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Unite de Diabetologie et Nutrition, Universite Catholique de Louvain (M.D., R.V.B., N.P., P.L., J.V., J.-M.K., J.-P.T.), B-1200 Brussels, Belgium; and Department of Pediatrics, University of North Carolina (L.U.), Chapel Hill, North Carolina 27599-7039
Address all correspondence and requests for reprints to: Dr. Jean-Paul Thissen, Unite de Diabetologie et Nutrition, Universite Catholique de Louvain, 54 avenue Hippocrate, B-1200 Brussels, Belgium. E-mail: thissen{at}diab.ucl.ac.be.
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Introduction |
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Materials and Methods |
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Animals
Five-week-old male Wistar rats (150–170 g) were obtained from Janvier Breeding (Le Genest-St. Isle, France). They were housed in cages at 22 C under controlled lighting. Animals had free access to laboratory chow and water. The experiments were conducted and the animals were cared for in accordance with the directives of the institutional animal care and use committee of University of Louvain.
Experimental design
Fasting.
After a 7-d adaptation period, animals were allocated to five groups of eight rats each: one group was fed ad libitum (Ctrl group), a second group was fasted for 51 h (Fasted group), and the three other groups were fasted for 51 h and injected with 125, 250, or 375 µg IGF-I/100 g body weight·d (Fasted + IGF-I 125, 250, or 375 µg). IGF-I-treated animals received the first IGF-I injection 12 h before food withdrawal and subsequent injections at 12-h intervals, with the last injection 3 h before death. In the high dose IGF-I treatment group, one animal died, probably from hypoglycemia. After death, blood and tibialis anterior muscles were collected for analysis.
Diabetes.
After a 7-d adaptation period, diabetes was induced by injecting under ether anesthesia 60 mg STZ (freshly prepared in 0.01 M citrate buffer, pH 4.5)/kg body weight (0.5 ml/injection) into the tail vein. The Ctrl group was injected with an equivalent volume of saline (0.9% NaCl). Pilot experiments were performed to determine the optimal dose of STZ needed to produce moderate to severe diabetes. Glycemia and glucosuria were measured daily to monitor for the development of diabetes. Only rats showing polydipsia, polyuria, glucosuria, and glycemia over 400 mg/dl were included in the experiment. Three days after STZ injection, diabetic animals were randomized in two groups: one was infused with IGF-I (STZ+IGF-I group; n = 8), and the second group was not treated (STZ group; n = 7). The three groups of rats (Ctrl, STZ, and STZ+IGF-I) were anesthetized with a mixture of 75 mg/kg ketamine (Ketalar, Pfizer, New York, NY) and 15 mg/kg xylazine hydrochloride (Rompun, Bayer, Leverkussen, Germany) administered by an ip injection. To limit the risk of infection that might result from the large number of injections and manipulations, IGF-I was administrated by two miniosmotic pumps implanted in the interscapular region of STZ+IGF-I rats. Each pump contained 10 mg IGF-I/ml, so as to deliver 200 µg/100 g body weight·d. The rate of infusion from the pumps was 1 µl/h. To ensure immediate delivery of peptide at the target rate, the pumps were placed in sterile saline solution at 37 C for 4 h before implantation. The untreated diabetic (STZ) and control animals (Ctrl) were sham-operated. At the end of the 7-d infusion period, animals were killed, and blood and gastrocnemius muscles were collected for analysis.
C2C12 cell culture.
Myoblasts from the muscle-derived C2C12 cell line were obtained from American Type Culture Collection (Manassas, VA). The seeding density used throughout the experiments was 0.4 x 106 cells/plate (10-cm diameter plate). Undifferentiated cells were grown in DMEM supplemented with 10% fetal calf serum, 1% penicillin/streptomycin (100 U/100 µg·ml), 1% nonessential amino acids, and 2% L-glutamine at 37 C in 5% CO2. When cells reached 90–100% confluence (after 3 d), the 10% heat-inactivated fetal calf serum was replaced by 2% heat-inactivated horse serum to induce myogenic differentiation. Muscle cells were examined for evidence of myotube formation and growth. The medium was changed every 48 h, and differentiation was allowed to continue for 96 h. Experiments were carried out on the fourth day of differentiation. To preserve the original characteristics of the C2C12 cell line, splitting of the cells was limited to a maximum of seven times.
Total RNA extraction and RT
Total RNA was isolated from the gastrocnemius, tibialis anterior muscle, and myoblasts derived from the C2C12 muscle cell line using TRIzol reagent as described by the manufacturer. Total RNA recovery was approximately 1 µg/mg tissue (for muscle) and approximately 100 µg/plate (for cells). RT using 1 µg total RNA was performed as described previously (14).
Primer design
Specific primers were selected using Primer Express software (Applied Biosystems, Foster City, CA). Forward and reverse oligonucleotides used were as follows: Ub, 5'-GAT-CCA-GGA-CAA-GGA-GGG-C-3' and 5'-CAT-CTT-CCA-GCT-GCT-TGC-CT-3'; E214 kDa, 5'-TCC-TGC-AGA-ACC-GAT-GGA-G-3' and 5'-CGG-CTC-ATC-CAA-CAG-AGA-CTG-3'; atrogin-1/MAFbx, 5'-CCA-TCA-GGA-GAA-GTG-GAT-CTA-TGT-T-3' and 5'-GCT-TCC-CCC-AAA-GTG-CAG-TA-3'; IGF-I, 5'-GCT-ATG-GCT-CCA-GCA-TTC-G-3' and 5'-TCC-GGA-AGC-AAC-ACT-CAT-CC-3'; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-TGC-ACC-ACC-AAC-TGC-TTA-3' and 5'-GGA-TGC-AGG-GAT-GAT-GTT-C-3'. Accession number for the sequences used were NM017314 (Ub), M62388 (E214 kDa), AY059628 (atrogin-1/MAFbx), AH 002176 (IGF-I), and AF106860 (GAPDH, used as a reporter gene). The muscle expression of this last gene was not affected by fasting or diabetes in our experiments, allowing its use as a reporter gene. Primers were tested to avoid primer dimers, self-priming formation, or unspecific amplification.
Real-time quantitative (RTQ)-PCR
RTQ-PCR was carried out using the following cycle parameters: 10 min at 95 C, followed by 40 cycles of 1 min at 60 C and 15 sec at 95 C. For each gene, RTQ-PCR was conducted in duplicate with a 25-µl reaction volume of 1 ng cDNA. To ensure the quality of the measurements, each plate included for each gene a negative control and a positive control. For each sample, a value or threshold cycle (Ct) was calculated based on the time (measured by the number of PCR cycles) at which the reporter fluorescent emission increased beyond a threshold level (based on the background fluorescence of the system). The samples were diluted in such a manner that the Ct value was observed between 15–30 cycles. The Ct from a positive sample was used to calculate the interassay coefficient of variation (CV). For each gene, the CV was calculated as the SD/mean of the Ct determined on five different plates and with different mixes. The CVs obtained for Ub, E214 kDa, atrogin-1/MAFbx, IGF-I, and GAPDH were 4%, 4%, 8%, 8%, and 2%, respectively. Results were expressed using the comparative Ct method as described in User Bulletin2 (Applied Biosystems). Briefly, the 
Ct values were calculated in every sample for each gene of interest as follows: Ctgene of interest – Ctreporter gene, with GAPHD as the reporter gene. Calculation of relative changes in the expression level of one specific gene (Ct) was performed by subtraction of Ct from the Ctrl group to the corresponding Ct from the treated groups (14, 21). The values and ranges given in different figures were determined as followed: 2–Ct with Ct + SEM and Ct – SEM, where SEM is the standard error of the mean of the Ct value (User Bulletin 2, Applied Biosystems).
Determination of mRNA half-lives
The rate of degradation of atrogin-1/MAFbx mRNA was determined by measuring its rate of disappearance in the presence of an inhibitor of RNA synthesis. Initial studies measuring the half-life of atrogin-1/MAFbx mRNA in the presence of actinomycin D (1 µg/ml) showed that there was a half-life of less than 180 min. After preincubation of C2C12 myotubes with or without IGF-I for 3 h, actinomycin D was added to the medium, and incubation was continued with collection of cells every 20 min for up to 180 min. Atrogin-1/MAFbx mRNA was quantified as described above.
IGF-I RIA
After serum extraction, circulating IGF-I was measured by RIA as described previously (22, 23).
Glucose concentration
Serum glucose concentrations were determined using an oxygen/peroxide electrode method (Synchron LX20, Beckman Coulter, Fullerton, CA).
Statistical analysis
Results are presented as the mean ± SEM. For in vivo experiments, treatment effects were assessed using one-way ANOVA. When the treatment effect was significant (P < 0.05), multiple comparisons among groups were performed using the Student-Newman-Keuls test. A log transformation of the glycemia and IGF-I values was used in the analysis to improve homogeneity of variance. For in vitro experiments, two-way ANOVA was used for analyzing time effect, treatment effect, and interaction between the two.
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Results |
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). Atrogin-1/MAFbx mRNA increased 5.8-fold (P < 0.001), whereas the expression of Ub and E214 kDa, two other components of the Ub-proteasome system, were not increased (Fig. 1A). In contrast, the IGF-I mRNA content of muscle was reduced in response to fasting (–66%; P < 0.001). As expected, circulating concentrations of IGF-I and glycemia were reduced (respectively, –56% and –49%; both P < 0.001; Fig. 1B and Table 1).
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), but did not produce a significant attenuation of loss of muscle weight (Table 1). IGF-I injection blunted (
57%) the fasting-induced stimulation of atrogin-1/MAFbx in tibialis anterior muscle (2.5- vs. 5.8-fold; P < 0.05; Fig. 1A). This effect was not dose dependent. In the fasted animals treated with IGF-I, glycemia decreased commensurate with the IGF-I dose (Table 1).
Diabetes
STZ-induced diabetes caused a 37% decline in the weight of the gastrocnemius muscles (P < 0.001; Table 1) and a 15.1-fold increase in atrogin-1/MAFbx mRNA (P < 0.001). Ub and E214 kDa mRNAs were increased 4.2- and 2.8-fold, respectively (P < 0.001; Fig. 2A). In contrast, muscle IGF-I mRNA was reduced, albeit not significantly in response to diabetes (–41%). Circulating concentrations of IGF-I were reduced (–60%; P < 0.001; Fig. 2B), whereas glycemia was markedly increased (600 vs. 136 mg/dl; P < 0.001; Table 1). Normalization of the circulating concentrations of IGF-I by infusion attenuated, but not significantly, the gastrocnemius weight loss (Table 1).
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). Although glycemia was reduced slightly by IGF-I infusion in diabetic animals, serum glucose concentrations in these animals were still much higher than those in controls (Table 1).
Regulation of atrogin-1/MAFbx gene expression by IGF-I in cultured muscle cells
Having observed that IGF-I inhibits atrogin-1/MAFbx gene expression in vivo, we attempted to further analyze its effect in an in vitro model. Four days after differentiation of the C2C12 cells, myotubes were exposed to increasing doses of IGF-I (0.5–100 nM) for 24 h. IGF-I decreased atrogin-1/MAFbx mRNA with a 50% effective dose (ED50) of approximately 5 nM. A maximal inhibitory effect was observed at 50 nM (–70%; P < 0.01; Fig. 3A). The reduction of atrogin-1/MAFbx mRNA by the 50-nM concentration of IGF-I was time dependent (Fig. 3B).
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). This observation, therefore, suggests that IGF-I inhibits atrogin-1/MAFbx mRNA by reducing its rate of transcription.
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), consistent with its in vivo stimulatory effect (11). The stimulation of atrogin-1/MAFbx mRNA by DEXA (10–6 M) was time dependent [1.9-fold after 6 h (P < 0.001) and 2.5-fold after 12 h (P < 0.001)]. Even in the presence of DEXA (10–6 M), IGF-I (50 nM) retained its inhibitory effect on atrogin-1/MAFbx mRNA (Fig. 6).
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Discussion |
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Our observations indicate that IGF-I exerts its inhibitory effect on atrogin-1/MAFbx expression independently of glycemia. Indeed, this effect is observed in hypoglycemic fasted rats and in hyperglycemic diabetic animals. Previous studies have shown that IGF-I treatment of diabetic rats increased body weight and restored normal growth (25, 26). However, skeletal muscle weights were not reported in these earlier experiments. We showed a small, albeit not significant, weight gain of gastrocnemius muscle after IGF-I treatment. This could be compatible with a reduction in the proteolysis linked to the reduced atrogin-1/MAFbx expression. However, muscle proteolysis has not been measured in the present study. The relative importance of the IGF-I-dependent regulation of muscle protein synthesis and degradation in diabetes as well as in fasting in the maintenance of muscle mass will require additional consideration in relation to changes in atrogin-1/MAFbx expression.
The observation that IGF-I inhibits atrogin-1/MAFbx in muscle cells in culture supports a direct role of IGF-I in the regulation of this gene. The ED50 for inhibition of atrogin-1/MAFbx mRNA by IGF-I in vitro is in the range of 1–5 nM, a concentration corresponding to the IGF-I receptor affinity (5 nM) (27), and this lends physiological relevance to our findings in animal models. This concentration is similar to the IGF-I concentrations measured in rat skeletal muscle (10 ng/g muscle) (28) and to the free concentrations of IGF-I in the circulation, assuming a maximum of 5% IGF-I in the free form (29). Furthermore, we observed that the inhibition of atrogin-1/MAFbx mRNA by IGF-I is not due to an acceleration of its degradation, because IGF-I did not decrease the half-life of atrogin-1/MAFbx mRNA in cultured muscle cells. This suggests that the down-regulation of atrogin-1/MAFbx mRNA by IGF-I results from inhibition of gene transcription. Such an IGF-I-dependent inhibitory effect on atrogin-1/MAFbx gene transcription has recently been demonstrated in vitro to be due to inactivation of Foxo transcriptional factors by phosphorylation through PI3kinase/Akt pathway (30, 31). One of these studies showed, in addition, that the inhibition by IGF-I of the overexpression of atrogin-1/MAFbx in a model of muscle denervation was associated with inhibition of muscle loss. Another study described a similar pathway for the regulation of atrogin-1/MAFbx by insulin, a hormone sharing some metabolic actions with IGF-I (32). Taken together, these results show for the first time that both IGF-I and insulin regulate atrogin-1/MAFbx gene expression through a common sequence of events. It remains to be determined whether such a mechanism is involved in vivo in the inhibition by IGF-I of atrogin-1/MAFbx expression in fasted and diabetic rats or in other catabolic models. This mode of atrogin-1/MAFbx expression regulation contrasts with that described for E214 kDa, whose mRNA degradation is enhanced by IGF-I in L6 myotubes (24).
Inhibition of atrogin-1/MAFbx mRNA by exogenous IGF-I, however, was not complete in either fasted or diabetic animals. Failure to normalize circulating IGF-I levels might explain the incomplete inhibition of atrogin-1/MAFbx gene expression in fasted animals. The presence of hypoglycemia in these animals, however, precluded the use of higher doses of IGF-I. In diabetic animals, circulating IGF-I levels were normalized. Three explanations may be proposed for the failure of IGF-I to normalize atrogin-1/MAFbx. First, it is possible that the increase in muscle atrogin-1/MAFbx in response to diabetes might be related more to the decline in local than in systemic IGF-I levels, and that muscle IGF-I levels were not normalized by IGF-I injections. The importance of local IGF-I has been revealed in a model of liver IGF-I-deficient mice, in which locally produced IGF-I plays a more important role in longitudinal growth than circulating IGF-I (33). It is also possible that besides decreased IGF-I, other factors might be responsible for the induction of atrogin-1/MAFbx in fasting and diabetes. This possibility is supported by observations that insulin, which is decreased in response to diabetes, regulates several other genes of the Ub-proteasome system (34). The role of insulin in the regulation of atrogin-1/MAFbx mRNA has not been reported. Glucocorticoids, whose production is increased in fasting (35) and diabetes (36), could also contribute to the induction of atrogin-1/MAFbx expression, because DEXA injection has been shown to stimulate atrogin-1/MAFbx expression in vivo (11).
When a direct stimulatory effect of glucocorticoids was investigated in a cell culture model, we observed that pharmacological concentrations of glucocorticoids were necessary to stimulate atrogin-1/MAFbx expression in cultured myocytes, whereas IGF-I inhibited atrogin-1/MAFbx mRNA at physiological concentrations. This suggests that under physiological conditions, atrogin-1/MAFbx mRNA is inhibited by IGF-I and is not stimulated by glucocorticoids. In cell culture, the opposite actions of glucocorticoids and IGF-I on atrogin-1/MAFbx are reminiscent of their opposite actions on proteolysis (19, 20, 37, 38).
The observation of a stimulatory effect of DEXA on atrogin-1/MAFbx gene expression in vitro supports its direct action on myotubes. This findings contrasts with the absence of glucocorticoid-responsive elements in the promoter of the atrogin-1/MAFbx gene (39). Although glucocorticoids at high dose regulate atrogin-1/MAFbx mRNA in vitro and in vivo, additional studies are needed to determine whether their increase in fasting and diabetes is responsible for the induction of atrogin-1/MAFbx, as has been reported in sepsis (13). It could be hypothesized that exposure to very high concentrations of glucocorticoids in fasting and diabetes might contribute to the failure of IGF-I to fully inhibit the induction of atrogin-1/MAFbx. Our in vitro results, however, do not support this possibility, because even in the presence of DEXA (10–6 M), IGF-I (50 nM) retained its full inhibitory effect on atrogin-1/MAFbx mRNA levels. Moreover, IGF-I has also been shown to inhibit glucocorticoid induction of several genes of the Ub-proteasome system in vivo (40, 41) and proteolysis mediated by this system in vitro (42).
The expressions of E214 kDa and Ub are increased in response to diabetes (8, 43) and are inhibited by IGF-I, as previously described in other catabolic conditions (18, 40, 41, 44). The inhibition of E214 kDa by IGF-I agrees with the in vitro observations made for L6 myotubes (24). In fasting, we observed no induction of Ub and E214 kDa, in contrast to previous studies (34, 45, 46). Comparisons between studies, however, showed that the animals used previously (45, 46) were much younger (body weight, 60–80 g) than those used in our and other experiments (47) where Ub and E214 kDa fail to increase after fasting. We observed that fasting in young, in contrast to mature, animals increases Ub and E214 kDa mRNA [4.8-fold (P < 0.001) and 1.7-fold (P < 0.01), respectively], as described by others (45, 46). This indicates that induction of muscle Ub mRNA in response to fasting is age dependent. The failure, however, of muscle Ub mRNA to increase during fasting in mature rats does not make its involvement in muscle proteolysis uncertain. There is indeed no systematic correlation between the rate of proteolysis and expression of the Ub gene in the muscle. Furthermore, it is accepted that Ub protein is present in large amounts in the cell and is recycled after the degradation of target protein by the proteasome (10). The amount of free Ub, therefore, is not believed to be rate-limiting for Ub conjugation (48), making stimulation of Ub gene transcription probably unnecessary to support increased proteolysis.
In conclusion, we found that atrogin-1/MAFbx mRNA in skeletal muscle of fasted and diabetic rats is reduced by administration of IGF-I, and that addition of IGF-I to cultured muscle cells lowers atrogin-1/MAFbx mRNA levels. We observed that IGF-I and glucocorticoids have direct and opposite effects on regulation of atrogin-1/MAFbx. We speculate that the inhibition of atrogin-1/MAFbx expression is instrumental in the suppressive action of IGF-I on muscle proteolysis. Verification of this hypothesis will be possible only when the muscle protein(s) specifically targeted by this Ub-ligase is identified.
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Footnotes |
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Abbreviations: Ctrl, Control; Ct, threshold cycle; CV, coefficient of variation; DEXA, dexamethasone; ED50, 50% effective dose; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RTQ-PCR, real-time quantitative PCR; STZ, streptozotocin; Ub, ubiquitin.
Received March 29, 2004.
Accepted for publication July 15, 2004.
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) or without (
) IGF-I (50 nM) for 3 h. Actinomycin D was then added at a final concentration of 1 µg/ml. At the times indicated, cells were collected, and mRNA was processed as described in Materials and Methods. Atrogin-1/MAFbx mRNA levels were compared with those in the Ctrl baseline group (time zero), which were arbitrarily fixed at 1. Results shown are the mean ± SEM of three independent experiments. The atrogin-1/MAFbx mRNA half-lives under the two conditions were calculated based on the linear regression equation.
