Immunization against IGF-I prevents increases in protein synthesis in diabetic rats after resistance exercise

Mark J. Fedele, Charles H. Lang, and Peter A. Farrell

Noll Physiological Research Center, Pennsylvania State University, University Park 16802; and Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

These studies examined whether passive immunization against insulin-like growth factor I (IGF-I) would prevent increases in rates of protein synthesis in skeletal muscle of diabetic rats after resistance exercise. Male Sprague-Dawley rats were pancreatectomized and randomly assigned to either an exercise or a sedentary group. Animals in each of these groups received either an IGF-I antibody or a nonspecific IgG from a subcutaneous osmotic pump. Exercise did not change plasma or gastrocnemius IGF-I concentrations in nondiabetic rats. However, plasma and muscle IGF-I concentrations were higher in IgG-treated diabetic rats that exercised compared with respective sedentary groups (P < 0.05). Passively immunized diabetic rats did not exhibit the same exercise-induced increase in IGF-I concentrations. In nondiabetic rats, protein synthesis rates were higher after exercise in both control and immunized groups. In diabetic rats, exercise increased protein synthesis in the IgG-treated animals but not in those treated with IGF-I antibody. There was also a significant positive correlation between both plasma and gastrocnemius IGF-I concentrations and rates of protein synthesis in diabetic (P < 0.01), but not nondiabetic, rats. These results suggest that IGF-I is compensatory for insulin in hypoinsulinemic rats by facilitating an anabolic response after acute resistance exercise.

anabolism; growth factors; hypoinsulinemia


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INSULIN HAS PROFOUND EFFECTS on skeletal muscle protein metabolism. Among the deleterious consequences of hypoinsulinemia is a reduction in the rate of protein synthesis in fast-twitch skeletal muscle (18). Fractional rates of protein synthesis are normalized when insulin is administered to streptozotocin-diabetic rats, and low doses of insulin are as effective as high doses in achieving this anabolic response (33). Thus the actions of insulin on skeletal muscle protein synthesis are typically considered permissive as opposed to stimulatory.

Resistance exercise is known to increase rates of protein synthesis in both rats and humans during the recovery period in the muscles directly involved in the locomotor activity (41, 42). Fluckey et al. (20) demonstrated with bilateral hindlimb perfusions that rats that had engaged in acute resistance exercise had higher rates of protein synthesis. However, a prerequisite for this elevation was the presence of insulin in the perfusion media. Moderately diabetic rats also maintain the ability to elicit an anabolic response to resistance exercise (14-16). However, the elevations in rates of protein synthesis in diabetic rats are contingent upon the exercise intensity not being too severe (15) as well as the degree of hypoinsulinemia (17). Severely diabetic rats (plasma insulin less than congruent 80 pM) are not able to increase rates of protein synthesis after moderate-intensity resistance exercise, suggesting that there is a low but critical concentration of circulating insulin below which appropriate anabolic responses are inhibited (17).

These findings showed that the extent of insulin availability necessary to regulate skeletal muscle protein metabolism after acute resistance exercise is different from what is required to maintain normal glucose homeostasis. Alternatively, it is conceivable that mild hypoinsulinemia per se does not prevent this anabolic response to the resistance exercise stimulus because of other factors that can compensate for inadequate insulin. One potential candidate to serve in this role is insulin-like growth factor I (IGF-I). IGF-I is a potent anabolic hormone that appears to function as both a classic endocrine hormone and a paracrine/autocrine regulator. Administration of IGF-I is known to stimulate muscle protein synthesis and the accretion of lean body mass (2, 3, 25, 35). Furthermore, in our previous studies we demonstrated that mildly diabetic rats that engaged in moderate-intensity acute resistance exercise had an almost threefold increase in intramuscular IGF-I and an approximate doubling in plasma IGF-I concentrations compared with diabetic sedentary rats. There was no significant difference in either plasma or intramuscular IGF-I between exercised and sedentary rats that were nondiabetic (16).

The present study investigated the potential compensatory role for IGF-I in facilitating anabolic processes in diabetic rats in response to acute resistance exercise. If a compensatory increase in IGF-I is functional, then, at least in principle, removal of IGF-I should impair the anabolic response to exercise under hypoinsulinemic conditions. Decreasing IGF-I availability was achieved by passively immunizing rats against IGF-I by the administration of a neutralizing antibody for 7 days via a subcutaneously implanted osmotic pump. After these rats engaged in acute resistance exercise, in vivo rates of protein synthesis were assessed in skeletal muscle to determine whether the immunoneutralization of circulating IGF-I would inhibit the expected exercise-induced increases in protein synthesis.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Pennsylvania State University. Male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were used in all experiments. They were individually housed in wire-bottom cages in temperature- and humidity-controlled holding facilities with lights on at 0700 and off at 1900. Rats were fed ad libitum a standard rodent diet (diet 5001, PMI Feeds, Richmond, IN) that contained 24% protein, 12% fat, 50% carbohydrate, 7% ash, 6% fiber, and vitamins. A total of 48 rats were used for this study (8 groups with n = 6 per group). The experimental design was a 2 × 2 × 2 comparison designated as follows: nondiabetic/diabetic × sedentary/exercised × control (nonspecific IgG) osmotic pump/anti-IGF-I osmotic pump. The timetable for the experimental procedures is illustrated in Fig. 1.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1.   Timetable and experimental design.

Partial pancreatectomy. On the basis of previous work (13), a partial pancreatectomy (PPX) procedure was used, and the method was modified to include rats weighing 110-140 g as opposed to the weight (90-110 g) suggested by Foglia (21). We find that a larger percentage (congruent 80%) of the animals becomes diabetic when heavier rats are pancreatectomized (unpublished observations). We also used a microcauterizer to eliminate small pancreatic blood vessels and reduce bleeding during surgery. Rats were anesthetized using isoflurane and were placed on a heated surgical pad, where sterile surgery was performed. The procedure requires the physical removal of pancreatic tissue from the splenic, duodenal, and pyloric regions while major blood vessels are left intact. This is accomplished using sterile cotton Q-tips. Pancreatic tissue between the bile duct and the duodenum is not removed, since this approximates 10% of the original total pancreatic tissue. At the conclusion of surgery, rats were given ampicillin (5 mg/100 g body wt sc; Sigma Chemical, St. Louis, MO) as an antimicrobial agent. Two weeks after PPX, a tail vein blood sample was obtained with animals in the fed state to determine with a Beckman Glucose Analyzer 2 the plasma glucose concentration. Rats that were not diabetic (<300 mg/dl) or too severely diabetic (>600 mg/dl) were eliminated from the study. The remaining diabetic rats were randomly assigned to exercise or sedentary groups. Age-matched nondiabetic sedentary and exercised control rats were housed and handled in a manner that was identical to diabetic rats, with the exception of surgery and tail vein sampling to verify diabetic status. We previously had determined that there was no difference in the rate of muscle protein synthesis and plasma insulin concentrations between rats undergoing the sham pancreatectomy procedure and naïve control animals (15). Therefore, we subsequently used rats with no surgical manipulation for nondiabetic controls.

Passive immunization. Rats were passively immunized against IGF-I by use of an affinity purified polyclonal antibody (sc-7144; Santa Cruz Biotechnology, Santa Cruz, CA) specific for human, rat, and mouse IGF-I, as determined by epitope mapping to the carboxy terminus of the human IGF-I precursor. The antibody was continuously administered over 7 days via a subcutaneously placed osmotic pump (Alzet model 2ML1; Alza, Palo Alto, CA). The antibody was first dialyzed into 0.9% NaCl, and a total of 75 µg of antibody in a volume of 2.0 ml was added to each pump. This dose of anti-IGF-I was determined in preliminary studies to be effective in decreasing circulating IGF-I concentrations and attenuating the increase in rates of protein synthesis in exercised diabetic rats (unpublished data). The osmotic pumps were inserted 7 days before tissue procurement. The rats were first anesthetized with isoflurane, and a small incision was made between the scapulae. With a blunt dissector, a cavity was formed by separating the subcutaneous connective tissues. The pump was inserted into this pocket, with the flow modulator pointing away from the site of incision. The incision was closed with nylon sutures, and the wound was powdered with chlorhexidine (Sigma Chemical). The antibody was delivered from the pump at a constant rate of 10 µl/h for the 7-day period. Control rats had osmotic pumps containing an equal amount of nonspecific IgG (sc-2028; Santa Cruz Biotechnology). The timing of pump insertion and surgical procedures was identical between control and experimental animals.

Resistance exercise. Details of the exercise protocol have been described previously (19). Briefly, rats were operantly conditioned to stand and touch an illuminated bar located high on a Plexiglas exercise cage. The height of the light was adjusted so that the movement required full extension and flexion of the hindlimbs while eliciting both concentric and eccentric muscle contractions. Electrical foot shock (<2 mA, 60 Hz) was used to reinforce these movements. Once the learning process was completed (3-4 sessions), weighted vests were strapped over the scapulae, and the rats were required to touch the high bar 50 times during one acute exercise session. We defined acute resistance exercise as four separate sessions with 1 day of rest between sessions. The rats performed 50 repetitions during each exercise session with a 0.2 (day 1)-, 0.4 (days 2 and 3)-, and 0.6 (day 4)-g weighted vest per g body wt. We had previously verified (unpublished data) that electromyographic activity in the gastrocnemius muscle recorded during the act of the resistance exercise increased when the rats lifted these weights during both the concentric and eccentric phases of lifting. The exercise sessions occurred in the dark (red light) in the late afternoon. Rats that did not perform exercise (sedentary) were placed in the lifting cages twice during the week of acute exercise and were given five electric shocks to simulate some of the stress experienced by the exercised groups. One of these shock control sessions occurred 16 h before the determination of rates of protein synthesis.

Rates of protein synthesis. All measurements of protein synthesis occurred 16 h after the fourth session of acute resistance exercise, which was 7 days after osmotic pump insertion. Food was withdrawn from the rats during the last 5 h of this 16-h period. Rats were anesthetized with isoflurane, and the left carotid artery and right jugular vein were cannulated. Rats remained unconscious after the placement of catheters and during the measurement of protein synthesis. Total time between the onset of anesthesia and completion of surgery was 10-15 min. Approximately 1.5 ml of arterial blood were collected to determine plasma concentrations of insulin, IGF-I, IGF binding proteins (IGFBP), and glucose. A flooding dose (22) of L-[2,3,4,5,6-3H]phenylalanine (1 mCi/rat; Amersham Life Science, Arlington Heights, IL) in unlabeled phenylalanine (150 mM; l ml/100 g body wt total volume) was injected into the venous catheter over a 15-s period. Arterial blood (1 ml) was taken at 6 and 10 min, and then the soleus and gastrocnemius muscles were excised. The superficial white muscle was removed from the gastrocnemius, and the remaining muscle was immediately frozen in liquid nitrogen. The gastrocnemius from the contralateral leg was used for the measurement of muscle IGF-I concentration. Frozen muscles were stored at -80°C until phenylalanine incorporation into TCA-precipitable protein was analyzed using dabsylation of the amino acid and measurement on an HPLC (10). Radioactivity in the phenylalanine peak was measured by liquid scintillation counting, with appropriate correction for quench. Protein determinations were made using a Lowry protein assay kit (Sigma Chemical), and the rate of muscle protein synthesis was calculated using the method of Garlick et al. (22). Hematocrit was determined manually with microhematocrit tubes spun for 6 min at 8,500 rpm on a Readcrit microhematocrit centrifuge (Clay Adams, New York, NY) and read with a microcapillary reader (Damon/IEC, Needham Heights, MA). Hemoglobin was determined by the cyanomethemoglobin method (11).

Hormone and binding protein assays. For each hormone or binding protein, all samples were assayed randomly and simultaneously. Plasma insulin concentrations were determined by radioimmunoassay (RIA) (32). The antibody (no. 1013, Linco Research, St. Charles, MO) used in the rat insulin assay also recognizes other mammalian insulin isoforms but does not cross-react with glucagon, pancreatic polypeptide, somatostatin, or IGF-I. Plasma IGF-I was also determined by RIA after first being extracted using a modified acid-ethanol (0.25 N HCl-87.5% ethanol) procedure with cryoprecipitation (12, 30). The IGF-I antibody (lot no. AFP4892898, National Hormone and Pituitary Program) does not cross-react with either insulin or IGF-II, and the assay has a sensitivity of 0.03-0.08 ng/tube. The effect of elevated circulating IGF-1 antibody, due to pump administration, on the IGF-I RIA was evaluated by polyethylene glycol stripping of plasma before RIA. A randomly selected subset of samples from the same nondiabetic animals whose data are provided in Fig. 3 was used. Those results (not shown) demonstrated the same responses as shown in Fig. 3, with the exception that the absolute concentrations were somewhat lower than for nonstripped samples, which can be attributed to a slight dilution of the plasma samples. Gastrocnemius muscles used for IGF-I determinations were extracted using acid homogenization and Sep-Pak C18 extraction (12, 30) and then measured by RIA.

IGFBP-3 in plasma was determined by Western ligand blot analysis, as described by Hossenlopp et al. (24) and slightly modified by our laboratory (40). Samples were separated on a 15% (wt/vol) SDS-polyacrylamide gel without reduction of disulfide bonds. The electrophoresed proteins were transferred onto nitrocellulose in Tris-methanol-glycine buffer. Nitrocellulose sheets were washed and then incubated overnight with radiolabeled IGF-I. The nitrocellulose sheets were washed extensively in Tween 20, dried, and autoradiographed with X-ray film (Kodak X-Omat AR; Eastman Kodak, Rochester, NY) and intensifying screens (Du Pont, Wilmington, DE) at -70°C for 2-4 days. Plasma IGFBP-1 was determined by Western blot analysis. Briefly, plasma samples were separated on a 12.5% (wt/vol) SDS-PAGE gel under nonreducing conditions, as previously described (27). Separated proteins were electroblotted onto nitrocellulose and blocked for 2 h at room temperature with Tris-buffered saline containing 1% nonfat dry milk. The membranes were then incubated with a 1:2,000 dilution of antiserum against rat IGFBP-1 at room temperature for 2 h. Antigen-antibody complexes were identified with goat anti-rabbit IgG conjugated with horseradish peroxidase (Sigma Chemical) and exposed to the enhanced chemiluminescence detection system (Amersham) for 1 min and to X-ray film for 10-30 s. Bands were scanned (Microtek ScanMaker IV) and quantitated using NIH Image 1.6 software. Representative samples from all experimental groups were electrophoresed on the same gel.

Statistical analysis. Statistical differences among groups were assessed by repeated-measures ANOVA with the PROC GLM procedure of SAS (SAS Institute, Cary, NC). The design was a two (nondiabetic/diabetic) by two (sedentary/exercised) by two (IgG osmotic pump/anti-IGF-I osmotic pump) group by treatments comparison. Each variable was tested separately, with all groups included, which allowed for comparisons on the effects of diabetes, exercise, and anti-IGF-I separately and combined. The number of comparisons was limited by the degrees of freedom based on the number of groups and the number of animals tested. When significant F ratios were present, a Student-Newman-Keuls post hoc procedure was used to evaluate differences among means. P < 0.05 was chosen a priori as statistically significant. Values are presented as means ± SE. The number of rats in each group was 6.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Table 1 provides the physical and physiological characteristics of the rats used in the study. Diabetic rats weighed significantly less (290 ± 10 g) than nondiabetic rats (363 ± 4 g, P < 0.05). There were no differences in body weight between exercised and the corresponding sedentary rats or between the IgG-treated and anti-IGF-I-treated groups for either diabetic or nondiabetic rats. Plasma glucose concentrations were significantly greater (P < 0.05) in diabetic rats (452 ± 26 mg/dl) than in nondiabetic rats (241 ± 8 mg/dl). Hematocrit and hemoglobin values were similar among all groups.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Physical and physiological characteristics of the rats

Arterial plasma insulin concentrations (Fig. 2) were significantly lower in diabetic rats compared with nondiabetic rats (P < 0.01). Anti-IGF-I treatment caused an enigmatic decrease in plasma insulin concentrations in the nondiabetic sedentary rats. These animals had lower (P < 0.05) insulin concentrations (210 ± 70 pmol/l) than the nondiabetic sedentary IgG-treated rats (588 ± 132 pmol/l) and both groups of nondiabetic exercised rats. The reasons for this effect are unclear, because this was the only group that exhibited this response, and this finding has not been previously reported. For all other groups, neither exercise nor the type of osmotic pump changed plasma insulin concentrations.


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2.   Arterial plasma insulin concentrations. Values are means ± SE; n = 6 per group. Sed, sedentary; Ex, acute resistance exercise. IGF-I, insulin-like growth factor I; anti-IGF-I pump refers to rats that had anti-IGF-I delivered from a subcutaneously placed osmotic pump for 7 days; IgG pump refers to control rats that had nonspecific IgG administered. *Concentrations for diabetic rats are lower than respective nondiabetic rats (P < 0.01); #anti-IGF-I reduced the insulin concentrations in nondiabetic sedentary rats vs. other groups of nondiabetic rats (P < 0.05).

We have previously reported that the plasma concentration of IGFBP-1 is increased, whereas the concentration of IGFBP-3 is decreased, in diabetic rats compared with nondiabetic control animals (17). These data are consistent with the large majority of both the clinical and preclinical literature. Furthermore, there was no detectable effect of the same exercise regimen on the plasma concentration of these particular IGFBPs. Therefore, in the present study we only examined the effect of exercise and IGF-I neutralization on diabetic rats. Data in Table 2 indicate that the plasma concentrations of IGFBP-3 and IGFBP-1 were not significantly altered by either exercise, compared with the sedentary condition, or IGF-I neutralization, compared with IgG treatment.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Effect of exercise and/or IGF antibody on the plasma concentration of IGFBP-3 and IGFBP-1

Plasma IGF-I concentrations (Fig. 3) were lower in all diabetic groups compared with their corresponding nondiabetic groups (P < 0.01). In both the sedentary and exercised nondiabetic rats, those that were infused with anti-IGF-I had lower plasma IGF-I concentrations than the respective IgG-treated rats (P < 0.01). Exercise did not change plasma IGF-I concentrations in the nondiabetic rats. In diabetics, passive immunization against IGF-I did not significantly lower plasma IGF-I concentrations. Diabetic rats infused with IgG that engaged in acute resistance exercise had higher plasma IGF-I concentrations (564 ± 59 ng/ml) than the corresponding sedentary group (348 ± 55 ng/ml, P < 0.05). However, plasma IGF-I did not significantly increase as a result of exercise in the diabetic rats infused with the anti-IGF-I. Thus passive immunization prevented the increase in plasma IGF-I concentrations that is normally exhibited in hypoinsulinemic rats after resistance exercise.


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 3.   Plasma IGF-I concentrations. Values are means ± SE; n = 6 per group. *Concentrations for diabetic rats are lower than respective nondiabetic rats (P < 0.01). #Passive immunization against IGF-I reduced plasma IGF-I concentrations vs. respective control groups infused with IgG (P < 0.01). , Exercise increased plasma IGF-I concentrations compared with respective sedentary group (P < 0.05).

Figure 4 illustrates the IGF-I peptide content in gastrocnemius muscle. Passive immunization against IGF-I increased muscle IGF-I concentrations in the nondiabetic rats. This increase was significant for sedentary nondiabetic rats (7.6 ± 0.4 ng/g muscle vs. 6.0 ± 0.6 ng/g for anti-IGF-I and IgG-treated rats, respectively, P < 0.05), whereas the increase was not significant for the exercised nondiabetic rats (P = 0.1). Exercise did not change muscle IGF-I content in the nondiabetic rats. In the sedentary diabetic rats, passive immunization increased muscle IGF-I concentrations (P < 0.05); however, passive immunization had the opposite effect in the diabetic exercised rats that exhibited lower muscle IGF-I concentrations (P < 0.05). Exercise increased muscle IGF-I in the diabetic rats infused with IgG (3.9 ± 0.3 vs. 6.9 ± 0.4 ng/g muscle for sedentary and exercised, respectively, P < 0.01). In contrast, the same resistance exercise-induced elevations in muscle IGF-I content did not occur in the diabetic rats that were passively immunized against IGF-I. Thus, as was the case for plasma IGF-I, treatment with anti-IGF-I also negated the exercise-induced increase in gastrocnemius IGF-I content in the diabetic rats.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 4.   Gastrocnemius muscle IGF-I peptide concentrations. Values are means ± SE; n = 6 per group. *Concentrations for nondiabetic rats are higher than respective diabetic rats (P < 0.05). #Anti-IGF-I-treated rats had different gastrocnemius IGF-I concentrations than respective IgG-treated group (P < 0.05). , Exercise increased muscle IGF-I concentrations vs. respective sedentary group (P < 0.05).

In vivo rates of protein synthesis, expressed as nanomoles of phenylalanine incorporated per gram of muscle per hour for the gastrocnemius and soleus muscles, are shown in Fig. 5. In the gastrocnemius, nondiabetic rats had higher (P < 0.05) rates of protein synthesis than the corresponding diabetics, with the one exception of the diabetic sedentary anti-IGF-I-treated rats, whose synthesis rates were not different from the respective nondiabetic group (129 ± 9 vs. 124 ± 16 nmol Phe · g muscle-1 · h-1 for nondiabetic and diabetic, respectively). Exercised nondiabetic rats had higher rates of protein synthesis compared with sedentary animals in both the anti-IGF-I and IgG control groups (P < 0.05). In the diabetics, the exercised IgG-treated rats had higher rates of protein synthesis (141 ± 23 nmol · g-1 · h-1) than the sedentary control rats (101 ± 14 nmol · g-1 · h-1); however, this increase was not statistically significant (P = 0.08). There was no essential difference in the rate of muscle protein synthesis between sedentary (124 ± 16 nmol · g-1 · h-1) and exercised (129 ± 18 nmol · g-1 · h-1) passively immunized diabetic rats.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 5.   In vivo rates of protein synthesis in gastrocnemius and soleus muscles. Values are means ± SE; n = 6 per group. *Rates for nondiabetic rats are higher than respective diabetic rats (P < 0.05); #rates of protein synthesis are higher in the exercised rats than the respective sedentary rats (P < 0.05).

Rates of protein synthesis in the soleus muscle were higher in the nondiabetics compared with the diabetics; however, none of these differences was significant. Passive immunization did not change rates of protein synthesis in the soleus in the diabetic rats. Passively immunized diabetic sedentary rats had higher rates of protein synthesis (191 ± 25 nmol · g-1 · h-1) than IgG-treated diabetic sedentary rats (134 ± 16 nmol · g-1 · h-1), but this difference did not reach statistical significance (P = 0.07). Exercise increased soleus rates of protein synthesis in nondiabetic IgG-treated rats (P < 0.05) but not in the nondiabetic anti-IGF-I-treated rats. In the diabetics, protein synthesis was higher in exercised control rats (205 ± 21 nmol · g-1 · h-1) than in sedentary control animals (134 ± 16 nmol · g-1 · h-1, P < 0.05). Exercise did not change rates of protein synthesis in the passively immunized diabetic rats. Thus, in both the gastrocnemius and soleus muscles of diabetic rats, elevations in rates of protein synthesis after acute resistance exercise were prevented as a result of passive immunization against IGF-I.

There was a positive linear relationship between plasma IGF-I concentrations and gastrocnemius rates of protein synthesis in the diabetics (P < 0.01) but not in the nondiabetic animals (Fig. 6). For these comparisons, the rats were differentiated only by diabetic status regardless of whether they exercised or were passively immunized against IGF-I. The number of nondiabetic plasma samples was 23, because one sample was lost during the assay procedures. Likewise, Fig. 7 shows that there is also a significant positive linear relationship between gastrocnemius IGF-I concentrations and rates of protein synthesis (P < 0.01). These findings suggest that IGF-I has a more important role in the diabetic than in the nondiabetic rats in facilitating increases in protein synthesis after acute resistance exercise.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6.   Correlation between gastrocnemius rates of protein synthesis and plasma IGF-I concentrations. For both diabetic and nondiabetic animals, exercised and sedentary, as well as anti-IGF-I and IgG-treated, rats were graphed together. For the diabetics, the slope of the line was significantly different from zero (P < 0.01). Equations for lines based on least squares linear analysis are y = 0.013x + 153 for nondiabetic rats and y = 0.146x + 70 for diabetic rats.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 7.   Correlation between gastrocnemius rates of protein synthesis and gastrocnemius muscle IGF-I peptide concentrations. For both diabetic and nondiabetic animals, exercised and sedentary as well as anti-IGF-I and IgG-treated rats were graphed together. For diabetics, the slope of the line was significantly different from zero (P < 0.01). Equations for lines based on least squares linear analysis are y = -4.78x + 193 for nondiabetic rats and y = 15.4x + 47 for diabetic rats.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The most important finding of these studies is that passive immunization against IGF-I prevents the postresistance exercise increase in skeletal muscle protein synthesis in diabetic rats. We previously demonstrated that moderately diabetic rats that engaged in resistance exercise were able to elevate in vivo rates of protein synthesis to the same extent as nondiabetic rats (16). Both plasma and gastrocnemius IGF-I concentrations were unchanged in the exercised nondiabetic rats. In contrast, diabetic rats that exercised had an approximate doubling of plasma IGF-I and a threefold increase in muscle IGF-I compared with the sedentary diabetic rats (16). Those previous findings suggested that the resistance exercise-induced increase in IGF-I in the hypoinsulinemic rats may be a compensatory mechanism to facilitate the anabolic response to the resistance exercise stimulus.

On the basis of the presumption that the resistance exercise-induced increase in IGF-I in hypoinsulinemic rats is functioning as a compensatory mechanism to facilitate the anabolism, we hypothesized that preventing this increase might abolish the anabolic response. Diabetic rats that were passively immunized did not have higher rates of protein synthesis after acute resistance exercise in either the gastrocnemius or the soleus muscles (Fig. 5). In contrast, IgG-treated diabetic rats did have higher protein synthesis rates after resistance exercise. In addition, acute resistance exercise did not induce an increase in either plasma (Fig. 3) or gastrocnemius (Fig. 4) IGF-I concentrations in the passively immunized diabetic rats, whereas it did in the control diabetic rats. These findings are consistent with previous studies in which moderately diabetic rats also had elevated rates of protein synthesis after resistance exercise, along with concomitant increases in plasma and muscle IGF-I concentrations (16). Thus it appears that passive immunization prevented the exercise-induced compensatory increase of IGF-I in the diabetic rats. Passive immunization did not prevent higher protein synthesis rates in the exercised nondiabetic rats despite reducing plasma IGF-I concentrations, compared with nondiabetic rats that were not immunized. Moreover, the nondiabetic rats had higher plasma insulin, plasma IGF-I, and muscle IGF-I concentrations than the diabetics. Thus the deleterious effects of immunoneutralizing IGF-I were not evident in the nondiabetic rats, presumably because IGF-I was not compensating for insulin under normal conditions.

The present study provides additional evidence that IGF-I is relatively more important in diabetic than in nondiabetic rats in facilitating an anabolic response. We found a significant positive correlation between both plasma (Fig. 6) and gastrocnemius IGF-I protein content (Fig. 7) concentrations and rates of protein synthesis in the diabetic but not the nondiabetic rats. A similar relationship was previously found in septic rats that also had a positive correlation between rates of protein synthesis and gastrocnemius IGF-I (28). Thus IGF-I appears to be acting in a stimulatory manner, as opposed to the permissive action of insulin in augmenting rates of protein synthesis under these conditions. These findings suggest that changes in the concentration of IGF-I as a result of various perturbations appear to be a fundamental determinant that affects skeletal muscle protein metabolism.

Extreme hypoinsulinemia in streptozotocin-diabetic rats is known to decrease protein synthesis in skeletal muscle under resting conditions (4). If pancreatectomized rats likewise become severely diabetic, then basal protein synthesis rates are also lower than those observed in nondiabetics (17). This condition of severe hypoinsulinemia also inhibits the increased protein synthesis rates after acute resistance exercise (17) that are normally observed in moderately diabetic and nondiabetic rats (14-16). Thus, for this study, moderately diabetic rats were used to determine whether the increases in protein synthesis rates are facilitated by the compensatory increase in IGF-I. The degree of hypoinsulinemia in the diabetic rats from this study was actually between what we have previously considered severe (17) and moderate (14-16). Despite not being statistically significant at the conventional level of acceptance, the IgG-treated diabetic rats that exercised had a 39% increase in the rate of protein synthesis in the gastrocnemius muscle compared with sedentary diabetic rats. Moderately diabetic rats that performed the same acute resistance exercise protocol in previous studies had increases in protein synthesis rates of 25% (16) and 38% (14), both of which were statistically significant. Taken together, there appears to be a graded relationship between the severity of hypoinsulinemia and the ability to increase rates of protein synthesis in response to the anabolic stimulus of resistance exercise.

Other studies have also investigated the effects of passive immunization against IGF-I and its effects on growth and protein metabolism. One study in lambs found that a bolus injection of IGF-I antiserum increased net protein catabolism, albeit this effect only lasted 1 h (26). A study in mice found that denervation-devascularization of the extensor digitorum longus followed by local administration of anti-IGF-I reduced the number and diameter of regenerating myofibers. These data suggest a delay in the proliferation of activated satellite cells under conditions in which IGF-I availability is reduced (29). In contradiction, some studies have found that IGF-I antibodies actually potentiate the effects of IGF-I (23, 37). The authors suggested that the particular antibodies might function in a manner similar to an enhancing IGF-I binding protein and thus increase the bioavailability and bioactivity of IGF-I. Our data do not necessarily support their suggestion, because passive immunization did not increase protein synthesis rates.

An examination of the IGF-I system is incomplete without an analysis of its binding proteins. IGFBP actions have often been described as being both stimulatory and inhibitory in relation to IGF-I-mediated processes (5). Approximately 80% of the IGFs in plasma are associated with IGFBP-3 in a large ternary complex that restricts their movement across the capillary endothelium, thereby retaining IGF-I in the circulation (6). A small amount of IGF-I is also bound to the lower molecular weight IGFBP-1 and is believed to be responsible for the acute regulation of IGF-I bioavailability and bioactivity (31). We have previously shown that plasma concentrations of IGFBP-1 and IGFBP-3 are not changed as a result of the acute resistance exercise in diabetic and nondiabetic rats (17), although hypoinsulinemia does result in a reduction in circulating levels of IGFBP-3 and an increase in IGFBP-1 (7, 8, 17). In the present study, we determined whether IGF-I antibody infusion and/or exercise altered IGFBP-1 and IGFBP-3 in diabetic rats. These treatments did not alter circulating concentrations of IGFBPs, suggesting that the IGF-I antibody was not indirectly affecting protein synthesis via a secondary alteration in one of the major IGFBPs.

It is difficult to differentiate between plasma and muscle IGF-I in terms of their relative importance in facilitating this compensatory response. A growing body of literature has shown that locally produced IGF-I, acting in a paracrine/autocrine manner, may be more important than circulating (endocrine) IGF-I for facilitating muscle development, hypertrophy, and responses to loading (1). Because the IGF-I antibody in this study was administered systemically, it is possible that this treatment regimen was not effective in neutralizing intramuscular IGF-I. An important consideration was whether the antibodies would be effective in binding and neutralizing circulating IGF-I and thereby hinder its physiological actions. Passive immunization was successful in lowering plasma IGF-I concentrations (Fig. 3). Interestingly, this treatment produced a concomitant increase in gastrocnemius IGF-I content in the nondiabetic rats (Fig. 4), which we have previously found (unpublished data). This increase in intramuscular IGF-I suggests that the systemically administered antibodies may have induced an upregulation of muscle IGF-I. This finding requires further study to determine whether this adaptation occurred at the level of gene expression and also to differentiate between the endocrine functions of hepatic-produced IGF-I and the paracrine/autocrine functions of locally produced tissue IGF-I.

IGF-I may be able to compensate for insulin because these two hormones share several key intracellular signaling intermediates (9). It is generally accepted that the pleiotropic effects of IGF-I are similar to those produced by insulin. In rats, IGF-I infusion alone can stimulate skeletal muscle protein synthesis under conditions that maintain insulin and amino acids near postabsorptive levels (25). Humans with type 1 diabetes have been successfully treated by IGF-I to achieve a correction of metabolic disorders and markedly improved insulin sensitivity, although this treatment has side effects (39). These authors hypothesized that IGF-I may function effectively as an insulin surrogate, presumably because insulin and IGF-I share several signaling intermediates and the IGF-I receptor and postreceptor signaling functions are still intact. With use of rodents as a model of type 1 diabetes, chronic administration of IGF-I to streptozotocin-diabetic rats leads to near-normal growth and improved nitrogen retention without influencing the abnormal carbohydrate metabolism (36, 38). Rossetti et al. (34) showed in partially pancreatectomized rats that the metabolic response to IGF-I was intact and the potency of IGF-I to stimulate glucose utilization was greater in diabetic compared with nondiabetic rats. All of these findings suggest that, under conditions of hypoinsulinemia, IGF-I may compensate for insulin by facilitating some of its physiological actions. Likewise, the increase in IGF-I that was exhibited in the exercised diabetic rats was perhaps an adaptation that enabled them to elicit a physiological response that is normally mediated by insulin. This ability of IGF-I to function in an "insulin-like" manner under hypoinsulinemic conditions supports the potential clinical use of IGF-I for humans with diabetes.

In conclusion, passive immunization against IGF-I prevented the compensatory increases of IGF-I in diabetic rats exhibited after acute resistance exercise. This lack of increased plasma and skeletal muscle IGF-I content in diabetic rats was associated with the inability of these animals to increase muscle protein synthesis after resistance exercise. There was also a positive relationship between plasma and gastrocnemius IGF-I concentrations and rates of protein synthesis in the diabetic, but not the nondiabetic, rats. Thus IGF-I appears to have a more important role in regulating skeletal muscle protein synthesis in the diabetic than in the nondiabetic rats. Mechanisms distinguishing the endocrine vs. the paracrine/autocrine actions of IGF-I and their significance in controlling skeletal muscle protein metabolism remain to be elucidated. Future studies are also needed to determine the potential therapeutic applications of IGF-I for ameliorating the metabolic disorders and muscle atrophy that are associated with type 1 diabetes and other muscle degenerative diseases.


    ACKNOWLEDGEMENTS

We thank Marlin Druckenmiller, Steve Bloomer, Neil Kubica, Dennis Koch, Fred Weyandt, and Doug Johnson for their superb technical efforts. We also thank Ofer Harel for helping with the statistical analysis.


    FOOTNOTES

These studies were supported by National Institutes of Health Grants AR-43127 (P. A. Farrell) and GM-38032 (C. H. Lang).

Address for reprint requests and other correspondence: M. J. Fedele, Univ. of Illinois, Chicago School of Kinesiology (m/c 194), 901 W. Roosevelt Rd., Chicago, IL 60608 (E-mail: mfedele{at}uic.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 25 August 2000; accepted in final form 5 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adams, GR. Role of insulin-like growth factor-I in the regulation of skeletal muscle adaptation to increased loading. In: Exercise and Sport Science Reviews, edited by Holloszy JO.. Baltimore: Williams & Wilkins, 1998, p. 31-60.

2.   Adams, GR, and Haddad F. The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy. J Appl Physiol 81: 2509-2516, 1996[Abstract/Free Full Text].

3.   Adams, GR, and McCue SA. Localized infusion of IGF-I results in skeletal muscle hypertrophy in rats. J Appl Physiol 84: 1716-1722, 1998[Free Full Text].

4.   Ashford, AJ, and Pain VM. Effect of diabetes on the rates of synthesis and degradation of ribosomes in rat muscle and liver in vivo. J Biol Chem 261: 4059-4065, 1986[Abstract/Free Full Text].

5.   Baxter, RC. Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities. Am J Physiol Endocrinol Metab 278: E967-E976, 2000[Abstract/Free Full Text].

6.   Baxter, RC, and Martin JL. Structure of the Mr 140,000 growth hormone-dependent insulin-like growth factor binding protein complex: determination by reconstitution and affinity-labeling. Proc Natl Acad Sci USA 86: 6898-6902, 1989[Abstract].

7.   Bereket, A, Lang CH, Blethen SL, Fan J, Frost RA, and Wislon TA. Insulin-like growth factor binding protein (IGFBP)-3 proteolysis in children with insulin dependent diabetes mellitus: a possible role for insulin in the regulation of IGFBP-3 protease activity. J Clin Endocrinol Metab 80: 2282-2288, 1995[Abstract].

8.   Bereket, A, Lang CH, Blethen SL, Gelato MC, Fan J, Frost RA, and Wilson TA. Effect of insulin on the insulin-like growth factor system in children with new-onset insulin-dependent diabetes mellitus. J Clin Endocrinol Metab 80: 1312-1317, 1995[Abstract].

9.   DeMeyts, P, Urso B, Christoffersen CT, and Shymko RM. Mechanism of insulin and IGF-I receptor activation and signal transduction specificity. Ann NY Acad Sci 766: 388-401, 1995[ISI][Medline].

10.   Drenevich, D, and Vary TC. Analysis of physiological amino acids using dabsyl derivatization and reversed-phase liquid chromatography. J Chromatogr 613: 137-144, 1993[Medline].

11.   Eilers, RJ. Notification of final adoption of an international method and standard solution for hemoglobinometry specifications for preparation of standard solution. Am J Clin Pathol 47: 212-214, 1967[ISI][Medline].

12.   Fan, J, Molina PE, Gelato MC, and Lang CH. Differential tissue regulation of insulin-like growth factor-I content and binding proteins after endotoxin. Endocrinology 134: 1685-1692, 1994[Abstract].

13.   Farrell, PA, Caston AL, and Rodd D. Changes in insulin response to glucose after exercise training in partially pancreatectomized rats. J Appl Physiol 70: 1563-1569, 1991[Abstract/Free Full Text].

14.   Farrell, PA, Fedele MJ, Hernandez J, Fluckey JD, Miller JL, III, Lang CH, Vary TC, Kimball SR, and Jefferson LS. Hypertrophy of skeletal muscle in diabetic rats in response to chronic resistance exercise. J Appl Physiol 87: 1075-1082, 1999[Abstract/Free Full Text].

15.   Farrell, PA, Fedele MJ, Vary TC, Kimball SR, and Jefferson LS. Effects of intensity of acute-resistance exercise on rates of protein synthesis in moderately diabetic rats. J Appl Physiol 85: 2291-2297, 1998[Abstract/Free Full Text].

16.   Farrell, PA, Fedele MJ, Vary TC, Kimball SR, Lang CH, and Jefferson LS. Regulation of protein synthesis after acute resistance exercise in diabetic rats. Am J Physiol Endocrinol Metab 276: E721-E727, 1999[Abstract/Free Full Text].

17.   Fedele, MJ, Hernandez JM, Lang CH, Vary TC, Kimball SR, Jefferson LS, and Farrell PA. Severe diabetes prohibits elevations in muscle protein synthesis after acute resistance exercise in rats. J Appl Physiol 88: 102-108, 2000[Abstract/Free Full Text].

18.   Flaim, KE, Copenhaver ME, and Jefferson LS. Effects of diabetes on protein synthesis in fast- and slow-twitch rat skeletal muscle. Am J Physiol Endocrinol Metab 239: E88-E95, 1980[Abstract/Free Full Text].

19.   Fluckey, JD, Kraemer WJ, and Farrell PA. Pancreatic islet insulin secretion is increased after resistance exercise in rats. J Appl Physiol 79: 1100-1105, 1995[Abstract/Free Full Text].

20.   Fluckey, JD, Vary TC, Jefferson LS, and Farrell PA. Augmented insulin action on rates of protein synthesis after resistance exercise in rats. Am J Physiol Endocrinol Metab 270: E313-E319, 1996[Abstract/Free Full Text].

21.   Foglia, VA. Caracteristicas del la diabetes en la rata. Rev. Soc Argentina Biol 20: 21-37, 1944.

22.   Garlick, PJ, McNurlan MA, and Preddy VR. A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [3H]phenylalanine. Biochem J 192: 719-723, 1980[ISI][Medline].

23.   Hill, RA, and Pell JM. Regulation of insulin-like growth factor I (IGF-I) bioactivity in vivo: further characterization of an IGF-I-enhancing antibody. Endocrinology 139: 1278-1287, 1998[Abstract/Free Full Text].

24.   Hossenlopp, P, Seurin D, Segonia-Quinson B, Hardouin S, and Binous M. Analysis of serum insulin-like growth factor binding proteins using Western blotting: use of the method for titration of the binding proteins and competitive bindings sites. Anal Biochem 154: 138-143, 1986[ISI][Medline].

25.   Jacob, R, Hu X, Niederstock D, Hasan S, McNulty PH, Sherwin RS, and Young LH. IGF-I stimulation of muscle protein synthesis in the awake rat: permissive role of insulin and amino acids. Am J Physiol Endocrinol Metab 270: E60-E66, 1996[Abstract/Free Full Text].

26.   Koea, JB, Gallaher BW, Breier BH, Douglas RD, Hodgkinson S, Shaw JHF, and Gluckman PD. Passive immunization against circulating insulin-like growth factor-I (IGF-I) increases protein catabolism in lambs: evidence for a physiological role for circulating IGF-I. J Endocrinol 135: 279-284, 1992[Abstract].

27.   Lang, CH, Fan J, Cooney R, and Vary TC. IL-1 receptor antagonist attenuates sepsis-induced alterations in the IGF system and protein synthesis. Am J Physiol Endocrinol Metab 270: E430-E437, 1996[Abstract/Free Full Text].

28.   Lang, CH, Frost RA, Jefferson LS, Kimball SR, and Vary TC. Endotoxin-induced decrease in muscle protein synthesis is associated with changes in eIF2B, eIF4E, and IGF-I. Am J Physiol Endocrinol Metab 278: E1133-E1143, 2000[Abstract/Free Full Text].

29.   Lefaucheur, J, and Sebille A. Muscle regeneration following injury can be modified in vivo by immune neutralization of basic fibroblast gowth factor, transforming growth factor beta 1 or insulin-like growth factor I. J Neuroimmunol 57: 85-91, 1995[ISI][Medline].

30.   Li, YH, Fan J, and Lang CH. Differential role of glucocorticoids in mediating endotoxin-induced changes in IGF-I and IGFBP-1. Am J Physiol Regulatory Integrative Comp Physiol 272: R1990-R1997, 1997[Abstract/Free Full Text].

31.   McCusker, RH, and Clemmons DR. The Insulin-Like Growth Factor Binding Proteins: Structure and Biological Functions. New York: Oxford, 1992, p. 110-150.

32.   Morgan, CR, and Lazarow A. Immunoassay of insulin: two antibody system. Plasma levels of normal, subdiabetic and diabetic rats. Diabetes 12: 115-126, 1963[ISI].

33.   Pain, VM, and Garlick PJ. Effect of streptozotocin diabetes and insulin treatment on the rate of protein synthesis in tissues of the rat in vivo. J Biol Chem 249: 4510-4514, 1974[Abstract/Free Full Text].

34.   Rossetti, L, Frontoni S, DiMarchi R, DeFronzo RA, and Giaccari A. Metabolic effects of IGF-I in diabetic rats. Diabetes 40: 444-448, 1991[Abstract].

35.   Russell-Jones, DL, Umpleby AM, Hennessy TR, Bowes SB, Shojaee-Moradie F, Hopkins KD, Jackson NC, Kelly JM, Jones RH, and Sönksen PH. Use of a leucine clamp to demonstrate that IGF-I actively stimulates protein synthesis in normal humans. Am J Physiol Endocrinol Metab 267: E591-E598, 1994[Abstract/Free Full Text].

36.   Scheiwiller, E, Guler HP, Merryweather J, Scandella C, Maerki W, Zapf J, and Froesch ER. Growth restoration of insulin-deficient diabetic rats by recombinant human insulin-like growth factor I. Nature 323: 169-171, 1986[ISI][Medline].

37.   Stewart, CEH, Bates PC, Calder TA, Woodall SM, and Pell JM. Potentiation of insulin-like growth factor-I (IGF-I) activity by an antibody: supportive evidence of IGF-I bioavailability in vivo by IGF binding proteins. Endocrinology 133: 1462-1465, 1993[Abstract].

38.   Tomas, FM, Knowles SE, Owens PC, Read LC, Chandler CS, Gargosky SE, and Ballard FJ. Increased weight gain, nitrogen retention and muscle protein synthesis following treatment of diabetic rats with insulin-like growth factor (IGF)-I and des(1-3) IGF-I. Biochem J 276: 547-554, 1991[ISI][Medline].

39.   Usala, AL, Madigan T, Burguera B, Sinha MK, Caro JF, Cunningham P, Powell JG, and Butler PC. Brief report: treatment of insulin-resistant diabetic ketoacidosis with insulin-like growth factor I in an adolescent with insulin-dependent diabetes. N Engl J Med 327: 853-857, 1992[ISI][Medline].

40.   Wojnar, MM, Fan J, Li YH, and Lang CH. Endotoxin-induced changes in IGF-I differ in rats provided enteral vs. parenteral nutrition. Am J Physiol Endocrinol Metab 276: E455-E464, 1999[Abstract/Free Full Text].

41.   Wong, TS, and Booth FW. Protein metabolism in rat gastrocnemius muscle after stimulated chronic concentric exercise. J Appl Physiol 69: 1709-1717, 1990[Abstract/Free Full Text].

42.   Yarasheski, KE, Zachwieja JJ, and Bier DM. Acute effects of resistance exercise on muscle protein synthesis rate in young and elderly men and women. Am J Physiol Endocrinol Metab 265: E210-E214, 1993[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 280(6):E877-E885
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society