Reduced Skeletal Muscle Inhibitor of B Content Is Associated With Insulin Resistance in Subjects With Type 2 Diabetes
http://www.100md.com
糖尿病学杂志 2006年第3期
1 Diabetes Division, University of Texas Health Science Center at San Antonio, San Antonio, Texas
2 Texas Diabetes Institute, San Antonio, Texas
AMPK, AMP-activated protein kinase; FFA, free fatty acid; GCRC, General Clinical Research Center; IB, inhibitor of B; IKK, IB kinase; IL, interleukin; NFB, nuclear factor B; TNF, tumor necrosis factor
ABSTRACT
Skeletal muscle insulin resistance plays a key role in the pathogenesis of type 2 diabetes. It recently has been hypothesized that excessive activity of the inhibitor of B (IB)/nuclear factor B (NFB) inflammatory pathway is a mechanism underlying skeletal muscle insulin resistance. However, it is not known whether IB/NFB signaling in muscle from subjects with type 2 diabetes is abnormal. We studied IB/NFB signaling in vastus lateralis muscle from six subjects with type 2 diabetes and eight matched control subjects. Muscle from type 2 diabetic subjects was characterized by a 60% decrease in IB protein abundance, an indicator of increased activation of the IB/NFB pathway. IB abundance directly correlated with insulin-mediated glucose disposal (Rd) during a hyperinsulinemic (40 mU · meC2 · mineC1)-euglycemic clamp (r = 0.63, P = 0.01), indicating that increased IB/NFB pathway activity is associated with muscle insulin resistance. We also investigated whether reversal of this abnormality could be a mechanism by which training improves insulin sensitivity. In control subjects, 8 weeks of aerobic exercise training caused a 50% increase in both IB and IB protein. In subjects with type 2 diabetes, training increased IB and IB protein to levels comparable with that of control subjects, and these increments were accompanied by a 40% decrease in tumor necrosis factor muscle content and a 37% increase in insulin-stimulated glucose disposal. In summary, subjects with type 2 diabetes have reduced IB protein abundance in muscle, suggesting excessive activity of the IB/NFB pathway. Moreover, this abnormality is reversed by exercise training.
Evidence has accumulated in recent years indicating that type 2 diabetes and other insulin-resistant disorders are characterized by chronic inflammation (1,2). Specifically, it has been postulated that excessive activity of the inhibitor B (IB)/nuclear factor B (NFB) inflammatory pathway may be an important molecular mechanism responsible for skeletal muscle insulin resistance (1,3eC7). NFB is a family of transcription factors that regulate the expression of proinflammatory genes. In unstimulated cells, NFB is predominantly localized in the cytoplasm, associated with an inhibitory protein, IB. Several stimuli, including cytokines (1), reactive oxygen species, hyperglycemia, and free fatty acids (FFAs) (5), activate IB kinase (IKK), the upstream kinase of IB. Upon activation by inflammatory factors, IKK phosphorylates IB, causing rapid IB polyubiquitination and degradation by proteosomes. Following release from IB, NFB translocates from the cytoplasm to the nucleus, where it binds to target genes to stimulate transcription of inflammatory mediators such as tumor necrosis factor (TNF), interleukin (IL)-1, and IL-6 (1,8,9).
IBs are members of a gene family that contain seven mammalian members, including IB, IB, IB, IB, Bcl-3, and the precursor Rel proteins p100 and p105. IB and IB share common properties to interact with NFB and inhibit DNA binding. NFB proteins consist of five members, including p65, p50, p52, RelB, and c-Rel. Dimerization of two NFB family members is necessary for their DNA-binding properties. The predominant activating NFB dimer in skeletal muscle is the p50-p65 heterodimer (10). NFB p65 contains a COOH-terminal transcriptional domain that is crucial for its ability to activate inflammatory gene expression (9).
Interventions aimed at blocking the IB/NFB pathway, such as genetic deletion of IKK in mice (4) and the administration of salicylates, which inhibit IKK, to subjects with type 2 diabetes (11) significantly improve peripheral insulin sensitivity. These findings strongly suggest that activation of the IB/NFB pathway plays an important role in the pathogenesis of insulin resistance. However, it is not known whether the IB/NFB pathway is excessively active in skeletal muscle from subjects with type 2 diabetes. The goal of the present study was to evaluate whether the activity of the IB/NFB pathway is increased in skeletal muscle from subjects with type 2 diabetes. We also examined whether an intervention known to improve insulin sensitivity, such as exercise training, could reverse abnormalities in the IB/NFB inflammatory pathway in skeletal muscle from subjects with type 2 diabetes. We hypothesized that inhibition of IB/NFB signaling in muscle is a mechanism by which training improves insulin sensitivity.
RESEARCH DESIGN AND METHODS
Six subjects with type 2 diabetes and eight healthy control subjects participated in the study. The metabolic data from these subjects have been previously reported (12). Each subject underwent a complete history, physical examination, screening laboratory tests, and a 75-g oral glucose tolerance test to determine the presence or absence of diabetes using established American Diabetes Association criteria. Three of six type 2 diabetic subjects were taking glyburide, which was withdrawn 3 days before the oral glucose tolerance test and insulin clamp studies. The remaining three type 2 diabetic subjects were treated with diet. The control subjects did not have a family history of type 2 diabetes and had normal glucose tolerance. Other than glyburide, no subject was taking any medication known to affect glucose metabolism. The study was approved by the institutional review board of the University of Texas Health Science Center at San Antonio, and all subjects gave written consent.
Following a 10- to 12-h overnight fast, subjects came to the General Clinical Research Center (GCRC) at the South Texas Veterans Health Care Hospital for a screening visit and glucose tolerance test. Within 3eC7 days, subjects returned to the GCRC for determination of their peak aerobic capacity (VO2peak) (12). Within 3eC7 days after the baseline VO2peak measurement, subjects returned to the GCRC at 8 A.M. after an overnight fast for a vastus lateralis muscle biopsy (13), followed by a 120-min euglycemic-hyperinsulinemic (40 mU · meC2 · mineC1) insulin clamp study, which was performed with infusion of 3-3H-glucose (prime = 25 e藽i, continuous infusion = 0.25 e藽i/min) to determine rates of glucose appearance and disposal, as previously described (12). In control subjects, the 3-3H-glucose glucose was given as a prime (25 e藽i)-continuous (0.25 e藽i/min) infusion starting 120 min before insulin. In type 2 diabetic subjects, the prime was increased (25 e藽i x fasting plasma glucose/100) and began 180 min before insulin. In diabetic subjects, following the start of insulin, the plasma glucose concentration was allowed to decrease to 100 mg/dl, at which level it was maintained. One week after the insulin clamp study, the subjects began an 8-week aerobic exercise training program on a stationary bicycle. The exercise intensity, duration, and frequency were progressively increased to 70% of VO2peak for 45 min four times per week, during the 8-week course of the exercise program. Once the training program was completed, the subjects returned to the GCRC for a repeat determination of VO2peak. A posttraining muscle biopsy and insulin clamp study was performed 24eC36 h after the last exercise bout.
Laboratory analyses.
Plasma insulin concentrations were measured by radioimmunoassay (Diagnostic Products, Los Angeles, CA), glucose was measured using the oxidase method and a Beckman analyzer, and HbA1c (A1C) was measured using a DCA 2000 analyzer (Bayer, Tarrytown, NY). FFA concentrations were determined by an enzymatic colorimetric method (Wako Chemicals, Nuess, Germany), and plasma IL-6 and TNF concentrations were measured using an enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN).
Muscle processing.
Muscle samples were homogenized as previously described (12). Briefly, muscle samples were weighted while still frozen and were homogenized in ice-cold lysis buffer (1:10, wt/vol) containing 50 mmol/l HEPES (pH 7.6), 150 mmol/l NaCl, 20 mmol/l sodium pyrophosphate, 20 mmol/l -glycerophosphate, 10 mmol/l sodium fluoride, 2 mmol/l sodium orthovanadate (Na3VO4), 2 mmol/l EDTA (pH 8.0), 1% Nonidet P-40, 10% glycerol, 1 mmol/l phenylmethylsulfonylfluoride, 1 mmol/l MgCl2, 1 mmol/l CaCl2, 10 e蘥/ml leupeptin, and 10 e蘥/ml aprotinin. Homogenates were incubated on ice for 20 min and then centrifuged at 15,000g for 20 min at 4°C. The supernatants were collected, and protein concentrations were measured by the Lowry method. Supernatants were stored at eC80°C until used.
Western blotting.
Muscle proteins (40 e蘥) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes, except for TNF, for which 150 e蘥 protein were used. After blocking in Tris-buffered saline with 5% nonfat dry milk, the membranes were incubated overnight at 4°C with antibodies against IB (Cell Signaling, Beverly, MA), IB (Santa Cruz Biotechnology, Santa Cruz, CA), NFB p50 (Cell Signaling), NFB p65 (Santa Cruz Biotechnology), IKK (Cell Signaling), phospho-IKK (Ser177/181) (Cell Signaling), and TNF (Santa Cruz Biotechnology) (14,15). For all these proteins, a 0.45-e蘭 nitrocellulose membrane was used, except for TNF, for which a 0.2-e蘭 membrane was used. Bound antibodies were detected with anti-rabbit immunoglobulin horseradish peroxidaseeClinked whole antibody and by using enhanced chemiluminescent reagents (PerkinElmer Life Science, Boston, MA). The membranes were exposed to film, and band intensity was quantified using Image Tool Software (University of Texas Health Science Center at San Antonio, San Antonio, TX).
Calculations.
During the postabsorptive state, steady-state conditions prevail and the rate of endogenous glucose appearance, which equals the rate of total body glucose uptake, is calculated as the 3-3H-glucose infusion rate (disintegrations per minute) divided by the steady-state plasma 3-3H-glucose specific activity (disintegrations per minute per milligram). During the insulin clamp, noneCsteady-state conditions prevail, and the rate of glucose production (Ra) is calculated using Steele’s equation (16). The rate of endogenous glucose production during the insulin clamp equals the tracer-derived Ra minus the exogenous rate of glucose infusion. The rate of whole-body glucose disposal (Rd) equals the rate of residual endogenous glucose production plus the exogenous glucose infusion rate adjusting to maintain euglycemia (90- to 120-min time period).
Statistical analysis.
All data are expressed as means ± SE. Differences between the control and diabetic groups were determined using the unpaired Student’s t test. Differences within a group, pre- versus posttraining, were determined using the paired t test. Correlation analysis was performed by the Pearson product-moment method. For all analyses, a P < 0.05 was considered to be statistically significant.
RESULTS
Table 1 summarizes the subject’s clinical and laboratory characteristics. There was no significant difference in age, body weight, and BMI between the type 2 diabetic and control subjects. Subjects with type 2 diabetes had higher fasting plasma glucose and insulin concentrations (P < 0.05), higher A1C (P < 0.05), and lower aerobic capacity (Vo2peak) (P < 0.05). There were no differences in FFA (690 ± 100 vs. 660 ± 100 e蘭ol/l), IL-6 (2.3 ± 0.5 vs. 2.3 ± 0.6 pg/ml), and TNF (1.0 ± 0.1 vs. 1.1 ± 0.2 pg/ml) plasma concentrations between the control and type 2 diabetic groups.
Exercise training.
Eight weeks of exercise training had no effect on body weight, BMI, or fasting plasma glucose and insulin concentrations in either group (Table 1). Plasma FFA, IL-6, and TNF concentrations did not change with training. However, physical training significantly increased the Vo2peakby 17% (P < 0.05) and 31% (P < 0.05) in the control and diabetic subjects, respectively. In the diabetic subjects, the increase in Vo2peak after training also was accompanied by a tendency for a 19% decrease in A1C (P = 0.05).
Euglycemic-hyperinsulinemic clamp studies and insulin sensitivity.
Basal endogenous glucose production was similar between groups, whereas insulin-mediated glucose disposal (Rd) was 95% higher in the control group (P < 0.05) (Table 1). Exercise training significantly increased Rd in the type 2 diabetic subjects by 37% (P < 0.05). In the control subjects, Rd tended to increase after training (P = 0.1). The smaller effect of training in control subjects is in part explained by a higher baseline Rd, resulting in a modest effect of training. Differences in sex distribution between groups did not explain the lesser effect of training on Rd observed in the control subjects (not shown).
IB/NFB signaling.
To determine whether skeletal muscle from subjects with type 2 diabetes has excessive activity of IB/NFB pathway, we measured IB and IB protein abundance. Because IB sequesters NFB in the cytoplasm and increases in IB abundance inversely correlate with NFB DNA binding in human skeletal muscle (17), a reduction in IB abundance is considered to indicate activation of the IB/NFB pathway. In skeletal muscle from type 2 diabetic subjects, IB was reduced by 60% compared with control subjects (P < 0.05) (Fig. 1A). IB was not significantly different in type 2 diabetic subjects (Fig. 1B). This reduction in IB suggests that there is excessive activity of the IB/NFB axis in skeletal muscle from type 2 diabetic subjects. Importantly, there was a positive correlation between IB and the rate of insulin-mediated glucose disposal (r = 0.63, P = 0.01) (Fig. 2A) and with the Vo2peak (r = 0.6, P = 0.02) (Fig. 2B). Consistent with these findings, there was a negative correlation between IB and the A1C concentration (r = eC0.5, P = 0.05) (Fig. 2C). There was also a positive correlation between insulin-mediated glucose disposal and the Vo2peak (r = 0.54, P = 0.04) (Fig. 2D). The predominant activating NFB dimer in muscle is p50-p65 (9,10). We measured NFB p50 and p65 protein in skeletal muscle and found that NFB p50 protein was decreased by 30% in type 2 diabetic versus control subjects (P < 0.05) (Fig. 3A). There were no differences in p65 protein between groups (Fig. 3B). In addition, there was a positive correlation between NFB p50 protein content and insulin-mediated glucose disposal (r = 0.58, P = 0.04) (Fig. 3C).
Effect of training on the IB/NFB pathway.
Because the IB/NFB pathway has been implicated in the cellular mechanisms responsible for skeletal muscle insulin resistance, we hypothesized that exercise training in patients with type 2 diabetes would reverse abnormalities in IB/NFB signaling and that this is a mechanism by which training improves insulin sensitivity. In control subjects, training caused a 50% increase in both IB (P < 0.05) and IB (P < 0.05) protein content (Fig. 4A and B). After training, NFB p50 protein tended to increase by 100% (P = 0.05) (Fig. 4C) in the control subjects but had no effect on NFB p65 (Fig. 4D). Moreover, in the muscle from type 2 diabetic subjects, physical training increased IB and IB protein by 98% (P < 0.05) and 185% (P < 0.05), respectively (Fig. 5A and B). Exercise training also increased NFB p50 in subjects with type 2 diabetes by 140% (P < 0.05) (Fig. 5C), but similar to the control subjects, exercise had no effect on NFB p65 (Fig. 5D). Furthermore, these increments in IB and NFB p50 protein were accompanied by a 37% increase in insulin-mediated glucose disposal (Table 1). These findings indicate that training can restore abnormalities in IB and NFB p50 content in muscle from subjects with type 2 diabetes.
IKK phosphorylation.
We measured IKK phosphorylation to further examine the mechanism responsible for the reduction in IB in the type 2 diabetic group. There was no difference in IKK phosphorylation between groups, and training had no effect on IKK phosphorylation (Fig. 6) or protein content (not shown).
TNF muscle content.
It has been shown that type 2 diabetes is associated with higher muscle expression of TNF, an NFB-regulated gene (18). Accordingly, in the type 2 diabetic subjects the reduction in IB was accompanied by a tendency for higher basal TNF protein content by 25% compared with the control subjects (P = NS) (Fig. 7), although we could only measure TNF muscle content in five control and four diabetic subjects because a large amount of muscle protein is required to measure TNF muscle content by Western blotting and muscle tissue was no longer available for mRNA expression analysis. It is possible that this trend would have reached statistical significance with a higher number of samples. Consistent with previous reports (19), the increases in IB and NFB p50 caused by training were associated with a reduction in TNF muscle content in both groups (P < 0.05) (Fig. 7).
DISCUSSION
In this study, we found that type 2 diabetic subjects have decreased IB muscle content, suggesting enhanced IB/NFB signaling. Importantly, there was a positive correlation between IB protein and insulin-mediated glucose disposal in type 2 diabetic and control subjects. These findings support the hypothesis that enhanced activation of the IB/NFB pathway is associated with insulin resistance in human skeletal muscle.
The mechanism responsible for the apparent increase in IB/NFB signaling in muscle from the type 2 diabetic subjects is not clear. One hypothesis involves lipid-induced activation of IB/NFB signaling. Intramyocellular lipid metabolites, particularly long-chain fatty acyl CoAs, diacylglycerol, and ceramides, have been shown to induce insulin resistance (20eC25). Moreover, skeletal muscle of insulin-resistant subjects is characterized by increases in fatty acyl CoA (26) and ceramides (27). Recently, it was shown that lipid-induced insulin resistance in L6 myotubes (28) and muscle from rodents (3,4,29) and humans (5) is associated with activation of the IB/NFB pathway. A role for this pathway in mediating insulin resistance is further strengthened by the findings that inhibition of IB/NFB signaling improves insulin sensitivity (4,11). In support of our findings, Bhatt et al. (29) recently reported that diet-induced obesity in rats, an intervention that causes insulin resistance, lead to a decrease in muscle IB content. Interestingly, this group found that differences in IB muscle content between muscle fiber types were not explained by muscle triglyceride content (29), suggesting that intramyocellular lipids were not responsible for the reduction in IB. However, metabolites of triglycerides and fatty acids (i.e., fatty acyl CoAs, diacylglycerol, and ceramides), and not triglycerides per se, are believed to be responsible for the insulin resistance. We were unable to measure fatty acyl CoA, ceramide, or diacylglycerol muscle content because of the limited amount of tissue that can be obtained in humans using the percutaneous biopsy technique. Infusion of FFAs to humans (5) and rats (29) has been associated with activation of the IkB/NFkB pathway. In the present study, fasting plasma FFA concentrations were similar between groups. It should be noted, however, that fasting FFA levels do not reflect FFA turnover and day-long plasma FFA concentrations. Reaven et al. (30) studied type 2 diabetic subjects with similar blood glucose concentrations compared with the subjects included in the present study and found that the type 2 diabetic subjects had similar fasting FFA plasma levels compared with the nondiabetic control subjects, yet mean 24-h plasma FFA concentrations were significantly elevated.
Type 2 diabetes is characterized by low-grade chronic inflammation (1,31,32), and cytokines, including TNF and IL-6, have been shown to activate the IB/NFB pathway. TNF and IL-6 plasma concentrations were similar between groups. Therefore, it seems unlikely that these cytokines were responsible for the decrease in muscle IB. Nonetheless, there are many other exogenous inflammatory stimuli that were not measured in this study and could have caused IB/NFB axis activation. Reactive oxygen species are also known to simulate the IB/NFB pathway (33eC35), and hyperglycemia-induced reactive oxygen species generation could have contributed to the lower IB seen in the type 2 diabetic group. Consistent with our findings, Bandyopadhyay et al. (36) did not find increased IKK phosphorylation in muscle from insulin-resistant subjects. Because IB phosphorylation by IKK leads to IB ubiquination and subsequent degradation, the decrease in IB content would then have to be explained by IKK-independent phosphorylation. Protein kinase C and eIF2 kinase can also associate with and phosphorylate IB (37).
How excessive activity of the IB/NFB pathway leads to muscle insulin resistance has not been fully elucidated. Insulin infusion increased insulin receptor substrate-1eCassociated phosphoinositide 3-kinase activity by 33% (P < 0.05) in control subjects but not in type 2 diabetic subjects (12). One potential mechanism for these decreases in insulin-stimulated glucose disposal and phosphoinositide 3-kinase activity in the diabetic group involves insulin receptor substrate-1 serine phosphorylation by IKK (38eC40), but, as mentioned above, IKK phosphorylation was unchanged. Moreover, training-induced improvements in insulin sensitivity were not explained by changes in IKK phosphorylation or phosphoinositide 3-kinase activity. In insulin-resistant (lipid-treated) L6 muscle cells, inhibition of NFB using a selective inhibitory peptide improved insulin-stimulated glucose transport (28). This indicates that NFB, and genes that this transcription factor controls, may directly influence insulin sensitivity. Yet, overactivation of NFB in transgenic mice was not associated with insulin resistance (41). Because the phenotype of these animals was characterized by severe muscle wasting, it is unclear how the muscle wasting influenced insulin sensitivity measurements. It is also possible that lifelong overactivation of NFB could lead to compensatory mechanisms aimed at normalizing insulin sensitivity. Further studies will help clarify how the IB/NFB pathway modulates insulin sensitivity in humans.
Few studies have examined the effect of exercise on the IB/NFB pathway. In rats, acute exercise activates IB/NFB signaling in muscle (42,43), while acute fatiguing exercise in humans reduces NFB activity (44). In the present study, we found that training increased IB and reduced TNF muscle content, suggesting decreased IB/NFB signaling. This increase in IB was not associated with changes in plasma TNF, IL-6, adiponectin (not shown), or FFA concentrations. Stimuli intrinsic to muscle could also be responsible for the training-induced changes in IB and TNF. Training significantly increased AMP-activated protein kinase (AMPK) phosphorylation in both control and type 2 diabetic groups (N.M., L.J.M., unpublished observations). In endothelial cells, chemical AMPK activation with 5-aminoimidazole-4-carboxamide ribonucleoside has been shown to inhibit NFB activity (45). These results suggest that exercise-stimulated AMPK activation may be responsible for the inhibition of the IB/NFB axis. In contrast, AMPK activation with 5-aminoimidazole-4-carboxamide ribonucleoside had no effect on NFB activity in rat skeletal muscle (43), arguing against a role for AMPK as a regulator of IB/NFB signaling. A reduction in intramyocellular lipid content caused by training could help explain the changes in IB and TNF. However, several studies have shown that trained athletes have increased triglyceride muscle content (46,47). The greater triglyceride storage in the trained athlete represents an adaptive response to training and provides a readily available source of energy for the contracting muscle. In contrast, elevated lipid stores in type 2 diabetes result from an imbalance between plasma FFA availability and oxidation and have been implicated in the development of insulin resistance. Future studies will be needed to examine whether elevated levels of fatty acyl CoA, diacylglycerol, and ceramides are related with abnormalities in IB/NFB signaling and to assess the effect of training on the content of these metabolites.
Type 2 diabetic subjects had a more pronounced reduction in IB content compared with IB. While the IB and IB isoforms share many similarities, they also display differences. The IB gene has a B recognition sequence in its promoter region (48). Thus, activation of NFB can induce resynthesis of IB. In contrast, NFB does not induce IB synthesis (8,49). Since the muscle of type 2 diabetic subjects appears to have excessive NFB activity, this could lead to selective resynthesis of IB but not of IB. Although it is not known whether IB remains suppressed upon long-term NFB activation, in Jurkat cells cytokine-induce IB degradation is rapidly restored within 2 h, whereas IB remains low for at least 24 h (50). Whether this differential regulation between IB and IB occurs in other chronic diseases remains to be determined.
We found that NFB p50 subunit content was reduced in muscle of type 2 diabetic subjects, and this decrease correlated with reduced insulin sensitivity. This might appear counterintuitive since NFB stimulates transcription of inflammatory genes. While the p50-p65 heterodimer is the predominant activating NFB dimer, the p50 subunit also can dimerize with another p50 subunit to form p50-p50 homodimers. While the p50-p65 heterodimer activates transcription of inflammatory genes, the p50-p50 homodimer inhibits gene transcription (9,51eC53). In view of our findings of reduced p50, one might speculate that type 2 diabetic subjects could have decreased abundance of the p50-p50 repressor homodimers, resulting in excessive NFB p50-p65 activity, and that training inhibited NFB activity by increasing p50-p50 homodimers. Future investigations are needed to determine whether NFB p50-p50 DNA binding is indeed reduced in muscle from type 2 diabetic subjects.
The present study shows an important association between insulin sensitivity and the content of IB and NFB p50 in human muscle. Yet, these results do not prove causality. Studies using genetic and pharmacologic approaches to manipulate IB/NFB signaling will help establish the role of this pathway in insulin sensitivity regulation.
ACKNOWLEDGMENTS
This work was supported by grants DK47936 (to L.J.M.), DK66483 (to L.J.M.), and DK24092 (to R.A.D.) from the National Institutes of Health; Grant RR01346 (GCRC, Audie L. Murphy Veterans Hospital); an Executive Research Committee Research Award from the University of Texas Health Science Center at San Antonio (to N.M.); a VA Merit Award (to R.A.D.); an American Diabetes Association Junior Faculty Award (to N.M.); and a grant from the Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand (to A.S.).
We also thank all the study participants.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Evans JL, Goldfine ID, Maddux BA, Grodsky GM: Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocr Rev 23:599eC622, 2002
Garg R, Tripathy D, Dandona P: Insulin resistance as a proinflammatory state: mechanisms, mediators, and therapeutic interventions. Curr Drug Targets 4:487eC492, 2003
Kim JK, Kim YJ, Fillmore JJ, Chen Y, Moore I, Lee J, Yuan M, Li ZW, Karin M, Perret P, Shoelson SE, Shulman GI: Prevention of fat-induced insulin resistance by salicylate. J Clin Invest 108:437eC446, 2001
Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M, Shoelson SE: Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta. Science 293:1673eC1677, 2001
Itani SI, Ruderman NB, Schmieder F, Boden G: Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and IB-. Diabetes 51:2005eC2011, 2002
Shoelson SE, Lee J, Yuan M: Inflammation and the IKK beta/I kappa B/NF-kappa B axis in obesity- and diet-induced insulin resistance. Int J Obes Relat Metab Disord 27 (Suppl. 3):S49eCS52, 2003
Ghanim H, Garg R, Aljada A, Mohanty P, Kumbkarni Y, Assian E, Hamouda W, Dandona P: Suppression of nuclear factor-kappaB and stimulation of inhibitor kappaB by troglitazone: evidence for an anti-inflammatory effect and a potential antiatherosclerotic effect in the obese. J Clin Endocrinol Metab 86:1306eC1312, 2001
Barnes PJ, Karin M: Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336:1066eC1071, 1997
Yamamoto Y, Gaynor RB: IkappaB kinases: key regulators of the NF-kappaB pathway. Trends Biochem Sci 29:72eC79, 2004
Jackman RW, Kandarian SC: The molecular basis of skeletal muscle atrophy. Am J Physiol Cell Physiol 287:C834eCC843, 2004
Hundal RS, Petersen KF, Mayerson AB, Randhawa PS, Inzucchi S, Shoelson SE, Shulman GI: Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J Clin Invest 109:1321eC1326, 2002
Christ-Roberts CY, Pratipanawatr T, Pratipanawatr W, Berria R, Belfort R, Kashyap S, Mandarino LJ: Exercise training increases glycogen synthase activity and GLUT4 expression but not insulin signaling in overweight nondiabetic and type 2 diabetic subjects. Metabolism 53:1233eC1242, 2004
Cusi K, Maezono K, Osman A, Pendergrass M, Patti ME, Pratipanawatr T, DeFronzo RA, Kahn CR, Mandarino LJ: Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle. J Clin Invest 105:311eC320, 2000
Gardiner TA, Gibson DS, de Gooyer TE, de la Cruz VF, McDonald DM, Stitt AW: Inhibition of tumor necrosis factor-alpha improves physiological angiogenesis and reduces pathological neovascularization in ischemic retinopathy. Am J Pathol 166:637eC644, 2005
Raina N, Jeejeebhoy KN: Effect of low-protein diet and protein supplementation on the expressions of TNF-alpha, TNFR-I, and TNFR-II in organs and muscle of LPS-injected rats. Am J Physiol Endocrinol Metab 286:E481eCE487, 2004
Steele R: Influences of glucose loading and of injected insulin on hepatic glucose output. Ann N Y Acad Sci 82:420eC430, 1959
Agusti C, Rano A, Rovira M, Filella X, Benito N, Moreno A, Torres A: Inflammatory response associated with pulmonary complications in non-HIV immunocompromised patients. Thorax 59:1081eC1088, 2004
Saghizadeh M, Ong JM, Garvey WT, Henry RR, Kern PA: The expression of TNF alpha by human muscle: relationship to insulin resistance. J Clin Invest 97:1111eC1116, 1996
Greiwe JS, Cheng B, Rubin DC, Yarasheski KE, Semenkovich CF: Resistance exercise decreases skeletal muscle tumor necrosis factor alpha in frail elderly humans. FASEB J 15:475eC482, 2001
Shulman GI: Cellular mechanisms of insulin resistance. J Clin Invest 106:171eC176, 2000
Ellis BA, Poynten A, Lowy AJ, Furler SM, Chisholm DJ, Kraegen EW, Cooney GJ: Long-chain acyl-CoA esters as indicators of lipid metabolism and insulin sensitivity in rat and human muscle. Am J Physiol Endocrinol Metab 279:E554eCE560, 2000
Hajduch E, Balendran A, Batty IH, Litherland GJ, Blair AS, Downes CP, Hundal HS: Ceramide impairs the insulin-dependent membrane recruitment of protein kinase B leading to a loss in downstream signalling in L6 skeletal muscle cells. Diabetologia 44:173eC183, 2001
Cooney GJ, Thompson AL, Furler SM, Ye J, Kraegen EW: Muscle long-chain acyl CoA esters and insulin resistance. Ann N Y Acad Sci 967:196eC207, 2002
Schmitz-Peiffer C, Craig DL, Biden TJ: Ceramide generation is sufficient to account for the inhibition of the insulin-stimulated PKB pathway in C2C12 skeletal muscle cells pretreated with palmitate. J Biol Chem 274:24202eC24210, 1999
Schubert KM, Scheid MP, Duronio V: Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine 473. J Biol Chem 275:13330eC13335, 2000
Hulver MW, Berggren JR, Cortright RN, Dudek RW, Thompson RP, Pories WJ, MacDonald KG, Cline GW, Shulman GI, Dohm GL, Houmard JA: Skeletal muscle lipid metabolism with obesity. Am J Physiol Endocrinol Metab 284:E741eCE747, 2003
Adams JM, 2nd, Pratipanawatr T, Berria R, Wang E, DeFronzo RA, Sullards MC, Mandarino LJ: Ceramide content is increased in skeletal muscle from obese insulin-resistant humans. Diabetes 53:25eC31, 2004
Sinha S, Perdomo G, Brown NF, O’Doherty RM: Fatty acid-induced insulin resistance in L6 myotubes is prevented by inhibition of activation and nuclear localization of nuclear factor kappa B. J Biol Chem 279:41294eC41301, 2004
Bhatt BA, Dube JJ, Dedousis N, Reider JA, O’Doherty RM: Diet-induced obesity and acute hyperlipidemia reduce IkappaBalpha levels in rat skeletal muscle in a fiber-type dependent manner. Am J Physiol Regul Integr Comp Physiol 290:R233eCR240, 2006
Reaven GM, Hollenbeck C, Jeng CY, Wu MS, Chen YD: Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM. Diabetes 37:1020eC1024, 1988
Pickup JC, Crook MA: Is type II diabetes mellitus a disease of the innate immune system Diabetologia 41:1241eC1248, 1998
Dandona P: The link between insulin resistance syndrome and inflammatory markers. Endocr Pract 9 (Suppl. 2):53eC57, 2003
Mercurio F, Manning AM: NF-kappaB as a primary regulator of the stress response. Oncogene 18:6163eC6171, 1999
Droge W: Free radicals in the physiological control of cell function. Physiol Rev 82:47eC95, 2002
Dalton TP, Shertzer HG, Puga A: Regulation of gene expression by reactive oxygen. Annu Rev Pharmacol Toxicol 39:67eC101, 1999
Bandyopadhyay GK, Yu JG, Ofrecio J, Olefsky JM: Increased p85/55/50 expression and decreased phosphotidylinositol 3-kinase activity in insulin-resistant human skeletal muscle. Diabetes 54:2351eC2359, 2005
Ghosh S, Baltimore D: Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B. Nature 344:678eC682, 1990
Gao Z, Zuberi A, Quon MJ, Dong Z, Ye J: Aspirin inhibits serine phosphorylation of insulin receptor substrate 1 in tumor necrosis factor-treated cells through targeting multiple serine kinases. J Biol Chem 278:24944eC24950, 2003
Gao Z, Hwang D, Bataille F, Lefevre M, York D, Quon MJ, Ye J: Serine phosphorylation of insulin receptor substrate 1 by inhibitor kappa B kinase complex. J Biol Chem 277:48115eC48121, 2002
de Alvaro C, Teruel T, Hernandez R, Lorenzo M: Tumor necrosis factor alpha produces insulin resistance in skeletal muscle by activation of inhibitor kappaB kinase in a p38 MAPK-dependent manner. J Biol Chem 279:17070eC17078, 2004
Cai D, Frantz JD, Tawa NE, Jr, Melendez PA, Oh BC, Lidov HG, Hasselgren PO, Frontera WR, Lee J, Glass DJ, Shoelson SE: IKKbeta/NF-kappaB activation causes severe muscle wasting in mice. Cell 119:285eC298, 2004
Ji LL, Gomez-Cabrera MC, Steinhafel N, Vina J: Acute exercise activates nuclear factor (NF)-kappaB signaling pathway in rat skeletal muscle. FASEB J 18:1499eC1506, 2004
Ho RC, Hirshman MF, Li Y, Cai D, Farmer JR, Aschenbach WG, Witczak CA, Shoelson SE, Goodyear LJ: Regulation of IkappaB kinase and NF-kappaB in contracting adult rat skeletal muscle. Am J Physiol Cell Physiol 289:C794eCC801, 2005
Durham WJ, Li YP, Gerken E, Farid M, Arbogast S, Wolfe RR, Reid MB: Fatiguing exercise reduces DNA binding activity of NF-kappaB in skeletal muscle nuclei. J Appl Physiol 97:1740eC1745, 2004
Cacicedo JM, Yagihashi N, Keaney JF Jr, Ruderman NB, Ido Y: AMPK inhibits fatty acid-induced increases in NF-kappaB transactivation in cultured human umbilical vein endothelial cells. Biochem Biophys Res Commun 324:1204eC1209, 2004
van Loon LJ: Use of intramuscular triacylglycerol as a substrate source during exercise in humans. J Appl Physiol 97:1170eC1187, 2004
Goodpaster BH, He J, Watkins S, Kelley DE: Skeletal muscle lipid content and insulin resistance: evidence for a paradox in endurance-trained athletes. J Clin Endocrinol Metab 86:5755eC5761, 2001
Ito CY, Kazantsev AG, Baldwin AS Jr: Three NF-kappa B sites in the I kappa B-alpha promoter are required for induction of gene expression by TNF alpha. Nucleic Acid Res 22:3787eC3792, 1994
Karin M, Delhase M: The I kappa B kinase (IKK) and NF-kappa B: key elements of proinflammatory signalling. Semin Immunol 12:85eC98, 2000
Thompson JE, Phillips RJ, Erdjument-Bromage H, Tempst P, Ghosh S: I kappa B-beta regulates the persistent response in a biphasic activation of NF-kappa B. Cell 80:573eC582, 1995
Udalova IA, Richardson A, Denys A, Smith C, Ackerman H, Foxwell B, Kwiatkowski D: Functional consequences of a polymorphism affecting NF-kappaB p50-p50 binding to the TNF promoter region. Mol Cell Biol 20:9113eC9119, 2000
Driessler F, Venstrom K, Sabat R, Asadullah K, Schottelius AJ: Molecular mechanisms of interleukin-10-mediated inhibition of NF-kappaB activity: a role for p50. Clin Exp Immunol 135:64eC73, 2004
Tong X, Yin L, Washington R, Rosenberg DW, Giardina C: The p50-p50 NF-kappaB complex as a stimulus-specific repressor of gene activation. Mol Cell Biochem 265:171eC183, 2004(Reduced Skeletal Muscle I)
2 Texas Diabetes Institute, San Antonio, Texas
AMPK, AMP-activated protein kinase; FFA, free fatty acid; GCRC, General Clinical Research Center; IB, inhibitor of B; IKK, IB kinase; IL, interleukin; NFB, nuclear factor B; TNF, tumor necrosis factor
ABSTRACT
Skeletal muscle insulin resistance plays a key role in the pathogenesis of type 2 diabetes. It recently has been hypothesized that excessive activity of the inhibitor of B (IB)/nuclear factor B (NFB) inflammatory pathway is a mechanism underlying skeletal muscle insulin resistance. However, it is not known whether IB/NFB signaling in muscle from subjects with type 2 diabetes is abnormal. We studied IB/NFB signaling in vastus lateralis muscle from six subjects with type 2 diabetes and eight matched control subjects. Muscle from type 2 diabetic subjects was characterized by a 60% decrease in IB protein abundance, an indicator of increased activation of the IB/NFB pathway. IB abundance directly correlated with insulin-mediated glucose disposal (Rd) during a hyperinsulinemic (40 mU · meC2 · mineC1)-euglycemic clamp (r = 0.63, P = 0.01), indicating that increased IB/NFB pathway activity is associated with muscle insulin resistance. We also investigated whether reversal of this abnormality could be a mechanism by which training improves insulin sensitivity. In control subjects, 8 weeks of aerobic exercise training caused a 50% increase in both IB and IB protein. In subjects with type 2 diabetes, training increased IB and IB protein to levels comparable with that of control subjects, and these increments were accompanied by a 40% decrease in tumor necrosis factor muscle content and a 37% increase in insulin-stimulated glucose disposal. In summary, subjects with type 2 diabetes have reduced IB protein abundance in muscle, suggesting excessive activity of the IB/NFB pathway. Moreover, this abnormality is reversed by exercise training.
Evidence has accumulated in recent years indicating that type 2 diabetes and other insulin-resistant disorders are characterized by chronic inflammation (1,2). Specifically, it has been postulated that excessive activity of the inhibitor B (IB)/nuclear factor B (NFB) inflammatory pathway may be an important molecular mechanism responsible for skeletal muscle insulin resistance (1,3eC7). NFB is a family of transcription factors that regulate the expression of proinflammatory genes. In unstimulated cells, NFB is predominantly localized in the cytoplasm, associated with an inhibitory protein, IB. Several stimuli, including cytokines (1), reactive oxygen species, hyperglycemia, and free fatty acids (FFAs) (5), activate IB kinase (IKK), the upstream kinase of IB. Upon activation by inflammatory factors, IKK phosphorylates IB, causing rapid IB polyubiquitination and degradation by proteosomes. Following release from IB, NFB translocates from the cytoplasm to the nucleus, where it binds to target genes to stimulate transcription of inflammatory mediators such as tumor necrosis factor (TNF), interleukin (IL)-1, and IL-6 (1,8,9).
IBs are members of a gene family that contain seven mammalian members, including IB, IB, IB, IB, Bcl-3, and the precursor Rel proteins p100 and p105. IB and IB share common properties to interact with NFB and inhibit DNA binding. NFB proteins consist of five members, including p65, p50, p52, RelB, and c-Rel. Dimerization of two NFB family members is necessary for their DNA-binding properties. The predominant activating NFB dimer in skeletal muscle is the p50-p65 heterodimer (10). NFB p65 contains a COOH-terminal transcriptional domain that is crucial for its ability to activate inflammatory gene expression (9).
Interventions aimed at blocking the IB/NFB pathway, such as genetic deletion of IKK in mice (4) and the administration of salicylates, which inhibit IKK, to subjects with type 2 diabetes (11) significantly improve peripheral insulin sensitivity. These findings strongly suggest that activation of the IB/NFB pathway plays an important role in the pathogenesis of insulin resistance. However, it is not known whether the IB/NFB pathway is excessively active in skeletal muscle from subjects with type 2 diabetes. The goal of the present study was to evaluate whether the activity of the IB/NFB pathway is increased in skeletal muscle from subjects with type 2 diabetes. We also examined whether an intervention known to improve insulin sensitivity, such as exercise training, could reverse abnormalities in the IB/NFB inflammatory pathway in skeletal muscle from subjects with type 2 diabetes. We hypothesized that inhibition of IB/NFB signaling in muscle is a mechanism by which training improves insulin sensitivity.
RESEARCH DESIGN AND METHODS
Six subjects with type 2 diabetes and eight healthy control subjects participated in the study. The metabolic data from these subjects have been previously reported (12). Each subject underwent a complete history, physical examination, screening laboratory tests, and a 75-g oral glucose tolerance test to determine the presence or absence of diabetes using established American Diabetes Association criteria. Three of six type 2 diabetic subjects were taking glyburide, which was withdrawn 3 days before the oral glucose tolerance test and insulin clamp studies. The remaining three type 2 diabetic subjects were treated with diet. The control subjects did not have a family history of type 2 diabetes and had normal glucose tolerance. Other than glyburide, no subject was taking any medication known to affect glucose metabolism. The study was approved by the institutional review board of the University of Texas Health Science Center at San Antonio, and all subjects gave written consent.
Following a 10- to 12-h overnight fast, subjects came to the General Clinical Research Center (GCRC) at the South Texas Veterans Health Care Hospital for a screening visit and glucose tolerance test. Within 3eC7 days, subjects returned to the GCRC for determination of their peak aerobic capacity (VO2peak) (12). Within 3eC7 days after the baseline VO2peak measurement, subjects returned to the GCRC at 8 A.M. after an overnight fast for a vastus lateralis muscle biopsy (13), followed by a 120-min euglycemic-hyperinsulinemic (40 mU · meC2 · mineC1) insulin clamp study, which was performed with infusion of 3-3H-glucose (prime = 25 e藽i, continuous infusion = 0.25 e藽i/min) to determine rates of glucose appearance and disposal, as previously described (12). In control subjects, the 3-3H-glucose glucose was given as a prime (25 e藽i)-continuous (0.25 e藽i/min) infusion starting 120 min before insulin. In type 2 diabetic subjects, the prime was increased (25 e藽i x fasting plasma glucose/100) and began 180 min before insulin. In diabetic subjects, following the start of insulin, the plasma glucose concentration was allowed to decrease to 100 mg/dl, at which level it was maintained. One week after the insulin clamp study, the subjects began an 8-week aerobic exercise training program on a stationary bicycle. The exercise intensity, duration, and frequency were progressively increased to 70% of VO2peak for 45 min four times per week, during the 8-week course of the exercise program. Once the training program was completed, the subjects returned to the GCRC for a repeat determination of VO2peak. A posttraining muscle biopsy and insulin clamp study was performed 24eC36 h after the last exercise bout.
Laboratory analyses.
Plasma insulin concentrations were measured by radioimmunoassay (Diagnostic Products, Los Angeles, CA), glucose was measured using the oxidase method and a Beckman analyzer, and HbA1c (A1C) was measured using a DCA 2000 analyzer (Bayer, Tarrytown, NY). FFA concentrations were determined by an enzymatic colorimetric method (Wako Chemicals, Nuess, Germany), and plasma IL-6 and TNF concentrations were measured using an enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN).
Muscle processing.
Muscle samples were homogenized as previously described (12). Briefly, muscle samples were weighted while still frozen and were homogenized in ice-cold lysis buffer (1:10, wt/vol) containing 50 mmol/l HEPES (pH 7.6), 150 mmol/l NaCl, 20 mmol/l sodium pyrophosphate, 20 mmol/l -glycerophosphate, 10 mmol/l sodium fluoride, 2 mmol/l sodium orthovanadate (Na3VO4), 2 mmol/l EDTA (pH 8.0), 1% Nonidet P-40, 10% glycerol, 1 mmol/l phenylmethylsulfonylfluoride, 1 mmol/l MgCl2, 1 mmol/l CaCl2, 10 e蘥/ml leupeptin, and 10 e蘥/ml aprotinin. Homogenates were incubated on ice for 20 min and then centrifuged at 15,000g for 20 min at 4°C. The supernatants were collected, and protein concentrations were measured by the Lowry method. Supernatants were stored at eC80°C until used.
Western blotting.
Muscle proteins (40 e蘥) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes, except for TNF, for which 150 e蘥 protein were used. After blocking in Tris-buffered saline with 5% nonfat dry milk, the membranes were incubated overnight at 4°C with antibodies against IB (Cell Signaling, Beverly, MA), IB (Santa Cruz Biotechnology, Santa Cruz, CA), NFB p50 (Cell Signaling), NFB p65 (Santa Cruz Biotechnology), IKK (Cell Signaling), phospho-IKK (Ser177/181) (Cell Signaling), and TNF (Santa Cruz Biotechnology) (14,15). For all these proteins, a 0.45-e蘭 nitrocellulose membrane was used, except for TNF, for which a 0.2-e蘭 membrane was used. Bound antibodies were detected with anti-rabbit immunoglobulin horseradish peroxidaseeClinked whole antibody and by using enhanced chemiluminescent reagents (PerkinElmer Life Science, Boston, MA). The membranes were exposed to film, and band intensity was quantified using Image Tool Software (University of Texas Health Science Center at San Antonio, San Antonio, TX).
Calculations.
During the postabsorptive state, steady-state conditions prevail and the rate of endogenous glucose appearance, which equals the rate of total body glucose uptake, is calculated as the 3-3H-glucose infusion rate (disintegrations per minute) divided by the steady-state plasma 3-3H-glucose specific activity (disintegrations per minute per milligram). During the insulin clamp, noneCsteady-state conditions prevail, and the rate of glucose production (Ra) is calculated using Steele’s equation (16). The rate of endogenous glucose production during the insulin clamp equals the tracer-derived Ra minus the exogenous rate of glucose infusion. The rate of whole-body glucose disposal (Rd) equals the rate of residual endogenous glucose production plus the exogenous glucose infusion rate adjusting to maintain euglycemia (90- to 120-min time period).
Statistical analysis.
All data are expressed as means ± SE. Differences between the control and diabetic groups were determined using the unpaired Student’s t test. Differences within a group, pre- versus posttraining, were determined using the paired t test. Correlation analysis was performed by the Pearson product-moment method. For all analyses, a P < 0.05 was considered to be statistically significant.
RESULTS
Table 1 summarizes the subject’s clinical and laboratory characteristics. There was no significant difference in age, body weight, and BMI between the type 2 diabetic and control subjects. Subjects with type 2 diabetes had higher fasting plasma glucose and insulin concentrations (P < 0.05), higher A1C (P < 0.05), and lower aerobic capacity (Vo2peak) (P < 0.05). There were no differences in FFA (690 ± 100 vs. 660 ± 100 e蘭ol/l), IL-6 (2.3 ± 0.5 vs. 2.3 ± 0.6 pg/ml), and TNF (1.0 ± 0.1 vs. 1.1 ± 0.2 pg/ml) plasma concentrations between the control and type 2 diabetic groups.
Exercise training.
Eight weeks of exercise training had no effect on body weight, BMI, or fasting plasma glucose and insulin concentrations in either group (Table 1). Plasma FFA, IL-6, and TNF concentrations did not change with training. However, physical training significantly increased the Vo2peakby 17% (P < 0.05) and 31% (P < 0.05) in the control and diabetic subjects, respectively. In the diabetic subjects, the increase in Vo2peak after training also was accompanied by a tendency for a 19% decrease in A1C (P = 0.05).
Euglycemic-hyperinsulinemic clamp studies and insulin sensitivity.
Basal endogenous glucose production was similar between groups, whereas insulin-mediated glucose disposal (Rd) was 95% higher in the control group (P < 0.05) (Table 1). Exercise training significantly increased Rd in the type 2 diabetic subjects by 37% (P < 0.05). In the control subjects, Rd tended to increase after training (P = 0.1). The smaller effect of training in control subjects is in part explained by a higher baseline Rd, resulting in a modest effect of training. Differences in sex distribution between groups did not explain the lesser effect of training on Rd observed in the control subjects (not shown).
IB/NFB signaling.
To determine whether skeletal muscle from subjects with type 2 diabetes has excessive activity of IB/NFB pathway, we measured IB and IB protein abundance. Because IB sequesters NFB in the cytoplasm and increases in IB abundance inversely correlate with NFB DNA binding in human skeletal muscle (17), a reduction in IB abundance is considered to indicate activation of the IB/NFB pathway. In skeletal muscle from type 2 diabetic subjects, IB was reduced by 60% compared with control subjects (P < 0.05) (Fig. 1A). IB was not significantly different in type 2 diabetic subjects (Fig. 1B). This reduction in IB suggests that there is excessive activity of the IB/NFB axis in skeletal muscle from type 2 diabetic subjects. Importantly, there was a positive correlation between IB and the rate of insulin-mediated glucose disposal (r = 0.63, P = 0.01) (Fig. 2A) and with the Vo2peak (r = 0.6, P = 0.02) (Fig. 2B). Consistent with these findings, there was a negative correlation between IB and the A1C concentration (r = eC0.5, P = 0.05) (Fig. 2C). There was also a positive correlation between insulin-mediated glucose disposal and the Vo2peak (r = 0.54, P = 0.04) (Fig. 2D). The predominant activating NFB dimer in muscle is p50-p65 (9,10). We measured NFB p50 and p65 protein in skeletal muscle and found that NFB p50 protein was decreased by 30% in type 2 diabetic versus control subjects (P < 0.05) (Fig. 3A). There were no differences in p65 protein between groups (Fig. 3B). In addition, there was a positive correlation between NFB p50 protein content and insulin-mediated glucose disposal (r = 0.58, P = 0.04) (Fig. 3C).
Effect of training on the IB/NFB pathway.
Because the IB/NFB pathway has been implicated in the cellular mechanisms responsible for skeletal muscle insulin resistance, we hypothesized that exercise training in patients with type 2 diabetes would reverse abnormalities in IB/NFB signaling and that this is a mechanism by which training improves insulin sensitivity. In control subjects, training caused a 50% increase in both IB (P < 0.05) and IB (P < 0.05) protein content (Fig. 4A and B). After training, NFB p50 protein tended to increase by 100% (P = 0.05) (Fig. 4C) in the control subjects but had no effect on NFB p65 (Fig. 4D). Moreover, in the muscle from type 2 diabetic subjects, physical training increased IB and IB protein by 98% (P < 0.05) and 185% (P < 0.05), respectively (Fig. 5A and B). Exercise training also increased NFB p50 in subjects with type 2 diabetes by 140% (P < 0.05) (Fig. 5C), but similar to the control subjects, exercise had no effect on NFB p65 (Fig. 5D). Furthermore, these increments in IB and NFB p50 protein were accompanied by a 37% increase in insulin-mediated glucose disposal (Table 1). These findings indicate that training can restore abnormalities in IB and NFB p50 content in muscle from subjects with type 2 diabetes.
IKK phosphorylation.
We measured IKK phosphorylation to further examine the mechanism responsible for the reduction in IB in the type 2 diabetic group. There was no difference in IKK phosphorylation between groups, and training had no effect on IKK phosphorylation (Fig. 6) or protein content (not shown).
TNF muscle content.
It has been shown that type 2 diabetes is associated with higher muscle expression of TNF, an NFB-regulated gene (18). Accordingly, in the type 2 diabetic subjects the reduction in IB was accompanied by a tendency for higher basal TNF protein content by 25% compared with the control subjects (P = NS) (Fig. 7), although we could only measure TNF muscle content in five control and four diabetic subjects because a large amount of muscle protein is required to measure TNF muscle content by Western blotting and muscle tissue was no longer available for mRNA expression analysis. It is possible that this trend would have reached statistical significance with a higher number of samples. Consistent with previous reports (19), the increases in IB and NFB p50 caused by training were associated with a reduction in TNF muscle content in both groups (P < 0.05) (Fig. 7).
DISCUSSION
In this study, we found that type 2 diabetic subjects have decreased IB muscle content, suggesting enhanced IB/NFB signaling. Importantly, there was a positive correlation between IB protein and insulin-mediated glucose disposal in type 2 diabetic and control subjects. These findings support the hypothesis that enhanced activation of the IB/NFB pathway is associated with insulin resistance in human skeletal muscle.
The mechanism responsible for the apparent increase in IB/NFB signaling in muscle from the type 2 diabetic subjects is not clear. One hypothesis involves lipid-induced activation of IB/NFB signaling. Intramyocellular lipid metabolites, particularly long-chain fatty acyl CoAs, diacylglycerol, and ceramides, have been shown to induce insulin resistance (20eC25). Moreover, skeletal muscle of insulin-resistant subjects is characterized by increases in fatty acyl CoA (26) and ceramides (27). Recently, it was shown that lipid-induced insulin resistance in L6 myotubes (28) and muscle from rodents (3,4,29) and humans (5) is associated with activation of the IB/NFB pathway. A role for this pathway in mediating insulin resistance is further strengthened by the findings that inhibition of IB/NFB signaling improves insulin sensitivity (4,11). In support of our findings, Bhatt et al. (29) recently reported that diet-induced obesity in rats, an intervention that causes insulin resistance, lead to a decrease in muscle IB content. Interestingly, this group found that differences in IB muscle content between muscle fiber types were not explained by muscle triglyceride content (29), suggesting that intramyocellular lipids were not responsible for the reduction in IB. However, metabolites of triglycerides and fatty acids (i.e., fatty acyl CoAs, diacylglycerol, and ceramides), and not triglycerides per se, are believed to be responsible for the insulin resistance. We were unable to measure fatty acyl CoA, ceramide, or diacylglycerol muscle content because of the limited amount of tissue that can be obtained in humans using the percutaneous biopsy technique. Infusion of FFAs to humans (5) and rats (29) has been associated with activation of the IkB/NFkB pathway. In the present study, fasting plasma FFA concentrations were similar between groups. It should be noted, however, that fasting FFA levels do not reflect FFA turnover and day-long plasma FFA concentrations. Reaven et al. (30) studied type 2 diabetic subjects with similar blood glucose concentrations compared with the subjects included in the present study and found that the type 2 diabetic subjects had similar fasting FFA plasma levels compared with the nondiabetic control subjects, yet mean 24-h plasma FFA concentrations were significantly elevated.
Type 2 diabetes is characterized by low-grade chronic inflammation (1,31,32), and cytokines, including TNF and IL-6, have been shown to activate the IB/NFB pathway. TNF and IL-6 plasma concentrations were similar between groups. Therefore, it seems unlikely that these cytokines were responsible for the decrease in muscle IB. Nonetheless, there are many other exogenous inflammatory stimuli that were not measured in this study and could have caused IB/NFB axis activation. Reactive oxygen species are also known to simulate the IB/NFB pathway (33eC35), and hyperglycemia-induced reactive oxygen species generation could have contributed to the lower IB seen in the type 2 diabetic group. Consistent with our findings, Bandyopadhyay et al. (36) did not find increased IKK phosphorylation in muscle from insulin-resistant subjects. Because IB phosphorylation by IKK leads to IB ubiquination and subsequent degradation, the decrease in IB content would then have to be explained by IKK-independent phosphorylation. Protein kinase C and eIF2 kinase can also associate with and phosphorylate IB (37).
How excessive activity of the IB/NFB pathway leads to muscle insulin resistance has not been fully elucidated. Insulin infusion increased insulin receptor substrate-1eCassociated phosphoinositide 3-kinase activity by 33% (P < 0.05) in control subjects but not in type 2 diabetic subjects (12). One potential mechanism for these decreases in insulin-stimulated glucose disposal and phosphoinositide 3-kinase activity in the diabetic group involves insulin receptor substrate-1 serine phosphorylation by IKK (38eC40), but, as mentioned above, IKK phosphorylation was unchanged. Moreover, training-induced improvements in insulin sensitivity were not explained by changes in IKK phosphorylation or phosphoinositide 3-kinase activity. In insulin-resistant (lipid-treated) L6 muscle cells, inhibition of NFB using a selective inhibitory peptide improved insulin-stimulated glucose transport (28). This indicates that NFB, and genes that this transcription factor controls, may directly influence insulin sensitivity. Yet, overactivation of NFB in transgenic mice was not associated with insulin resistance (41). Because the phenotype of these animals was characterized by severe muscle wasting, it is unclear how the muscle wasting influenced insulin sensitivity measurements. It is also possible that lifelong overactivation of NFB could lead to compensatory mechanisms aimed at normalizing insulin sensitivity. Further studies will help clarify how the IB/NFB pathway modulates insulin sensitivity in humans.
Few studies have examined the effect of exercise on the IB/NFB pathway. In rats, acute exercise activates IB/NFB signaling in muscle (42,43), while acute fatiguing exercise in humans reduces NFB activity (44). In the present study, we found that training increased IB and reduced TNF muscle content, suggesting decreased IB/NFB signaling. This increase in IB was not associated with changes in plasma TNF, IL-6, adiponectin (not shown), or FFA concentrations. Stimuli intrinsic to muscle could also be responsible for the training-induced changes in IB and TNF. Training significantly increased AMP-activated protein kinase (AMPK) phosphorylation in both control and type 2 diabetic groups (N.M., L.J.M., unpublished observations). In endothelial cells, chemical AMPK activation with 5-aminoimidazole-4-carboxamide ribonucleoside has been shown to inhibit NFB activity (45). These results suggest that exercise-stimulated AMPK activation may be responsible for the inhibition of the IB/NFB axis. In contrast, AMPK activation with 5-aminoimidazole-4-carboxamide ribonucleoside had no effect on NFB activity in rat skeletal muscle (43), arguing against a role for AMPK as a regulator of IB/NFB signaling. A reduction in intramyocellular lipid content caused by training could help explain the changes in IB and TNF. However, several studies have shown that trained athletes have increased triglyceride muscle content (46,47). The greater triglyceride storage in the trained athlete represents an adaptive response to training and provides a readily available source of energy for the contracting muscle. In contrast, elevated lipid stores in type 2 diabetes result from an imbalance between plasma FFA availability and oxidation and have been implicated in the development of insulin resistance. Future studies will be needed to examine whether elevated levels of fatty acyl CoA, diacylglycerol, and ceramides are related with abnormalities in IB/NFB signaling and to assess the effect of training on the content of these metabolites.
Type 2 diabetic subjects had a more pronounced reduction in IB content compared with IB. While the IB and IB isoforms share many similarities, they also display differences. The IB gene has a B recognition sequence in its promoter region (48). Thus, activation of NFB can induce resynthesis of IB. In contrast, NFB does not induce IB synthesis (8,49). Since the muscle of type 2 diabetic subjects appears to have excessive NFB activity, this could lead to selective resynthesis of IB but not of IB. Although it is not known whether IB remains suppressed upon long-term NFB activation, in Jurkat cells cytokine-induce IB degradation is rapidly restored within 2 h, whereas IB remains low for at least 24 h (50). Whether this differential regulation between IB and IB occurs in other chronic diseases remains to be determined.
We found that NFB p50 subunit content was reduced in muscle of type 2 diabetic subjects, and this decrease correlated with reduced insulin sensitivity. This might appear counterintuitive since NFB stimulates transcription of inflammatory genes. While the p50-p65 heterodimer is the predominant activating NFB dimer, the p50 subunit also can dimerize with another p50 subunit to form p50-p50 homodimers. While the p50-p65 heterodimer activates transcription of inflammatory genes, the p50-p50 homodimer inhibits gene transcription (9,51eC53). In view of our findings of reduced p50, one might speculate that type 2 diabetic subjects could have decreased abundance of the p50-p50 repressor homodimers, resulting in excessive NFB p50-p65 activity, and that training inhibited NFB activity by increasing p50-p50 homodimers. Future investigations are needed to determine whether NFB p50-p50 DNA binding is indeed reduced in muscle from type 2 diabetic subjects.
The present study shows an important association between insulin sensitivity and the content of IB and NFB p50 in human muscle. Yet, these results do not prove causality. Studies using genetic and pharmacologic approaches to manipulate IB/NFB signaling will help establish the role of this pathway in insulin sensitivity regulation.
ACKNOWLEDGMENTS
This work was supported by grants DK47936 (to L.J.M.), DK66483 (to L.J.M.), and DK24092 (to R.A.D.) from the National Institutes of Health; Grant RR01346 (GCRC, Audie L. Murphy Veterans Hospital); an Executive Research Committee Research Award from the University of Texas Health Science Center at San Antonio (to N.M.); a VA Merit Award (to R.A.D.); an American Diabetes Association Junior Faculty Award (to N.M.); and a grant from the Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand (to A.S.).
We also thank all the study participants.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
REFERENCES
Evans JL, Goldfine ID, Maddux BA, Grodsky GM: Oxidative stress and stress-activated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocr Rev 23:599eC622, 2002
Garg R, Tripathy D, Dandona P: Insulin resistance as a proinflammatory state: mechanisms, mediators, and therapeutic interventions. Curr Drug Targets 4:487eC492, 2003
Kim JK, Kim YJ, Fillmore JJ, Chen Y, Moore I, Lee J, Yuan M, Li ZW, Karin M, Perret P, Shoelson SE, Shulman GI: Prevention of fat-induced insulin resistance by salicylate. J Clin Invest 108:437eC446, 2001
Yuan M, Konstantopoulos N, Lee J, Hansen L, Li ZW, Karin M, Shoelson SE: Reversal of obesity- and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta. Science 293:1673eC1677, 2001
Itani SI, Ruderman NB, Schmieder F, Boden G: Lipid-induced insulin resistance in human muscle is associated with changes in diacylglycerol, protein kinase C, and IB-. Diabetes 51:2005eC2011, 2002
Shoelson SE, Lee J, Yuan M: Inflammation and the IKK beta/I kappa B/NF-kappa B axis in obesity- and diet-induced insulin resistance. Int J Obes Relat Metab Disord 27 (Suppl. 3):S49eCS52, 2003
Ghanim H, Garg R, Aljada A, Mohanty P, Kumbkarni Y, Assian E, Hamouda W, Dandona P: Suppression of nuclear factor-kappaB and stimulation of inhibitor kappaB by troglitazone: evidence for an anti-inflammatory effect and a potential antiatherosclerotic effect in the obese. J Clin Endocrinol Metab 86:1306eC1312, 2001
Barnes PJ, Karin M: Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med 336:1066eC1071, 1997
Yamamoto Y, Gaynor RB: IkappaB kinases: key regulators of the NF-kappaB pathway. Trends Biochem Sci 29:72eC79, 2004
Jackman RW, Kandarian SC: The molecular basis of skeletal muscle atrophy. Am J Physiol Cell Physiol 287:C834eCC843, 2004
Hundal RS, Petersen KF, Mayerson AB, Randhawa PS, Inzucchi S, Shoelson SE, Shulman GI: Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J Clin Invest 109:1321eC1326, 2002
Christ-Roberts CY, Pratipanawatr T, Pratipanawatr W, Berria R, Belfort R, Kashyap S, Mandarino LJ: Exercise training increases glycogen synthase activity and GLUT4 expression but not insulin signaling in overweight nondiabetic and type 2 diabetic subjects. Metabolism 53:1233eC1242, 2004
Cusi K, Maezono K, Osman A, Pendergrass M, Patti ME, Pratipanawatr T, DeFronzo RA, Kahn CR, Mandarino LJ: Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle. J Clin Invest 105:311eC320, 2000
Gardiner TA, Gibson DS, de Gooyer TE, de la Cruz VF, McDonald DM, Stitt AW: Inhibition of tumor necrosis factor-alpha improves physiological angiogenesis and reduces pathological neovascularization in ischemic retinopathy. Am J Pathol 166:637eC644, 2005
Raina N, Jeejeebhoy KN: Effect of low-protein diet and protein supplementation on the expressions of TNF-alpha, TNFR-I, and TNFR-II in organs and muscle of LPS-injected rats. Am J Physiol Endocrinol Metab 286:E481eCE487, 2004
Steele R: Influences of glucose loading and of injected insulin on hepatic glucose output. Ann N Y Acad Sci 82:420eC430, 1959
Agusti C, Rano A, Rovira M, Filella X, Benito N, Moreno A, Torres A: Inflammatory response associated with pulmonary complications in non-HIV immunocompromised patients. Thorax 59:1081eC1088, 2004
Saghizadeh M, Ong JM, Garvey WT, Henry RR, Kern PA: The expression of TNF alpha by human muscle: relationship to insulin resistance. J Clin Invest 97:1111eC1116, 1996
Greiwe JS, Cheng B, Rubin DC, Yarasheski KE, Semenkovich CF: Resistance exercise decreases skeletal muscle tumor necrosis factor alpha in frail elderly humans. FASEB J 15:475eC482, 2001
Shulman GI: Cellular mechanisms of insulin resistance. J Clin Invest 106:171eC176, 2000
Ellis BA, Poynten A, Lowy AJ, Furler SM, Chisholm DJ, Kraegen EW, Cooney GJ: Long-chain acyl-CoA esters as indicators of lipid metabolism and insulin sensitivity in rat and human muscle. Am J Physiol Endocrinol Metab 279:E554eCE560, 2000
Hajduch E, Balendran A, Batty IH, Litherland GJ, Blair AS, Downes CP, Hundal HS: Ceramide impairs the insulin-dependent membrane recruitment of protein kinase B leading to a loss in downstream signalling in L6 skeletal muscle cells. Diabetologia 44:173eC183, 2001
Cooney GJ, Thompson AL, Furler SM, Ye J, Kraegen EW: Muscle long-chain acyl CoA esters and insulin resistance. Ann N Y Acad Sci 967:196eC207, 2002
Schmitz-Peiffer C, Craig DL, Biden TJ: Ceramide generation is sufficient to account for the inhibition of the insulin-stimulated PKB pathway in C2C12 skeletal muscle cells pretreated with palmitate. J Biol Chem 274:24202eC24210, 1999
Schubert KM, Scheid MP, Duronio V: Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine 473. J Biol Chem 275:13330eC13335, 2000
Hulver MW, Berggren JR, Cortright RN, Dudek RW, Thompson RP, Pories WJ, MacDonald KG, Cline GW, Shulman GI, Dohm GL, Houmard JA: Skeletal muscle lipid metabolism with obesity. Am J Physiol Endocrinol Metab 284:E741eCE747, 2003
Adams JM, 2nd, Pratipanawatr T, Berria R, Wang E, DeFronzo RA, Sullards MC, Mandarino LJ: Ceramide content is increased in skeletal muscle from obese insulin-resistant humans. Diabetes 53:25eC31, 2004
Sinha S, Perdomo G, Brown NF, O’Doherty RM: Fatty acid-induced insulin resistance in L6 myotubes is prevented by inhibition of activation and nuclear localization of nuclear factor kappa B. J Biol Chem 279:41294eC41301, 2004
Bhatt BA, Dube JJ, Dedousis N, Reider JA, O’Doherty RM: Diet-induced obesity and acute hyperlipidemia reduce IkappaBalpha levels in rat skeletal muscle in a fiber-type dependent manner. Am J Physiol Regul Integr Comp Physiol 290:R233eCR240, 2006
Reaven GM, Hollenbeck C, Jeng CY, Wu MS, Chen YD: Measurement of plasma glucose, free fatty acid, lactate, and insulin for 24 h in patients with NIDDM. Diabetes 37:1020eC1024, 1988
Pickup JC, Crook MA: Is type II diabetes mellitus a disease of the innate immune system Diabetologia 41:1241eC1248, 1998
Dandona P: The link between insulin resistance syndrome and inflammatory markers. Endocr Pract 9 (Suppl. 2):53eC57, 2003
Mercurio F, Manning AM: NF-kappaB as a primary regulator of the stress response. Oncogene 18:6163eC6171, 1999
Droge W: Free radicals in the physiological control of cell function. Physiol Rev 82:47eC95, 2002
Dalton TP, Shertzer HG, Puga A: Regulation of gene expression by reactive oxygen. Annu Rev Pharmacol Toxicol 39:67eC101, 1999
Bandyopadhyay GK, Yu JG, Ofrecio J, Olefsky JM: Increased p85/55/50 expression and decreased phosphotidylinositol 3-kinase activity in insulin-resistant human skeletal muscle. Diabetes 54:2351eC2359, 2005
Ghosh S, Baltimore D: Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B. Nature 344:678eC682, 1990
Gao Z, Zuberi A, Quon MJ, Dong Z, Ye J: Aspirin inhibits serine phosphorylation of insulin receptor substrate 1 in tumor necrosis factor-treated cells through targeting multiple serine kinases. J Biol Chem 278:24944eC24950, 2003
Gao Z, Hwang D, Bataille F, Lefevre M, York D, Quon MJ, Ye J: Serine phosphorylation of insulin receptor substrate 1 by inhibitor kappa B kinase complex. J Biol Chem 277:48115eC48121, 2002
de Alvaro C, Teruel T, Hernandez R, Lorenzo M: Tumor necrosis factor alpha produces insulin resistance in skeletal muscle by activation of inhibitor kappaB kinase in a p38 MAPK-dependent manner. J Biol Chem 279:17070eC17078, 2004
Cai D, Frantz JD, Tawa NE, Jr, Melendez PA, Oh BC, Lidov HG, Hasselgren PO, Frontera WR, Lee J, Glass DJ, Shoelson SE: IKKbeta/NF-kappaB activation causes severe muscle wasting in mice. Cell 119:285eC298, 2004
Ji LL, Gomez-Cabrera MC, Steinhafel N, Vina J: Acute exercise activates nuclear factor (NF)-kappaB signaling pathway in rat skeletal muscle. FASEB J 18:1499eC1506, 2004
Ho RC, Hirshman MF, Li Y, Cai D, Farmer JR, Aschenbach WG, Witczak CA, Shoelson SE, Goodyear LJ: Regulation of IkappaB kinase and NF-kappaB in contracting adult rat skeletal muscle. Am J Physiol Cell Physiol 289:C794eCC801, 2005
Durham WJ, Li YP, Gerken E, Farid M, Arbogast S, Wolfe RR, Reid MB: Fatiguing exercise reduces DNA binding activity of NF-kappaB in skeletal muscle nuclei. J Appl Physiol 97:1740eC1745, 2004
Cacicedo JM, Yagihashi N, Keaney JF Jr, Ruderman NB, Ido Y: AMPK inhibits fatty acid-induced increases in NF-kappaB transactivation in cultured human umbilical vein endothelial cells. Biochem Biophys Res Commun 324:1204eC1209, 2004
van Loon LJ: Use of intramuscular triacylglycerol as a substrate source during exercise in humans. J Appl Physiol 97:1170eC1187, 2004
Goodpaster BH, He J, Watkins S, Kelley DE: Skeletal muscle lipid content and insulin resistance: evidence for a paradox in endurance-trained athletes. J Clin Endocrinol Metab 86:5755eC5761, 2001
Ito CY, Kazantsev AG, Baldwin AS Jr: Three NF-kappa B sites in the I kappa B-alpha promoter are required for induction of gene expression by TNF alpha. Nucleic Acid Res 22:3787eC3792, 1994
Karin M, Delhase M: The I kappa B kinase (IKK) and NF-kappa B: key elements of proinflammatory signalling. Semin Immunol 12:85eC98, 2000
Thompson JE, Phillips RJ, Erdjument-Bromage H, Tempst P, Ghosh S: I kappa B-beta regulates the persistent response in a biphasic activation of NF-kappa B. Cell 80:573eC582, 1995
Udalova IA, Richardson A, Denys A, Smith C, Ackerman H, Foxwell B, Kwiatkowski D: Functional consequences of a polymorphism affecting NF-kappaB p50-p50 binding to the TNF promoter region. Mol Cell Biol 20:9113eC9119, 2000
Driessler F, Venstrom K, Sabat R, Asadullah K, Schottelius AJ: Molecular mechanisms of interleukin-10-mediated inhibition of NF-kappaB activity: a role for p50. Clin Exp Immunol 135:64eC73, 2004
Tong X, Yin L, Washington R, Rosenberg DW, Giardina C: The p50-p50 NF-kappaB complex as a stimulus-specific repressor of gene activation. Mol Cell Biochem 265:171eC183, 2004(Reduced Skeletal Muscle I)