Adaptive Medicine 3(2): 112-118, 2011 DOI: 10.4247/AM.2011.

ABB015

IGF-1 Partially Reproduces Beneficial Effect of Exercise Training on Glucose Tolerance in Normal Rats
Ching-Yu Tzeng1, 2, Yu-Chiang Lai1, Chien-Wen Hou1, Chung-Yu Chen1, Shin-Da Lee3, 6, Chih-Yang Huang4, Chiu-Chou Chen 1, Te-Chih Liu 1, Yuh-Feng Liou 1, Chung-Lan Kao 5, and Chia-Hua Kuo 1, 3
1 2

Laboratory of Exercise Biochemistry, Taipei Physical Education College, Taipei, Taiwan, ROC Department of Physical Education, Fu Jen Catholic University, Taipei, Taiwan, ROC 3 Department of Physical Therapy, Graduate Institute of Rehabilitation Science, China Medical University, Taichung, Taiwan, ROC 4 Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan, ROC 5 Department of Physical Medicine and Rehabilitation, Taipei Veterans General Hospital and National Yang Ming University, Taipei, Taiwan, ROC 6 Department of Healthcare Administration, Asia University, Taichung, Taiwan, Republic of China

Exercise transiently elevates the IGF-1 (insulin-like growth factor 1) level, but whether exogenous IGF-1 administration can reproduce exercise training benefit in glycemic control is currently unknown. This study compared the effect of IGF-1 administration and exercise training on glycogen storage, glucose tolerance, and muscle glucose transporter 4 (GLUT4) protein expression in normal rats. Forty rats were weight matched and evenly assigned to the following 4 groups: control (C), exercise trained (E), IGF-1 treated (I), and exercise-trained + IGF-1 (EI). Same volume of saline or IGF-1 (2 g/kg BW) was injected daily to the rats. Exercise training consisted of daily 90 min of swimming for the first week and gradually increased to 180 min, twice for the third week. Oral glucose tolerance test (OGTT) was performed in all rats under fasted condition. Muscle tissues were removed at the end of the 3-week treatments (3 days after OGTT). The levels of GLUT4 protein and mRNA were determined in red and white portions of the quadriceps muscle (RQ and WQ). Both exercise training and chronic IGF-1 administration increased GLUT4 expression and improved glucose tolerance without an observed additive effect. Exercise training increased glycogen level in RQ and WQ above control level. Despites chronic IGF-1 administration increased muscle GLUT4 expression above control level, glycogen increase was not observed. Our data suggest that IGF-1 can partially reproduce exercise training effect on improving glycemic control. Key Words: insulin-like growth factor, insulin resistance, diabetes, glycogen, aging

Introduction
Under postprandial conditions, skeletal muscle becomes the main site for glucose disposal. Thus, this tissue plays a pivotal role in regulating whole body glucose homeostasis (3, 9). Glucose transporter 4 (GLUT4) is the main glucose transporter isoform expressed in skeletal muscle, which can be rapidly recruited to the plasma membrane upon insulin stimulation. This protein translocation increases the membrane permeability to circulating glucose and thus increases glucose disposal in skeletal muscle. Several early reports found that the amount of GLUT4 protein is strongly correlated with maximal insulinstimulated glycogen storage in skeletal muscle (13, 17). Therefore, interventions that enhance muscle GLUT4 protein expression might be a possible method for treating patients with insulin resistance and type 2 diabetes. The beneficial consequence of regular exercise training on glucose tolerance, insulin sensitivity, and muscle glycogen storage has been previously reported in humans and animals (6, 7, 12). This improvement is thought, to some extent, associated with the increased GLUT4 protein expression in exercised muscle (6, 17, 27). The underlying mechanism for the exercise-induced increase in GLUT4 expression has not been fully understood. Available data suggest that exercise can transiently elevate the IGF-1 (insulin-

Corresponding author: Chia-Hua Kuo, Ph.D., Laboratory of Exercise Biochemistry, Taipei Physical Education College, 101, Sec. 2, Jhongcheng Rd., Shihlin District, Taipei City 111, Taiwan, Republic of China. Fax: +886-2-28753383, E-mail: kuochiahua@gmail.com *Chung-Lan Kao and Chia-Hua Kuo equally contributed to this work. Received: July 29, 2011; Revised: August 22, 2011; Accepted: August 29, 2011. 2011 by The Society of Adaptive Science in Taiwan and Airiti Press Inc. ISSN : 2076-944X. http://www.sast.org.tw

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like growth factor 1) level (5, 24, 28), and this signal has been suggested to be associated with exercise training-dependent improvement in muscle properties (5, 19). In this study, we compared the effect of IGF-1 administration and exercise training on GLUT4 protein expression in rat muscles, as well as its association to glycogen storage and whole-body glucose tolerance.

Materials and Methods

Animal Care and Experimental Design Forty male Sprague-Dawley rats from the National Animal Laboratory of the NSC (National Science Council, Taipei, Taiwan, ROC) weighing 200 g each were housed 3 per cage and were provided normal rat chow (PMI Nutrition International, Brentwood, MO, USA) and water ad libitum. The temperature of the animal room was maintained at 23C, with a 12-h light-dark cycle. After 1 week of familiarization, the rats were weight-matched and divided into 4 groups: control (C, n = 10), exercise (E, n = 10), IGF-1 (I, n = 10), and exercise + IGF-1 (EI, n = 10). Exercise training protocol consisted of 90 min of swimming for the first week and gradually increased to 180 2 min (1-h rest in between) for the third week. Swimming training started at 9 am every morning with a break on Sunday. The temperature of the water, 25 cm in depth in a plastic barrel, was maintained at 34 1C; 3 rats were placed in each barrel at the same time. For IGF1 administration, recombinant human IGF-1 was produced from eukaryotic cells (Leinco Technologies, St. Louis, MO, USA). IGF1 was dissolved in saline and was injected (ip) 2 g/kg 1 h after exercise training (15). At the end of the third week, rats were anesthetized at a time when they had recovered 18 h after the cessation of the last exercise bout, and red and white portion of the quadriceps muscles were excised and examined for GLUT4 protein level, glycogen content, and citrate synthase activity. Oral glucose tolerance test (OGTT) was performed 3 days before muscle sample excision; and the muscle tissue analysis for glycogen, GLUT4 protein, and citrate synthase was performed 3 days after the OGTT to avoid the potential interference effect of OGTT procedure on the muscle glycogen result (IGF-1 was continuously given during the next 3 days after OGTT). The same volume of saline was daily injected as a placebo into control animals. In the EI group, IGF-1 was dissolved in saline and injected (2 g/kg) 1 h after exercise training. Oral Glucose Tolerance Test (OGTT) OGTT was performed 18 h after the last bout of ex-

ercise, according to Hou et al. (11). This recovery period included a 12-h fast prior to the glucose intubation for OGTT. Blood samples were withdrawn from the tail at 0 (fasted sample), 15, and 45 min after the oral glucose load (1 g/kg BW) for blood glucose and serum insulin measurements, according to the procedure given in Cortez et al. (8). A glucose analyzer (Lifescan, Milpitas, CA, USA) was used for glucose concentration determination; the glucose oxidase method was used. The consistency of the analyzer was tested using real blood samples twice before use. Serum insulin levels were measured using enzyme-linked immunosorbent assay (ELISA) with anti-insulin monoclonal antibody. GLUT4 Protein Three days after the OGTT, and 18 h after the last exercise bout with an immediate glucose intubation (1 g/kg BW), muscles were surgically removed for analysis of glycogen, and GLUT4 protein levels, and citrate synthase activity assay. Muscle samples for GLUT4 protein were homogenized in ice-cold HES (20 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 1 mM EDTA, and 250 mM sucrose, pH 7.4) buffer (1: 20) with a Polytron homogenizer (Kinematica, Littau, Switzerland). Sample homogenates and standards were diluted 1: 1 with Laemmli sample buffer (125 mM Tris, 20% glycerol, 2% SDS, and 0.008% bromophenol blue, pH 6.8). The Western blotting procedure for GLUT4 analysis was followed the previously described method (11). Muscle homogenates containing 75 g (red gastrocnemius and plantaris muscles) of protein were subjected to SDSpolyacrylamide gel electrophoresis (PAGE) and electrophoretically transferred to a PVDF membrane. Two heart homogenates containing 15 and 30 g of protein were loaded in parallel with the muscle samples. GLUT4 antiserum (Chemicon, Temecula, CA, USA) was used for immunoblotting (directly against the carboxyl-terminus of the GLUT4 protein) in a dilution of 1: 5000. GLUT4 protein was visualized using an ECL Western blot detection kit (Amersham, Arlington Heights, IL, USA) on x-ray film according to the manufacturers instructions. GLUT4 mRNA For RNA extraction, muscle tissues were homogenized in guanidium isothiocyanate-beta-mercaptoethanol buffer with a Polytron. Total RNA was isolated from frozen tissue samples. For Northern blotting analysis, equal amounts of total RNA (20 g) were denatured by heating at 60C for 10 min and separated on 1% agarose-formaldehyde gels. Ethidium bromide staining of the formaldehyde gel and the transferred blots

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Area under Curve of Glucose

were used for determining the quality of the RNA sample. Treatment groups were always analyzed in parallel. GLUT-4 mRNA level was determined by hybridization with DIG-labeled anti-sense GLUT-4 cRNA. GLUT4 mRNA was quantified on the blots using densitometric analysis with NIH image software. Both 28S ribosomal RNA and beta-actin mRNA were used as an internal standard on each blot. The amount of GLUT-4 mRNA present in each sample was determined by comparing the intensity of the treatment band with control band on each membrane. Glycogen About 50 mg of muscle sample was dissolved in 1 N KOH at 70C for 30 min. Dissolved homogenate was neutralized by glacial acetic acid and incubated overnight in acetate buffer (0.3 M sodium acetate, pH 4.8) containing amyloglucosidase. The reaction mixture was neutralized with 1 N NaOH. Samples were then analyzed by measuring glucosyl units using the Trinder reaction (Sigma, St. Louis, MO, USA). Citrate Synthase Activity Citrate synthase (CS) activity was determined in the plantaris and red gastrocnemius muscles as originally described by Srere (25). Briefly, samples were homogenized in HES buffer in a 1: 40 dilution. The supernatant was assayed spectrophotometrically using DTNB. Assays were performed at 37C in a spectrophotometer (Beckman, Fullerton, CA, USA) equipped with a thermoelectric flow cell and a 1-cm light path. Statistical Analysis A two-way analysis of variance among the experimental groups was performed for all variables. Fishers protected least significance test, which holds the value of type I errors to 0.05 for each test, was used to distinguish significant differences between pairs of groups. P < 0.05 was considered statistically significant. All values are expressed as the means SE.

A
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Area under Curve of Insulin

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Fig. 1. Area under curve of glucose (A) and insulin (B) of oral glucose tolerance. C: control; E: exercise-training; I: IGF-1 administration; IE: IGF-1 administration + exercise training. *Significance against C group, P < 0.05.

Results
OGTT was performed under overnight fasted condition. Fig. 1 displays the mean value for area under curves (AUC) of glucose (Fig. 1A) and insulin (Fig. 1B) during OGTT, as indicators for the whole body glucose tolerance and insulin sensitivity. The AUC of glucose for the E, I, and IE groups was significantly lower than that in the C group (P < 0.05). No significant difference was found among the E, I, and IE groups. The AUC of insulin level was not different among all groups.

Data for muscle glycogen content are showed in Fig. 2. Exercise training significantly elevated glycogen content in RQ and WQ (P < 0.05), whether IGF1 was treated or not. IGF-1 administration alone did not cause significant difference in glycogen content from control for both muscles. For both RQ and WQ, no significant difference in glycogen content was observed between the E and IE groups. All GLUT4 proteins given are relative to mean control level. Data for muscle GLUT4 protein is displayed in Fig. 3 (3A for RQ; 3B for WQ). Both exercise training and IGF-1 administration elevated GLUT4 protein levels in RQ and WQ (P < 0.05). Muscles for the IE group also showed greater GLUT4 protein level than those for the C group (P < 0.05). For both muscles, IGF-1 administration, with exercise or not, significantly elevated GLUT4 protein level

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A
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RQ Glycogen (mmol/g)

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Fig. 2. Glycogen. RQ: red portion of quadriceps muscle; WQ: white portion of quadriceps muscle. *Significance against C group, P < 0.05.

Fig. 3. GLUT4 protein. RQ: red portion of quadriceps muscle; WQ: white portion of quadriceps muscle. *Significance against C group, P < 0.05. #Significance against E group, P < 0.05.

above control and exercise-trained levels (P < 0.05). There is no difference in GLUT4 protein level of RQ and WQ between the I and IE groups. All GLUT4 mRNA given are relative to mean control level. Data for muscle GLUT4 mRNA are displayed in Fig. 4 (4A for RQ; 4B for WQ). Both exercise training and IGF-1 administration elevated GLUT4 mRNA levels in RQ and WQ (P < 0.05). Muscles for the IE group also showed greater GLUT4 mRNA level than those of the C group (P < 0.05). For both muscles, the IGF-1 treated group, with exercise or not, displays greater GLUT4 protein level than those in the control and exercise-trained groups (P < 0.05). For RQ and WQ, no significant difference in GLUT4 protein level was observed between the I and IE groups.

Result for muscle CS activity as a mitochondria marker is shown in Fig. 5 (5A for RQ; 5B for WQ). The activity of CS in the E, I, and IE groups was significantly greater than that in both RQ and WQ (P < 0.05). There is no difference among the E, I, and IE groups.

Discussion
Previous studies on animal muscle have shown that following weight overload there is an increase in the expression of IGF-1 mRNA in muscle (21, 22). Recently, Bamman et al. (2) reported a 62% increase in IGF-1 mRNA concentration in human muscle 48 h after a single bout of exercise. It was unknown how much, or in what aspect, the training effect is mediated by exercise-induced IGF-1 production. Thus we hy-

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RQ GLUT4 mRNA (% control)

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Fig. 4. GLUT4 mRNA. RQ: red portion of quadriceps muscle; WQ: white portion of quadriceps muscle. *Significance against C group, P < 0.05. #Significance against E group, P < 0.05.

Fig. 5. Citrate synthase activity. RQ: red portion of quadriceps muscle; WQ: white portion of quadriceps muscle. *Significance against C group, P < 0.05.

pothesized that, without exercise training, chronic IGF-1 administration can simulate the exercise training effect on GLUT4 protein expression, glycogen storage, and thus affecting the whole-body glucose tolerance. This hypothesis was partially demonstrated by the current study. If exercise and IGF-1 mediate different mechanism to enhance GLUT4 protein expression, then additive effect should be observed. Here we found IGF-1 significantly elevated GLUT4 protein and mRNA level in both IGF-1 treated and exercisetrained muscle, and no additive effect was found. Both white and red skeletal muscles had similar training adaptation in GLUT4 increases, suggesting that both muscle groups were recruited by our exercise training protocol. Cortez has previously reported that exercise training-induced GLUT-4 gene and protein expression occurred only in the recruited muscle (8). It is noteworthy that the improvement in the wholebody glucose tolerance is associated with greater

GLUT4 protein expression, regardless of treatment of IGF-1 or exercise training. This result suggests that exercise training-induced GLUT4 elevation could be mediated by IGF-1 signaling pathway. Alternatively, a cross-talk between exercise-derived signal and IGF-1 signaling pathways to enhance GLUT4 protein expression was reciprocally inhibited to each other (14). Although the increased insulin AUC in the IGF1 injected rats did not reach statistical significance, we could not rule out the possibility that reduced glucose AUC was affected by greater insulin secretion. It has been reported that IGF-1 administration can stimulate beta-cell proliferation (16). To further clarify whether IGF-1 effect is completely mediated by GLUT4 protein expression requires data concerned insulin-stimulated glucose uptake and GLUT-4 protein translocation in skeletal muscle. IGF-1 can also increase the angiogenesis in skeletal muscle which may

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also play a role for the observed improvement in glucose tolerance (18). The generally observed post-exercise glycogen supercompensation (greater than pre-exercise glycogen storage) is partly associated with increased GLUT4 protein expression. The causal relationship was demonstrated in the muscle with greater GLUT4 protein expression by germ-line manipulation that leads to a greater insulin-stimulated glycogen storage (26). The present study that increased glycogen storage by exercise training was occurred in parallel with an increase in GLUT4 protein following a 16-h recovery also supports this idea. However, in the IGF-1 treated group glycogen level was not elevated even though we saw a greater GLUT4 protein level. This result implicates that, during the post-exercise recovery phase, metabolic need for glycogen utilization was also elevated by IGF-1 administration. IGF-1 is generally known as an enhancer for metabolism in skeletal muscle due to greater energy requirement for muscle growth or protein turnover (10). Therefore, the lower glycogen storage in IGF-1 treated muscle compared to the trained muscle could be due to greater glycogen turnover for the enhanced rate of synthesis for protein. The fact that glycogen supercompensation phenomenon was only observed in the exercise-trained muscle, also suggests that motor unit recruitment is essential for the training effect. It is known that the glucose transport across the plasma membrane for providing substrate for glycogen synthesis is relied on the number of GLUT4 protein presence on plasma membrane, which can be regulated by insulin (12). Brozinick et al. (4) has shown that exercise training can increase GLUT4 protein concentration in the proximity adjacent to plasma membrane, as observed 48-h post exercise. Therefore, increased distribution of GLUT4 protein to plasma membrane by exercise training could lead to a faster rate of glucose transport for glycogen synthesis after meal, which might in turn explain why greater glycogen level did not occur in IGF-1 treated muscle without exercise. The ability to maintain glucose homeostasis after carbohydrate ingestion relies on the efficient glucose metabolism in skeletal muscle, resulting from parallel expression of the proteins controlling glucose uptake and disposal, including glucose transporter and mitochondria enzymes for glucose oxidation. Therefore, alteration in the oxidative capacity of a muscle may also contribute a change in glucose disposal property. Many studies have reported that both citrate synthase and GLUT4 protein are regulated in parallel by exercise training (12). In the present study, we found that exercise training and IGF-1 administration significantly elevated citrate synthase activity (as a generic mitochondrial marker) concurrent with

an improvement in insulin sensitivity. This result suggests that the elevation of muscle oxidative capacity with both treatments may also take part in improving glucose tolerance and insulin sensitivity. In addition, this result also implies that the generally observed exercise training effect can be partly simulated by IGF-1 treatment. The fact that no additive effect by exercise-training and IGF-1 also suggests that both stimulations share common signaling mechanism for inducing mitochondria biogenesis. Previous study has shown that the beneficial effect of exercise training on insulin sensitivity is attenuated in elderly and high body mass index individuals (20), the population generally displays lower IGF-1 level (1, 23). Apparently, IGF-1 signaling system is one anabolic signal that can be stimulated by exercise (24, 28). In this study, the evidence implicates that IGF-1 is relevant for upregulating GLUT4 protein expression and citrate synthase activity. Apparently, the benefit of IGF-1 administration could not simulate all the benefits of exercise training. Greater glycogen storage was only observed in exercise-trained muscle, which highlights the unique effect of exercise over the chronic IGF-1 administration. Glycogen is an important anaerobic substrate relevant for the body to encounter metabolic stress, and thus is important for survival under various challenge in normal life. We found that IGF-1 administration can partly mimic the effect of exercise training in enhancing GLUT4 protein expression, citrate synthase activity, and glucose tolerance. However, training-induced glycogen supercompensation was absent with IGF-1 treatment alone, suggesting the importance of muscle contraction component in the full benefit of exercise training.

Acknowledgments
The present work was partly sponsored by Ministry of Education.

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