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Centre for Integrated Systems Biology and Medicine (L.N., K.C., K.T.), School of Biomedical Sciences, Nottingham University Medical School, Queens Medical Centre, Nottingham, NG7 2UH, United Kingdom; Division of Nutritional Sciences (T.P., R.G.B.), School of Biosciences, Sutton Bonington Campus, University of Nottingham, Loughborough LE12 5RD, United Kingdom; Department of Medicine (H.Y., G.I.B.), University of Chicago, Chicago, Illinois 60637; and Department of Movement Sciences (M.M.A.L.P., L.J.C.v.L.), Maastricht University, 6211 KP Maastricht, The Netherlands
Address all correspondence and requests for reprints to: Dr. Kostas Tsintzas, School of Biomedical Sciences, Nottingham University Medical School, Queen’s Medical Centre, Nottingham, NG7 2UH, United Kingdom. E-mail: Kostas.Tsintzas{at}nottingham.ac.uk.
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Abstract |
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Research Design and Methods: In the first part of this study, 10 healthy subjects underwent hyperinsulinemic euglycemic (4.5 mmol/liter) clamps for 6 h with iv infusion of either saline or a 20% Intralipid emulsion (Fresenius Kabi AG, Bad Homburg, Germany). Skeletal muscle biopsies were taken before and after 3- and 6-h insulin infusion and analyzed for calpain-10 mRNA and protein expression. In the second part of the study, muscle samples obtained after an overnight fast in 10 long-standing, sedentary type 2 diabetes patients, 10 sedentary, weight-matched, normoglycemic controls, and 10 age-matched, endurance-trained cyclists were analyzed for calpain-10 mRNA and protein content.
Results: Intralipid infusion in healthy subjects reduced whole body glucose disposal by approximately 50% (P < 0.001). Calpain-10 mRNA (P = 0.01) but not protein content was reduced after 6-h insulin infusion in both the saline and Intralipid emulsion trials. Skeletal muscle calpain-10 mRNA and protein content did not differ between the type 2 diabetes patients and normoglycemic controls, but there was a strong trend for total calpain-10 protein to be greater in the endurance-trained athletes (P = 0.06).
Conclusions: These data indicate that skeletal muscle calpain-10 expression is not modified by insulin resistance per se and suggest that hyperinsulinemia and exercise training may modulate human skeletal muscle calpain-10 expression.
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Introduction |
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Despite the in vitro evidence linking calpain-10 to diabetes and altered glucose metabolism, the precise role of calpain-10 in the development of insulin resistance in humans in vivo remains to be resolved. In the presence of lipid-induced insulin resistance, skeletal muscle calpain-10 mRNA expression has been up-regulated by insulin in normal glucose tolerant (NGT) but not impaired glucose tolerant (IGT) subjects (5). These findings prompted the authors to suggest that up-regulation of calpain-10 may occur as a protective mechanism against the development of insulin resistance. However, our laboratory has previously reported that profound changes in whole body insulin sensitivity after starvation and subsequent refeeding in healthy humans had no effect on skeletal muscle calpain-10 mRNA or protein content (6), casting some doubt on this hypothesis.
One factor that has added to the complexity of elucidating the physiological role of calpain-10 is the expression of a number of protein isoforms. Analysis of human cDNA revealed a complex pattern of alternative splicing that may lead to the generation of up to eight different protein isoforms, ranging from approximately 672 (calpain-10a) to 138 (calpain-10 h) amino acids in length (1). The mRNA of calpain-10 is expressed ubiquitously in all adult and fetal tissues that have been examined (7), but the quantity of calpain-10a and 10f mRNA appears to represent the majority of the total calpain-10 transcript in skeletal muscle tissue (8). Of the studies that have considered isoform expression, some have noted an important role for the smaller calpain-10 proteins. For example, two putative calpain-10 protein isoforms of 54 and 60 kDa have been linked to insulin secretion from the pancreas (9, 10) and skeletal muscle differentiation (11), respectively, suggesting that these isoforms may play a more functional role than full-length calpain-10.
Surprisingly, there is currently no information regarding the characterization of calpain-10 expression in type 2 diabetic subjects or in subjects across the spectrum of insulin sensitivity. Clearly this remains an important area for investigation, given the inconsistent results surrounding the replication of the genetic association of calpain-10 with type 2 diabetes (12, 13), and the uncertainty surrounding the exact role of calpain-10 in the tissues that are important for glucose homeostasis. Based on previous findings demonstrating that calpain-10 mRNA is down-regulated in subjects who carry the at-risk calpain-10 single nucleotide polymorphism 43 (SNP-43) genotype (5, 14), it is possible that a long-term reduction in calpain-10 is a hallmark feature of insulin resistance and type 2 diabetes.
Therefore, the overall aim of this study was to investigate skeletal muscle calpain-10 expression in insulin-resistant states in humans. First, we examined whether acute induction of insulin resistance by lipid and heparin infusion (15, 16) modulates calpain-10 mRNA and protein expression in vivo in skeletal muscle. Second, we assessed whether a more prolonged presence of an insulin-resistant state is associated with a differential expression of calpain-10 mRNA and protein in skeletal muscle. For this purpose skeletal muscle tissue obtained from long-standing type 2 diabetes patients, age- and weight-matched normoglycemic controls, and age-matched endurance-trained subjects was assessed for calpain-10 mRNA and protein expression. In both arms of the study, we specifically focused on the protein expression of all observed calpain-10 isoforms. We hypothesized that both an acute (lipid-induced) and chronic (diabetes-related) insulin resistance state are associated with a reduction in muscle calpain-10 expression.
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Subjects and Methods |
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There were 10 healthy men (age 22 ± 1 yr, body mass 78 ± 3 kg, body mass index 24 ± 1 kg/m2) recruited from the students of the University of Nottingham to take part in this study. All procedures used in this study were performed according to the Declaration of Helsinki and approved by the University of Nottingham Medical School Ethics Committee. The details of this study protocol have been described in full elsewhere (17). On two separate occasions, at least 2 wk apart, subjects underwent a hyperinsulinemic (50 mU human Actrapid/m2·min) (Actrapid; Novo Nordisk, Bagsværd, Denmark) euglycemic (4.5 mmol/liter) clamp for 6 h with iv infusion of either 0.9% saline at a rate of 90 ml/h (CON) or a 20% Intralipid emulsion (Fresenius Kabi AG, Bad Homburg, Germany) at 90 ml/h plus heparin (200 U prime and 600 U/h) (LIPID). Blood samples were obtained every 5 min for the determination of blood glucose, and every 20 min for the determination of serum insulin and plasma free fatty acid concentrations as described previously (17). Glucose disposal was calculated as previously described (17) and normalized to lean body mass as determined with the use of dual energy x-ray absorptiometry (Hologic QDR-2000; Hologic, Waltham, MA). Skeletal muscle samples were obtained from the vastus lateralis using the percutaneous needle biopsy technique before and after 3- and 6-h insulin infusion.
Type 2 diabetes study protocol
There were 10 sedentary, overweight patients diagnosed with type 2 diabetes for 7 ± 1 yr, 10 sedentary, age- and weight-matched, normoglycemic controls, and 10 age-matched endurance-trained cyclists selected to participate in this study (Table 1
). All type 2 diabetes patients were using oral blood glucose lowering medication (metformin with or without sulfonylurea derivatives). Glucose tolerance status was verified with a 2-h oral glucose tolerance test according to World Health Organization criteria (Table 1). In addition, we calculated the insulin sensitivity index based on the basal and 2-h plasma glucose and insulin concentrations obtained in the oral glucose tolerance test as a valid estimate of whole body insulin sensitivity (18). Insulin sensitivity was substantially lower in the type 2 diabetes patients when compared with the sedentary and trained controls (48 ± 6 vs. 86 ± 12 and 133 ± 24, respectively; P < 0.01). All physiological and metabolic characteristics of the three groups of subjects studied have been reported previously (19). All muscle samples were obtained from the vastus lateralis using the percutaneous needle biopsy technique after an overnight fast. Subjects were instructed to refrain from strenuous physical activity for 2 d before muscle biopsy and received the same standardized meal (4.2 kJ/kg; consisting of 61 energy percent carbohydrate, 24 energy percent fat, and 15 energy percent protein) the evening before the trial.
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RNA extraction and real-time PCR
Total RNA was extracted from 10- to 20-mg frozen muscle tissue using TRIZOL reagent (Invitrogen, Paisley, UK) essentially as described by Chomczynski and Sacchi (20). TaqMan quantitative real-time RT-PCR was used for quantification of mRNA levels as previously described (6). TaqMan primers and probes were designed using Primer Express version 2.0 software (Perkin-Elmer, Norwalk, CT), and their sequences were reported previously (6). The probe and primers hybridized to an area common to multiple isoforms of calpain-10 mRNA, and, therefore, all mRNA data represent the summative expression of five potential calpain-10 mRNA transcripts (10a–10e). It is likely that isoform 10a represents the majority of the mRNA in skeletal muscle as has been previously demonstrated (8). Calpain-10 mRNA data were normalized to the housekeeping gene 
-actin expression in the Intralipid protocol, and the hydroxymethylbilane synthase expression in the type 2 diabetic protocol as -actin mRNA levels were found to be significantly altered in the diabetic group (data not shown).
Protein extraction and Western blotting Total protein extracts were prepared from 20- to 30-mg frozen biopsy tissue. Samples were homogenized for 30 sec on ice in 6 volumes buffer containing 50 mM HEPES, 1 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 10% glycerol, 150 mM NaCl, 1% Triton X-100 (pH 7.5), and 4 µl protease inhibitors cocktail (Sigma P8340; Sigma-Aldrich, St. Louis, MO) per ml buffer. The homogenates were centrifuged at 10,000 g for 20 min at 4 C, and calpain-10 expression was determined in the supernatant fraction using Western blotting as previously described (6). We confirmed that this method of calpain-10 protein extraction resulted in the quantification of the majority of the calpain-10 pool in the skeletal muscle supernatants (data not shown).
Statistics
Data were analyzed using a general linear model for repeated measures (SPSS, version 13.0; SPSS, Inc., Chicago, IL) and one-way ANOVA, where appropriate. All data are presented as mean ± SEM. The Student’s t test (two tailed) was used to compare paired data where appropriate, with Holm-Bonferroni step-wise corrections applied to avoid type 1 error.
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Results |
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Plasma free fatty acid concentrations were higher in the LIPID than the CON trial (2.3 ± 0.3 vs. 0.2 ± 0.0 mmol/liter, respectively; P < 0.01). The average blood glucose (CON 4.5 ± 0.02 vs. LIPID 4.6 ± 0.02 mmol/liter) and serum insulin (CON 89 ± 3 vs. LIPID 96 ± 2 mU/liter) concentrations during the clamps were not significantly different. Glucose disposal was reduced after 3 h in the LIPID trial, and during the last 30 min of the clamp, it was 50% lower than the CON trial (37.8 ± 4.4 vs. 79.6 ± 4.0 µmol/kg lean mass·min, respectively; P < 0.001; Fig. 1). We have previously reported that this marked development of lipid-induced insulin resistance is accompanied by significant perturbations in both whole body and skeletal muscle substrate metabolism (17).
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). To differentiate the effect of lipid infusion alone on calpain-10 mRNA expression, a separate group of subjects (n = 6) was infused with Intralipid for 3 h in the absence of insulin. Consistent with the aforementioned findings, Intralipid infusion alone had no significant effect on calpain-10 expression (before 2.8 ± 0.3 vs. after 3.4 ± 0.6; P = 0.49) (data not shown).
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). Individually, none of these bands were significantly altered by insulin infusion alone or by the infusion of insulin and Intralipid together (data not shown). Therefore, there was no difference in the mean total calpain-10 protein expression during the CON (before clamp 0.55 ± 0.09 vs. 6-h clamp 0.54 ± 0.07; P = 0.61) or LIPID clamp (before clamp 0.52 ± 0.08 vs. 6-h clamp 0.45 ± 0.06; P = 0.31) (Fig. 2B). We observed no correlation between calpain-10 mRNA and total calpain-10 protein levels at baseline (r = 0.31; P = 0.46). Type 2 diabetes study
Calpain-10 mRNA levels were not significantly different among the three groups (CON 0.89 ± 0.05 vs. diabetics 0.82 ± 0.05 vs. trained 0.90 ± 0.04; P = 0.38) (Fig. 3A). Consistent with the Intralipid study, immunoreactive bands were seen for calpain-10 protein at 75, 60, 48, and 35 kDa, and all were quantified in each group. Although there was no significant difference in the expression of any of the protein bands between each group, interesting trends in the expression of some calpain-10 isoforms were seen, as shown in Fig. 3B. In particular, the expression of the 60-kDa isoform was 3.7-fold higher in the trained control group when compared with the diabetic group (P = 0.07). Moreover, the expression of the 75-kDa (trained 0.63 ± 0.19 vs. diabetic 0.38 ± 0.05; P = 0.20) and 35-kDa (trained 0.96 ± 0.29 vs. diabetic 0.67 ± 0.07; P = 0.34) bands was also higher in the trained group vs. the diabetics but did not reach statistical significance. When the mean total calpain-10 protein levels were calculated for each group, there was a strong trend toward reduced total calpain-10 protein in the diabetes patients compared with the endurance-trained controls (diabetics 0.45 ± 0.05 vs. trained 0.67 ± 0.10; P = 0.06) (Fig. 3B). There was a similar trend between the type 2 diabetics and sedentary controls, although the difference was not as strong as observed with the endurance-trained controls (diabetics 0.45 ± 0.05 vs. CON 0.55 ± 0.05; P = 0.14).
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Discussion |
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One of the main findings from this study was that insulin infusion reduced calpain-10 mRNA content, and this response was not altered by lipid infusion. This finding appears to be in contrast to previous work by Carlsson et al. (5), who failed to observe any effect of 2-h insulin infusion only on calpain-10 mRNA levels in both NGT or IGT subjects. This may be explained by differences in the duration of each clamp (2 vs. 6 h) and/or the rate of insulin infusion (40 vs. 50 mU/m2·min) in the study by Carlsson et al. (5) and the present study, respectively. Indeed, we only observed a significant down-regulation of calpain-10 gene expression after 6-h, but not 3-h, insulin infusion. After 24-h Intralipid infusion, which resulted in a 25% reduction in insulin-stimulated glucose uptake, Carlsson et al. (5) observed a 3-fold increase in calpain-10 mRNA in response to 2-h insulin infusion in NGT but not in IGT subjects. This led the authors to speculate that in the latter subjects, the ability to up-regulate calpain-10 could be a protective mechanism secondary to sustained elevations in circulating lipids. We are not able to confirm this hypothesis because the addition of lipid infusion to the insulin clamp did not alter the response of calpain-10 mRNA to insulin infusion in healthy humans, despite a pronounced reduction (
50%) in insulin-stimulated glucose disposal. The conflicting results may be explained by methodological differences between the study by Carlsson et al. (5) and the present study. Because the effect of Intralipid infusion on insulin-mediated glucose uptake appears to be dose dependent (21), differences in the duration (24 vs. 6 h, respectively) and rate of lipid infusion (30 vs. 90 ml/h, respectively) may explain, at least in part, the differences between studies.
Although the observation that calpain-10 mRNA expression is regulated by hyperinsulinemia but not insulin resistance may appear paradoxical, it should be pointed out that short-term hyperinsulinemia alters the expression of approximately 800 genes in healthy human skeletal muscle, including a number of genes encoding proteolytic enzymes, e.g. many proteasome components and ubiquitin-ligases (22). The effect of insulin infusion on calpain-10 expression may reflect the potential role of calpain-10 in skeletal muscle protein turnover, which would be consistent with the role of the ubiquitous calpains in skeletal muscle (23). The physiological significance of the mRNA changes is not clear given that calpain-10 protein expression was unchanged. Currently, there is little information regarding the regulation of calpain-10 transcription (i.e. mRNA half-life) or translation (i.e. posttranslational modifications), but it is possible that chronic insulin resistance may lead to changes in calpain-10 protein expression.
To address further the issue of whether a more prolonged insulin-resistant state rather than an acute induction in healthy humans is associated with a reduction in the expression of calpain-10, we went on to measure for the first time calpain-10 mRNA and protein expression in type 2 diabetes patients, and compared these results to age-matched, normoglycemic sedentary and endurance-trained subjects. A major finding was that calpain-10 mRNA was not different across this spectrum of insulin sensitivity and was clearly not down-regulated in long-standing type 2 diabetic subjects. This result is particularly surprising given some of the results of previous in vivo and in vitro studies. For example, Baier et al. (14) demonstrated that in Pima Indians, reduced skeletal muscle calpain-10 mRNA content was found in carriers of the calpain-10 at-risk genotype (G/G) at SNP-43 and was linked to reduced whole body carbohydrate oxidation rates. Numerous in vitro studies have also provided evidence, albeit mostly indirect, that reduced calpain-10 expression negatively affects insulin-mediated glucose uptake (4) and that this can be accounted for by its interaction with glucose transporter 4 (GLUT4) (3). Nonetheless, the results of this part of the current study are consistent with the Intralipid infusion study and with our previous in vivo study, which demonstrated that a fasting-induced change in insulin sensitivity has no affect on calpain-10 expression in skeletal muscle. Together, these studies provide evidence that skeletal muscle calpain-10 mRNA levels are not modified by insulin resistance and/or type 2 diabetes.
In relation to the proposed role of calpain-10 in insulin resistance and type 2 diabetes, we cannot easily explain the differences between our findings and the results of some previous studies. Previous in vitro studies used calpain inhibitors to investigate the functional role of calpain-10, and these compounds had some profound effects on insulin-stimulated glucose uptake (3, 24). First, it is important to note that there is no direct biochemical evidence that calpain-10 possesses any calpain-like activity at all; in fact, careful analysis of its structure reveals a complete lack of the calcium binding sites that are typically required for the activation of the ubiquitous calpains. Thus, it cannot be assumed that the use of these inhibitors has any direct effect on calpain-10 activity. Second, the inhibitors used in previous studies are well known to inhibit the activity of not only members of the calpain system, but they also have diverse effects on numerous proteins of other proteolytic systems (25). In fact, it has been demonstrated that the effect of traditional calpain inhibitors on GLUT4 regulation may indeed be due to the nonspecific nature of these compounds (26). It is worth noting that specific inhibition of all calpain activity in mice via calpastatin overexpression leads to an increase in GLUT4 protein, a decrease in GLUT4 mRNA, but importantly, no change in insulin-stimulated glucose uptake (2). A recent study has addressed these problems and used short interfering RNAs to knock down calpain-10 expression in isolated skeletal muscle cells (4). Knockdown of calpain-10 expression resulted in a reduction in skeletal muscle insulin-mediated glucose uptake but had no affect on glycogen synthesis or protein kinase B/AKT phosphorylation, lending some support to the hypothesis that calpain-10 affects glucose uptake via interactions with GLUT4. However, these findings must be replicated in in vivo systems of reduced calpain-10 expression (i.e. calpain-10 knockout models) before firm conclusions can be drawn.
The current study does not exclude a role for calpain-10 in human skeletal muscle metabolism. There was a strong trend toward increased calpain-10 protein in the skeletal muscle of the endurance-trained control subjects when compared with the diabetes patients, particularly with a putative 60-kDa calpain-10 isoform. This band could represent isoforms 10b, 10c, or 10d because their estimated molecular masses are close to 60 kDa. This observation is interesting because a similar-sized calpain-10 protein is increased with skeletal muscle differentiation in vitro (11), and because physical training is associated with changes in muscle protein turnover (27) and greater oxidative capacity (28, 29). Indeed, the endurance-trained subjects had significantly greater maximal oxygen consumption and, thus, greater oxidative capacity that may account for some of the differences in calpain-10 protein expression in the trained vs. the diabetic subjects. These data would also be consistent with the more traditional role of calpains as regulators of skeletal muscle mass, and may reflect the potential role of calpain-10 in skeletal muscle protein turnover and/or adaptations to training. These findings also raise the interesting possibility that physical activity rather than insulin resistance may be an important physiological regulator of calpain-10 protein levels in human skeletal muscle. Clearly, more detailed studies are needed to confirm these findings, and it would be interesting to investigate the effects of an endurance-training program on skeletal muscle calpain-10 expression.
It is important to acknowledge the potential impact of genetic variation within the calpain-10 gene on the results obtained in the present study. In HepG2 cells in vitro, the SNP-43 G/G genotype appears to up-regulate calpain-10 gene expression (1), but in vivo it may have the opposite effect because skeletal muscle calpain-10 mRNA is down-regulated in healthy subjects homozygous for the G allele (14). We determined the SNP-43 genotype in the subjects that participated in the Intralipid (but not diabetic) arm of the present study and found that six of 10 subjects were homozygous for the at-risk G allele, and four were G/A heterozygotes (data not shown). Although there was no difference between the two groups for baseline or insulin-mediated calpain-10 mRNA or protein expression, the low number of subjects and the high frequency of the at-risk allele (30) make it difficult to exclude any effect of SNP-43 on calpain-10 expression.
In conclusion, the present study provides evidence showing that insulin resistance in vivo does not affect calpain-10 mRNA and protein levels in human skeletal muscle, whether acutely induced by lipid infusion or as part of long-standing type 2 diabetes. The current study also provides novel observations that imply that exercise training may be an important physiological regulator of calpain-10 protein levels in human skeletal muscle. These results need to be confirmed in studies using a larger number of subjects as well as in groups of subjects stratified by calpain-10 genotype.
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Footnotes |
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Present address for L.N.: Division of Diabetes, Department of Medicine, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78229.
Disclosure Statement: The authors have nothing to disclose.
First Published Online December 18, 2007
Abbreviations: CON, Saline; GLUT4, glucose transporter 4; IGT, impaired glucose tolerant; LIPID, Intralipid emulsion; NGT, normal glucose tolerant; SNP-43, single nucleotide polymorphism 43.
Received September 5, 2007.
Accepted December 6, 2007.
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, P = 0.06 vs. diabetes group.