscle, the data presented in this article lays groundwork for further avenues of research. Further studies addressing MT-I+II in type 2 diabetes will provide valuable insight into potential pharmacological or therapeutic interventions and contribute to the understanding of the metallothionein antioxidant defense system.
ACKNOWLEDGMENTS
This work was funded by Novo Nordisk Fonds Komitee, Novo Nordisk Fonden, Diabetesforeningen, jenforeningen, Ministerio de Ciencia y Tecnologe猘, Feder SAF 2002-01268, Direccie?General de Recerca 2001SGR 00203, Fonds de Recherche en Santee du Queebec, and Concordia University.
The authors acknowledge the excellent technical assistance of Regitze Kraunse, Jeppe Bach, Thomas Beck (F.D.’s lab), Hanne Hadberg, Pernille Froh, Ha Nguyen (M.P.’s lab), and Dr. Mercedes Giralt (J.H.’s lab). Invaluable administrative assistance was provided by Vibeke Hvass.
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