1 Exercise Metabolism Group, School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia
2 Skeletal Muscle Research Laboratory, School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia
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ABSTRACT |
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The pathogenesis of type 2 diabetes has not been fully elucidated; however, there is growing evidence linking this disease with oxidative stress (13). Oxidative stress, resulting from increased production or decreased removal of reactive oxygen species (ROS), plays a key role in the pathogenesis of late diabetes complications (2) and insulin-stimulated glucose uptake (4). To combat protein-related homeostatic disruption, such as oxidative stress, cells respond by synthesizing a family of highly conserved proteins termed heat shock proteins (HSPs). In skeletal muscle, the most widely studied family is the 70-kDa family, which contains the constitutive HSP73 and inducible HSP72 forms. HSP72 has been found to protect skeletal muscle against contraction-induced ROS formation (5). However, the 30-kDa family, also known as the heme oxygenases (HOs; HO-1 being the inducible form and HO-2 the constitutive form) is also expressed in skeletal muscle and plays an important role in the cellular defense against oxidative stress and the negative effects of proinflammatory cytokines (6). Recently, Kurucz et al. (7) observed a decreased expression of HSP72 mRNA in patients with type 2 diabetes, with this reduction being correlated with some markers of insulin resistance. This study (7) provided preliminary evidence that HSP72 is related to insulin resistance, but their results with respect to insulin sensitivity during a hyperinsulinemic clamp were not conclusive. Nonetheless, the authors hypothesized mechanisms by which stress proteins may be implicated in insulin resistance, and they suggested that further research in this area is warranted. Furthermore, in a preliminary study that compared >5,000 genes in skeletal muscle samples from patients with insulin resistance and healthy subjects, HSP72 was 1 of only 17 genes that were markedly lower in the patient population (8).
Little is known regarding the functional significance of HO-1 in skeletal muscle, although HO-1 provides protection against proinflammatory cytokines, which are known to be elevated in the skeletal muscle of patients with type 2 diabetes (9).
It is also not known whether insulin stimulates the expression of these genes and/or the potential mechanisms by which genes associated with cellular defense against oxidative stress may be affecting insulin sensitivity. It has been hypothesized that both GLUT4 and the peroxisome proliferatoractivated receptors (PPARs) interact with HSP72, and that this interaction may be responsible, in part, for the role of HSP72 expression in insulin resistance (7). The aim of the present study was, therefore, to investigate the role of selected genes associated with cellular defense against oxidative stress in the etiology of insulin resistance in human skeletal muscle. We performed a euglycemic-hyperinsulinemic clamp in patients with type 2 diabetes and control subjects, and we related this measure to HSP72 and HO-1 mRNA in skeletal muscle samples obtained before and after the clamp. In addition, we measured the mRNA content of GLUT4, PPAR-, and PPAR-
, as well as the maximal activity levels of the oxidative enzymes ß-hyroxyacyl-CoA dehydrogenase (ß-HAD) and citrate synthase (CS). We hypothesized that both HSP72 and HO-1 mRNA would be lower in patients with type 2 diabetes compared with healthy control subjects, and that their expression would correlate with an impaired glucose infusion rate during the clamp. We further hypothesized that the expression of these genes would be related to intramuscular oxidative capacity, but that GLUT4, PPAR-
, and PPAR-
gene expression would be unimpaired in the skeletal muscle of patients with type 2 diabetes.
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RESEARCH DESIGN AND METHODS |
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Blood biochemistry.
Blood glucose concentration was measured using an automated analyzer (2300 Stat Plus Glucose and L-Lactate analyzer; Yellow Springs Instruments, Yellow Springs, OH). Plasma insulin concentration was determined by radioimmunoassay (Phadeseph, Insulin RIA; Pharmacia & Upjohn Diagnostics, Uppsala, Sweden), HbA1c was determined by specific ion-exchange chromatography (Sigma Diagnostics, Castle Hill, New South Wales, Australia), and plasma free fatty acid (FFA) concentration was measured using an enzymatic colorimetric method (NEFA C test kit; Wako, Richmond, VA). To provide an indication of ROS formation, plasma collected from diabetic and age-matched control subjects were analyzed for the enzyme myeloperoxidase (MPO), which catalyzes the formation of hypochlorous acid, a powerful oxidant derived from chloride ions and hydrogen peroxide (10). This analysis was performed using an enzyme-linked immunoassay (Calbiochem, San Diego, CA). Because of technical difficulties, we were unable to perform this analysis on plasma from young control subjects.
Measurement of gene expression.
A portion of muscle (30 mg) was extracted for total RNA using a modification of an acid guanidium thiocyanate-phenol chloroform extraction method described elsewhere (11). To visualize the integrity of the total RNA, 0.5 µg was fractionated on a 1% denaturing agarose gel. The total RNA was subsequently quantified two to three more times before 1 ng of each total RNA sample was reverse-transcribed in a 10-µl reaction containing 1 x TaqMan reverse transcriptase buffer, 5.5 mmol/l MgCl2, 500 mmol/l each 2'-deoxynucleoside and 5'-triphosphate, 2.5 mmol/l random hexamers, 0.4 units/ml RNase inhibitor, 1.25 units/ml Multiscribe reverse transcriptase (Applied Biosystems, Foster City, CA) and made up to volume with H2O (0.05% diethylpyrocarbonate treated). Control samples were also analyzed, where all of the above reagents are added to RNA samples except the Multiscribe reverse transcriptase. The reverse transcription reactions were performed using a GeneAmp PCR system 2400 (PerkinElmer, Wellesley, MA) with conditions at 25°C for 10 min, 48°C for 30 min, and 95°C for 5 min. Then, 2 ml 0.5 mol/l EDTA (pH 8.0) was added to each sample, and they were stored at -20°C until further analysis. Real-time PCR was used to quantitate human HSP72, HO-1, PPAR-
, PPAR-
, and GLUT4 expression from the cDNA samples. Human probe and primers were designed (Primer Express version 1.0; Applied Biosystems) from the human gene sequence accessed from Gen-Bank/EMBL. To limit the possibility of nonspecific amplification of each gene, we designed the nucleic acid sequences to span an exon-exon junction when possible in order to limit the possibility of genomic DNA contamination. The probe and primer sequences are presented in Table 2.
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PCRs were carried out in 25-µl reactions of TaqMan universal PCR master mix (1 x), 50 nmol/l TaqMan 18S probe, 20 nmol/l 18S forward primer, 80 nmol/l 18S reverse primer, and probes and primers at specific concentrations ranging from 50 to 150 nmol/l (probes) and 50 to 900 nmol/l (primers) for each gene of interest. The specific concentrations for each gene were optimized in preliminary experiments. Based on preliminary experiments, 5 ng of cDNA was used for HSP72, PPAR-, PPAR-
, and GLUT4, and 20 ng of cDNA was used for HO-1. This resulted in similar CT thresholds for all genes (see RESULTS section). cDNA and control preparations not containing reverse transcriptase were amplified using the following conditions: 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min for HSP72, GLUT4, and PPAR-
. Conditions for HO-1 and PPAR-
were identical but were run for 50 cycles because of the low basal expression of these genes. This was determined from preliminary experiments. Furthermore, in preliminary experiments, polymerized products were run on a gel to visualize a single band. Of note, in previous experiments where we have followed these procedures, we have compared our gene expression data obtained from real-time PCR methodology with that obtained using a Northern blot, and we have demonstrated comparable results (12).
For each sample, a CT value was obtained by subtracting 18S CT values from the CT of the gene of interest. Therefore, a higher
CT value indicates a lower relative expression. To determine the basal mRNA expression when comparing groups, we used the young control subjects preclamp value as the control. Hence, a
CT value was obtained for each of the young control subjects and averaged. This value was subtracted from the
CT value for each subject preclamp to derive a
-
CT value. The expression of each gene was then evaluated by 2-
-
CT. Because the
CT value for each subject in young control subjects was 0, the 2-
-
CT for the young control subjects group preclamp became 1. To determine the effect of insulin on the mRNA expression in each group, a
CT value was obtained by subtracting 18S CT values from each gene of interest, using the preclamp value as the control. Preclamp values for each subject were subtracted from the postclamp samples for each subject to derive a
-
CT value. The expression was then evaluated by 2-
-
CT, with all preclamp values for each subject being 1.
Intramuscular triglycerides, CS, and ß-HAD activity.
Muscle (30 mg) was freeze-dried under vacuum for 24 h. The sample (
10 mg) was viewed under a microscope (6.3x) at room temperature for dissection and removal of all traces of adipose tissue, connective tissue, and blood contaminants. This procedure yielded
78 mg of dry-weight dissected muscle, from which a direct measure of intramuscular triglyceride (IMTG) content was determined, as previously described (13). The coefficient of variation for this measurement was
8%. Muscle (510 mg) was homogenized in 1:50 dilution (wt/vol) of a 175-mmol/l potassium buffer solution, and CS activity was assayed spectrophotometrically at 25°C. ß-HAD activity was assayed spectrophotometrically at 25°C, measuring the disappearance of NADH using the same homogenate as for CS (14).
Statistics.
Data are presented as the means ± SE. Differences between experimental groups were determined using a one-way ANOVA for all measures except the effect of insulin on mRNA expression. For this measure, a two-way (group x clamp) ANOVA was used. Significant differences were located using a Newman-Kuels post hoc test. To examine the relationship between gene expression and markers of insulin sensitivity, data from all groups were combined and examined with Pearson product correlations. Statistical significance was accepted at P < 0.05.
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RESULTS |
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Basal mRNA expression.
Both HSP72 and HO-1 were reduced (P < 0.05) in the diabetic group by 33 and 55%, respectively, compared with young control subjects. No differences were observed in basal gene expression when comparing age-matched control subjects with young control subjects for these genes (Fig. 1). No differences were observed in basal mRNA expression of GLUT4 (1.32 ± 0.21- and 0.69 ± 0.13-fold change for age-matched control subjects and the diabetic group relative to young control subjects = 1), PPAR- (1.66 ± 0.57- and 1.10 ± 0.59-fold change for age-matched control subjects and the diabetic group relative to young control subjects = 1), and PPAR-
(0.90 ± 0.38- and 1.07 ± 0.19-fold change for age-matched control subjects and the diabetic group relative to young control subjects = 1). The average CT value (means ± SD) for the five genes for young control subjects ranged from 27.1 ± 0.8 (HSP72) to 31.2 ± 2.1 (PPAR-
).
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DISCUSSION |
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HSP72 and insulin resistance.
Our observation of a reduction in the basal expression of HSP72 mRNA in the skeletal muscle of patients with type 2 diabetes supports previous investigations (7,8). Our data are novel with respect to the expression and induction of HSP72 in these patients and the possible mechanisms by which HSP72 could be exerting its effect. First, in a previous study (7), the relationship between HSP72 mRNA expression and insulin-stimulated glucose uptake during a hyperinsulinemic-euglycemic clamp in patients with type 2 diabetes and age-matched control subjects tended to be different but was not significant (r = 0.525, P = 0.081). The authors were therefore unable to conclude with conviction that this relationship was marked. Our data extend their findings by clearly demonstrating that the relationship is indeed marked. Even in young and age-matched control subjects who were not classed as insulin resistant, the relationship was nonetheless apparent (Fig. 4). This may have been related to the higher BMI of age-matched compared with young control subjects (Table 2). We further demonstrated a significant correlation between the expression of this gene and muscle oxidative capacity, as well as a moderate relationship between IMTG accumulation and HSP72 mRNA (Fig. 4).
In a previous study (7), the authors speculated on the mechanism by which HSP72 expression may be affecting insulin sensitivity. They suggested that GLUT4 may interact with HSP72 because both are known to interact with F-actin. In addition, these authors suggested that the PPARs may also play a role because HSP72 and the PPARs form a complex in vivo in the rat. In the present study, we found that the expression of these genes was not defective in the diabetic group relative to young and age-matched control subjects. In addition, we found no reduction in GLUT4 total protein (data not shown). Because we did not measure the trafficking of GLUT4 to the plasma membrane during the clamp or the total protein or ligand binding efficiency of the PPARs, our data cannot categorically rule out the possibility that these proteins may interact with HSP72. However, the results argue against such an hypothesis.
The fact that HSP72 mRNA was markedly associated with muscle oxidative capacity and moderately associated with IMTG accumulation raises two hypotheses. First, it is well known that HSP72 plays a pivotal role in mitochondrial biogenesis (15). Cytosolic HSP72 is a major precursor protein that interacts with the trans-outer mitochondrial membrane complex to import proteins into the mitochondria. Given the relationship between type 2 diabetes and impaired muscle oxidative capacity (16,17), along with the relationship in the present study, we propose that impaired HSP72 expression in patients with type 2 diabetes may play a role in impaired oxidative capacity. In addition, our observation that HSP72 was moderately associated with accumulation of IMTG raises an alternative hypothesis. It has recently been demonstrated that c-Jun NH2-terminal kinase (JNK) activity is abnormally elevated in obesity (18). Furthermore, an absence of JNK results in significantly improved insulin sensitivity and enhanced insulin receptor signaling capacity in two different models of mouse obesity (18). Of note, HSP72 inhibits JNK via inhibition of its dephosphorylation (19,20). Because an accumulation of IMTG is associated with insulin resistance (21), one role of intramuscular HSP72 may be to downregulate JNK, a mechanism that would be impaired in patients with type 2 diabetes.
A major limitation to the present study is that like previous investigations (7,8), we did not obtain sufficient muscle tissue to examine HSP72 protein content. However, we have previously demonstrated a remarkable similarity between HSP72 mRNA and protein expression in contracting human skeletal muscle (22), although this response is sometimes variable in subjects undergoing exercise (23). Nonetheless, our previous data (22), together with the evidence of a strong relationship between HSP72 gene transcription and protein translation (24), suggest that protein levels were also reduced in the muscles of the patients with type 2 diabetes.
It has been previously demonstrated that insulin activates HSP72 gene expression in a human hepatoma cell line (25). We have shown that HSP72 mRNA is induced by muscle contraction (11,22,23) and recombinant human interleukin-6 infusion (26). To our knowledge, however, no previous studies have examined the effect of a hyperinsulinemic-euglycemic clamp on HSP72 mRNA expression in human skeletal muscle. Our data demonstrate that HSP72 mRNA is induced to a small extent by this intervention, but that this induction is not defective in patients with type 2 diabetes (Table 3). It is not surprising that the clamp induced HSP72 mRNA in skeletal muscle because insulin activates signal transducer and activator of transcription (STAT) proteins (27), which are present in skeletal muscle (28) and activate HSP72. However, it was somewhat surprising that the fold induction of HSP72 by the clamp was uniform across groups, given the lower basal level in the diabetic group. We have no evidence for whether this small induction would lead to the translation of HSP72 protein in this time frame, and we therefore have difficulty interpreting these data. In addition, we cannot determine whether the increased expression was caused by the insulin or the enhanced glucose uptake associated with the clamp, although one would expect that the response was due to the former because, in a preliminary study, we have shown that increasing plasma glucose content reduces both circulating HSP72 protein and intramuscular HSP72 mRNA (29). Nonetheless, our findings may have implications for those presented by Kurucz et al. (7) because in that study a bolus of insulin was provided to the patients with type 2 diabetes before the muscle biopsy. This may have, in fact, reduced the difference in HSP72 mRNA when comparing the patient with the control group.
We cannot conclude from our data that the decrease in basal HSP72 mRNA mediates insulin resistance because we only present associations. However, our data, coupled with those presented previously in humans (7,8), as well as earlier studies in diabetic rodents showing that pharmacological increases in HSP72 expression improves insulin sensitivity (30), provide some evidence that HSP72 is directly involved in the pathogenesis of insulin resistance.
HO-1 and insulin resistance.
Comparatively little is known about the functional significance of HO-1 in skeletal muscle. To our knowledge, this is the first study to measure the expression of this gene in the muscles of patients with type 2 diabetes. Like HSP72, HO-1 expression was markedly reduced in the diabetic group (Fig. 1) and correlated with GDR (Fig. 5). However, it must be noted that the range in expression for HO-1 when comparing individuals was much greater compared with HSP72. Given that a decrease of 1 in the CT value corresponds to a halving of the copy number of the gene, the range in HSP72 was 1014 (Fig. 4), whereas the range for HO-1 was 1222 (Fig. 5), with the highest CT values in the patients in the diabetic group. Hence, our data show that patients with type 2 diabetes have markedly reduced HO-1 gene expression even relative to HSP72. It has been suggested that HO-1 provides protection against the effects of proinflammatory cytokines (6), and a polymorphism of the promoter region of the human HO-1 gene is associated with the susceptibility to pathogenesis associated with type 2 diabetes (31).
It is well known that the proinflammatory cytokine tumor necrosis factor (TNF)- is implicated in the etiology of insulin resistance and type 2 diabetes, primarily by reducing tyrosine phosphorylation of insulin receptor substrate-1 (rev. in 32). Although the relationship between HO-1 and TNF-
has not been investigated in skeletal muscle, HO-1 inhibits the deleterious effects of TNF-
in cultured fibroblasts (33). It is possible that there is a relationship between HO-1 and TNF-
in the muscles of patients with type 2 diabetes, and this warrants investigation. Interestingly, HO-1 is also induced in rat skeletal myoblasts by the nitric oxide (NO) donor sodium nitroprusside (34), and in neuronal NO synthase (NOS) protein, expression is reduced in the skeletal muscle of patients with type 2 diabetes (35). It is possible, therefore, that HO-1 mRNA expression is reduced in the skeletal muscle of patients with type 2 diabetes because of the reduced NOS expression. Of note, although HO-1 was not induced by insulin in young or age-matched control subjects, there was a
70-fold increase in insulin-induced gene expression in the diabetic group. Although this observation shows that insulin has an abnormal effect on HO-1 gene expression with type 2 diabetes, we are unable to shed light on this finding. We can only speculate that due to the aberrant insulin signaling cascade in patients with type 2 diabetes, insulin may have resulted in intramuscular stress or ROS production. Of note, MPO content was higher in the diabetic group compared with age-matched control subjects (Fig. 3). Stored in primary granules of neutrophils, MPO catalyzes the formation of hypochlorous acid, a powerful ROS (10). Although we were not able to measure MPO in young control subjects, our data in the diabetic group and age-matched control subjects provide further evidence that the pathogenesis of type 2 diabetes involves oxidant production.
In summary, we have provided evidence that the expression of genes associated with providing cellular defense against oxidative stress are markedly reduced in the skeletal muscles of patients with type 2 diabetes. Furthermore, the expression of these genes is associated with whole-body insulin sensitivity and muscle oxidative capacity. Our data cannot demonstrate any causal relationship between markers of oxidative stress and insulin resistance, and it must be noted that glucose disposal during the clamp was lower in age-matched control subjects compared with young control subjects, yet the expression of our measured genes was similar when comparing these groups. This demonstrates that the factors associated with insulin sensitivity are complex and multifactoral. Nonetheless, our data provide compelling evidence of a strong association between antioxidant defense and insulin sensitivity, therefore suggesting that reduced stress protein expression in skeletal muscle may be a primary factor in the etiology of type 2 diabetes. This hypothesis requires investigation, since stress proteins can be pharmacologically induced, suggesting a potential novel therapeutic target for muscle insulin resistance.
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ACKNOWLEDGMENTS |
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We acknowledge the assistance of Drs. Adamandia D. Kriketos and Gregory J. Cooney for measuring the IMTGs. We also thank Sally Clark, Kate Greenway, Mitchell Anderson, and Dr. David Newman for technical assistance.
Address correspondence and reprint requests to Mark A. Febbraio, PhD, Associate Professor Research, Skeletal Muscle Research Laboratory, School of Medical Sciences, RMIT University, P.O. Box 71, Bundoora 3083, Victoria, Australia. E-mail: mark.febbraio{at}rmit.edu.au
Received for publication March 24, 2003 and accepted in revised form June 6, 2003
CS, citrate synthase; CT, critical threshold; DEXA, dual-energy X-ray absorptiometry; FFA, free fatty acid; FFM, fat-free mass; GDR, glucose disposal rate; ß-HAD, ß-hyroxyacyl-CoA dehydrogenase; HO, heme oxygenase; HSP, heat shock protein; IMTG, intramuscular triglyceride; JNK, c-Jun NH2-terminal kinase; MPO, myeloperoxidase; NOS, nitric oxide synthase; PPAR, peroxisome proliferatoractivated receptor; ROS, reactive oxygen species; TNF, tumor necrosis factor
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REFERENCES |
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