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Renal Division (X.W., J.H.), Department of Medicine, Emory University, Atlanta, Georgia 30322; and Nephrology Division (Z.H., J.D., W.E.M.), Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Xiaonan Wang, M.D., Renal Division, WMB 338, Emory University School of Medicine, M/S 1930/001/1AG, 1639 Pierce Drive, Atlanta, Georgia 30322. E-mail: xwang03{at}emory.edu.
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Introduction |
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and possibly other cytokines can cause insulin resistance (9, 10). Another potential proteolytic trigger of muscle protein breakdown is a decrease in the responses to insulin or IGF-I. For example, there is evidence that insulin deficiency causes muscle protein breakdown by activating the UPP in processes that include transcription of genes encoding subunits of this system (11, 12). This is relevant because catabolic conditions that stimulate muscle protein degradation by the UPP such as aging, acidosis, chronic kidney disease (CKD), or acidosis are often associated with insulin resistance (13, 14, 15, 16). The presence of these complicating factors raises the question of whether insulin resistance by itself will stimulate protein metabolism and, if so, by what mechanisms. In a model of insulin deficiency, we showed that there is accelerated muscle proteolysis and that this is caused by a decrease in the activity of phosphatidylinositol 3-kinase (PI3K) (17, 18). We found that a decrease in PI3K activity reduces the level of phosphorylated Akt (pAkt), and a low pAkt has been shown to relieve the inhibition of the expression of specific E3 ubiquitin-conjugating enzymes atrogin-1/MAFbx and MuRF1 in muscle (17, 19, 20). This is relevant because expression of these E3 enzymes occurs in several conditions causing loss of lean body mass, suggesting there is a complex genetic program associated with activation of muscle protein degradation (21). Besides stimulating activity of the UPP, we found that a decrease in muscle PI3K activity also activates Bax, which stimulates the activity of caspase-3 leading to muscle protein loss by providing substrates for the UPP (17, 18).
In the present study, we explored the possibility that insulin resistance would cause muscle atrophy and examined potential proteolytic pathways that could cause accelerated loss of muscle protein. We took advantage of the well-established characteristics of db/db mice to test this possibility because kidney failure, systemic sepsis, etc. do not complicate this genetic model of insulin resistance. Thus, db/db mice provided us an opportunity to determine whether insulin resistance alone can stimulate the UPP and muscle protein degradation.
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Materials and Methods |
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At the end of different experimental manipulations, db/db and control mice were anesthetized with 12 mg/kg xylazine and 60 mg/kg ketamine, and the soleus, the extensor digitorum longus (EDL), and plantaris muscles were removed to measure protein degradation; gastrocnemius muscles were also removed, plunged into liquid nitrogen, and stored at –80 C.
Plasma insulin was measured using 1–2-3 ultrasensitive mouse insulin enzyme immunoassay (EIA) kit (American Lab Products, Windham, NH) by the Biochemistry Core Laboratory of Emory University. Blood glucose concentration was measured by the Accu-CHEK advantage blood glucose meter (Indianapolis, IN). Adiponectin concentration in serum was measured using a mouse adiponectin ELISA kit (American Lab Products).
Protein degradation
Protein degradation was measured as the rate of tyrosine release into the media because muscle neither synthesizes nor degrades tyrosine and it does not accumulate in the intracellular pool (22, 23). Soleus, EDL, and plantaris muscles were pinned to plastic supports to maintain muscles at resting length. They were incubated in standard Krebs-Henseleit bicarbonate buffer containing 10 mM glucose and 0.5 mM cycloheximide (to block tyrosine reuse), placed in individual flasks, and gassed with 95% O2/5% CO2 for 3 min. After an initial 30-min preincubation, each muscle was transferred to a flask containing fresh media, regassed with 95% O2/5% CO2 for 3 min, and incubated at 37 C for 2 h. Tyrosine in the media was measured (5, 22).
Immunoblotting
Gastrocnemius muscles were homogenized in RIPA buffer except when we measured the 14-kDa actin fragment arising from the activity of caspase-3 (18). To measure this actin fragment, muscles were harvested and homogenized in hypotonic buffer and protein concentration was measured using a PC protein assay kit (Bio-Rad, Hercules, CA). The levels of signaling proteins and the 14-kDa actin fragment were detected by standard Western blotting (17, 18). We used the following primary antibodies: an antiactin antibody (Sigma-Aldrich, St. Louis, MO), an anti-IRS-1 antibody (Upstate, Lake Placid, NY), and Cell Signaling (Danvers, MA) antibodies against phospho-Ser307 IRS-1, Akt, pAkt, and the forkhead transcription factors, FoxO1 and pFoxO1.
Muscle histology
To assess differences in the cross-sectional area of the plantaris muscle, we embedded them in TBS tissue freezing media (Fisher, Pittsburgh, PA) in isopentane cooled in dry ice. Cross-sections (10 µm) on gelatin-coated slides were treated with an anti-laminin antibody (Sigma-Aldrich), and the area of at least 500 individual myofibers per muscle was measured using the Micro-Suite Five Biological System (Olympus, Melville, NY).
Proteasome activity
To measure proteasome chymotryptic-like peptidase activity in vitro, gastrocnemius muscles were homogenized in a harvest buffer [50 µM Tris-HCl (pH 7.4), 5 mM MgCl2, 250 mM sucrose, 2 mM ATP, and 1 mM dithiothreitol]. The mixture was centrifuged (5 min at 400 x g) and clarified by sequential centrifugations of 10,000 x g for 20 min before centrifuging at 100,000 x g for 5 h to isolate the 20S and 26S proteasomes (24). After resuspension, proteasome chymotryptic-like activity was determined as the release of 7-amino-4-methylcoumarin (AMC) from the fluorogenic peptide substrate LLVY-AMC (N-Suc-Leu-Leu-Val-Tyr-AMC) using the Proteasome Activity Assay Kit (Chemicon International, Temecula, CA).
Cell culture and RT-PCR
Mouse 3T3-L1 preadipocytes, purchased from American Type Culture Collection (Manassas, VA) were studied between passages 3 and 10. Initially, they were grown in standard media containing DMEM (Invitrogen, Carlsbad, CA) containing 10% bovine calf serum (HyClone, Logan, UT), penicillin (200 U/ml), and streptomycin (200 µg/ml) in a humidified atmosphere (95% O2/5% CO2). After 2 d, cells were switched to differentiation media with 1 µM dexamethasone, 10 µg/ml insulin, and 0.5 mM 3-methyl-1-isobutylxanthine (Sigma-Aldrich) for 2 d and then grown in post-differentiation medium containing 10 µg/ml insulin for 5 d. Before being studied, cells were shown to be maximally differentiated morphologically (>90% of cells). The cells were then placed in standard media for another 2 d. Subsequently, we determined the influence of exposing cells to 10 µM rosiglitazone (Cayman Chemical, Ann Arbor, MI) for 16 h.
For measuring RT-PCR, we isolated total RNA from mouse muscles and from differentiated 3T3-L1 cells using Trizol reagent (Invitrogen). RT was performed using the GeneAmp RNA PCR Core kit (Applied Biosystems, Foster City, CA) and 2 µg denatured RNA according to the manufacturer’s instructions. Primers for specific genes were designed to cross intron-exon boundaries and used to generate amplicons in their linear ranges as follows: adiponectin (U37222; 532-bp product), forward, 5'-GATTCAACTTGCGCCTCATCTTAGGC-3', and reverse, 5'-CCGACGTTACTACAACTGAAGAGC-3'; TNF (NM_11731; 212-bp product), forward, 5'-TCTCATCAGTTCTATGGCCC-3', and reverse, 5'-GGGAGTAGACAAGGTACAAC-3'; and IL-6 (NM_031168; 343-bp product), forward, 5'-GCCAGAGTCCTTCAGAGAGATACAG-3', and reverse, 5'-CCCAACGATTCATATTGTCAG-3'. For each sample, 18S rRNA was used as an internal control using QuantumRNA 18S primers (Ambion, Austin, TX).
Real-time PCR
Total RNA was extracted using the Purelink RNA purification kit (Invitrogen) and 2 µg RNA sample was used for RT by oligo 9-mer primers and Superscript II (Invitrogen). Real-time PCR was performed with SYBR Green PCR reagents (Bio-Rad, Hercules, CA) and the Opticon DNA Engine (Bio-Rad) using the following cycle parameters: 94 C for 2 min and 40 cycles at 94 C for 15 sec, 55 C for 30 sec, and 72 C for 30 sec with a final extension at 72 C for 10 min. Primers used in this study were mouse atrogin-1/MAFbx, forward, 5'-GCAGAGAGTCGGCAAGTC-3', and reverse, 5'-CAGGTCGGTGAT CGTGAG-3'; mouse MuRF-1, forward, 5'-CAACCTGTGCCGCAAGTG-3', and reverse, 5'-CAACCTCGT GCCTACAAGATG-3'; and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward, 5'-ACCCCCAATGTAT CCGTTGT-3', and reverse, 5'-TACTCCTTGGAGGCCATGTA-3'. The threshold cycle (Ct) is defined as the number of cycles required for the fluorescence signal to exceed the detection threshold. mRNA expression was standardized to the GAPDH gene, and expression was calculated as the difference between the threshold values of the two genes (2 – 
Ct). Melting curve analysis was always performed during real-time quantitative PCR to analyze and verify the specificity of the reaction. The values are given as the means ± SE of three independent experiments.
Statistical analysis
Results are presented as mean ± SE. Comparisons of results between groups were by ANOVA using Systat Software ANOVA, and P < 0.05 was considered significant.
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), but after 9 wk, their weight was 65% greater (n = 12; P < 0.0001). At 9 wk of age, the blood sugar of db/db mice was 3.27-fold higher and plasma insulin levels were 15.5-fold higher than values in control mice (both n = 12; P < 0.0001).
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; n = 9; P < 0.05). Likewise, the ratio between the muscle weight and body weight in db/db mice was 50% below that of control mice (P < 0.001). The lower muscle mass of db/db mice at 70 d is shown in Fig. 1.
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, the cross-sectional area of muscle fibers in db/db mice (1407.7 ± 61.5 µm2) was significantly smaller than that in WT mice (2194.8 ± 106.3 µm2; P < 0.001; n = 9).
Muscle atrophy in db/db mice is associated with increased protein degradation
The rate of protein degradation in the soleus muscle of db/db mice was 28.1% higher than the rate measured in soleus muscles of control mice. Protein degradation in EDL muscles of db/db mice was 43.5% higher and the rate in plantaris muscles was 34.1% higher than that in muscle of control mice (Fig. 2A; n = 9; P < 0.05).
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; P < 0.05; n = 6).
Caspase-3 acts as an initial protease to cleave actomyosin/myofibrils, forming substrates that are rapidly degraded by the UPP (18). Activated caspase-3 in muscle also leaves a characteristic 14-kDa actin fragment as a footprint. The density of the 14-kDa actin fragment in gastrocnemius muscles of db/db mice was 2.1-fold higher, indicating that caspase-3 had cleaved actomyosin/myofibrils (Fig. 2C).
PI3K/Akt cellular signaling is impaired in muscle of db/db mice
IRS-1-associated PI3K activity was significantly depressed in muscle of db/db mice (Fig. 3A). The decrease in PI3K activity was accompanied by an increase in phosphorylation of IRS-1 at serine 307 (Fig. 3B). This change in serine phosphorylation has been linked to decreased insulin responses because of its interference with insulin receptor signaling (25, 26). The lower PI3K activity was also associated with a decrease in pAkt (Fig. 3B).
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). However, the weight gain was unaffected.
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). These improvements in the PI3K/Akt pathway were associated with a decrease in the rate of protein degradation measured in isolated muscles (Fig. 6A). There also was a decrease in the proteolytic activity of the proteasome; the level in muscle of db/db mice was not significantly different from that measured in proteasomes isolated from control mice (Fig. 6B).
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).
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). Administration of rosiglitazone for 35 d resulted in an increase in plasma adiponectin levels in both db/db and control mice. Because the balance between adiponectin and cytokines is associated with insulin resistance (32), we treated differentiated 3T3-L1 cells with rosiglitazone for 16 h and measured mRNAs of adiponectin, TNF, and IL-6. As shown in Fig. 8, rosiglitazone increased adiponectin mRNA by 31% but decreased the mRNAs of the inflammatory cytokines TNF and IL-6 by 34 and 20%, respectively. The improvement in the high rate of muscle proteolysis was accompanied by an increase in the muscle content of pFoxO1, a decrease in FoxO1, and a decrease in the expression of the E3 ubiquitin-conjugating enzymes atrogin-1/MAFbx and MuRF1 (Fig. 9).
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Discussion |
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The model of insulin resistance we studied, db/db mice, did not have complicating illnesses or differences in the duration of diabetes, exercise, etc., but their muscles weighed less than those of the control mice and there was an increase in protein degradation (Figs. 1 and 2). There also were abnormalities in the cellular signaling pathway that stimulates muscle protein breakdown (17, 19, 20). Specifically, we found an increase in phosphorylation of serine 307 of IRS-1, a decrease in the activity of PI3K, and a decrease in pAkt (Fig. 3). These changes were associated with evidence of increased proteolytic activities of both caspase-3 and the UPP (Figs. 2 and 9). Although these changes in PI3K/Akt signaling are consistent with abnormalities we have found to cause accelerated muscle atrophy in streptozotocin-treated rats (17), there are differences in the two models. First, the level of phosphorylated serine 307 of IRS-1 was much higher in muscle of db/db mice. Second, potential toxicity from streptozotocin was not present in db/db mice (41). Because db/db mice are routinely used as a model of type 2 diabetes, our results seem physiologically relevant. Finally, our results indicate there is a common set of abnormalities of the PI3K/Akt pathway that leads to muscle wasting.
Our results demonstrate a cause-effect relationship between insulin resistance and the stimulation of muscle protein degradation. Specifically, we found that with rosiglitazone, blood glucose and plasma insulin levels fell sharply, indicating an improvement in insulin resistance. Concomitantly, the abnormalities in cellular signaling were corrected and proteasome proteolytic activity decreased (Figs. 5 and 6). However, there was only partial recovery of the cross-sectional area and mass of muscle. Possibly, the recovery was not complete because rosiglitazone did not stimulate protein synthesis. Indeed, it has been reported that changes in protein synthesis and degradation in response to changes in amino acids can differ even though both processes act through a common cell signaling pathway (42).
Mechanisms that could cause the abnormalities in cellular signaling leading to accelerated muscle protein degradation include increased glucocorticoid production and a decrease in the adiponectin level. An increase in glucocorticoids can stimulate the expression of atrogin-1/MAFbx and MuRF1 by increasing phosphorylation of the forkhead transcription factor (17, 19, 20). Moreover, we have shown that both a high physiological amount of glucocorticoids and either insulin deficiency or acidosis are required to activate the UPP and protein degradation in muscle of adrenalectomized rats; the same dose of glucocorticoids does not cause these abnormalities in rats without acidosis or insulin deficiency (12, 28). In the present study, we found that glucocorticoid production by db/db mice was high and that rosiglitazone eliminated this response. In addition, rosiglitazone reduced the activation of FoxO1 and the expression of atrogin-1/MAFbx and MuRF1 (Fig. 9). These results are consistent with the suppression of muscle protein degradation.
Regarding adiponectin, low levels have been shown to be closely linked to the development of insulin resistance via a mechanism involving enhanced cytokine production by adipocytes (32). In the present study, we showed that rosiglitazone increased plasma adiponectin levels (Fig. 7B) and decreased the expression of the mRNA levels of the inflammatory cytokines TNF and IL-6 in adipocytes (Fig. 8). From these results, we cannot determine the contribution of inflammatory cytokines to insulin resistance, but others have shown that these cytokines are linked to the development of insulin resistance via phosphorylation of serine 307 of IRS-1 (9, 10).
In conclusion, we find that insulin resistance is associated with accelerated muscle protein degradation in studies of a model of insulin resistance that is uncomplicated by kidney failure, sepsis, etc. The cellular signaling mechanism that links insulin resistance directly to protein degradation in muscle is also present in other conditions such as kidney failure. Consequently, the increase in muscle wasting that occurs in conditions associated with insulin resistance could arise in part from defects in cellular signaling caused by insulin resistance.
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Footnotes |
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First Published Online June 15, 2006
Abbreviations: AMC, 7-Amino-4-methylcoumarin; CKD, chronic kidney disease; EDL, extensor digitorum longus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pAkt, phosphorylated Akt; PI3K, phosphatidylinositol 3 kinase; UPP, ubiquitin-proteasome pathway.
Received February 23, 2006.
Accepted for publication June 2, 2006.
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- and obesity-induced insulin resistance. Science 271:665–668[Abstract] stimulate phosphorylation of IRS-1 at inhibitory Ser307 via distinct pathways. J Clin Invest 107:181–189[CrossRef][Medline]This article has been cited by other articles:
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