Adaptive Medicine 3(2): 112-118, 2011DOI: 10.4247/AM.2011.ABB015IGF-1 Partially Reproduces Beneficial Effect of Exercise Training onGlucose Tolerance in Normal RatsChing-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, 31 Laboratory of Exercise Biochemistry, Taipei Physical Education College, Taipei, Taiwan, ROC2 Department of Physical Education, Fu Jen Catholic University, Taipei, Taiwan, ROC3 Department of Physical Therapy, Graduate Institute of Rehabilitation Science, China Medical University, Taichung, Taiwan, ROC4 Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan, ROC5 Department of Physical Medicine and Rehabilitation, Taipei Veterans General Hospital and National Yang Ming University, Taipei, Taiwan, ROC6 Department of Healthcare Administration, Asia University, Taichung, Taiwan, Republic of ChinaExercise transiently elevates the IGF-1 (insulin-like growth Introductionfactor 1) level, but whether exogenous IGF-1 adminis-tration can reproduce exercise training benefit in glycemic Under postprandial conditions, skeletal muscle be-control is currently unknown. This study compared the comes the main site for glucose disposal. Thus, thiseffect of IGF-1 administration and exercise training on tissue plays a pivotal role in regulating whole bodyglycogen storage, glucose tolerance, and muscle glucose glucose homeostasis (3, 9). Glucose transporter 4transporter 4 (GLUT4) protein expression in normal rats. (GLUT4) is the main glucose transporter isoformForty rats were weight matched and evenly assigned tothe following 4 groups: control (C), exercise trained (E), expressed in skeletal muscle, which can be rapidlyIGF-1 treated (I), and exercise-trained + IGF-1 (EI). Same recruited to the plasma membrane upon insulinvolume of saline or IGF-1 (2 µg/kg BW) was injected daily stimulation. This protein translocation increases theto the rats. Exercise training consisted of daily 90 min of membrane permeability to circulating glucose andswimming for the first week and gradually increased to thus increases glucose disposal in skeletal muscle.180 min, twice for the third week. Oral glucose tolerance Several early reports found that the amount of GLUT4test (OGTT) was performed in all rats under fasted protein is strongly correlated with maximal insulin-condition. Muscle tissues were removed at the end of stimulated glycogen storage in skeletal muscle (13,the 3-week treatments (3 days after OGTT). The levels of 17). Therefore, interventions that enhance muscleGLUT4 protein and mRNA were determined in red and GLUT4 protein expression might be a possible methodwhite portions of the quadriceps muscle (RQ and WQ). for treating patients with insulin resistance and typeBoth exercise training and chronic IGF-1 administration 2 diabetes.increased GLUT4 expression and improved glucose The beneficial consequence of regular exercisetolerance without an observed additive effect. Exercise training on glucose tolerance, insulin sensitivity, andtraining increased glycogen level in RQ and WQ abovecontrol level. Despites chronic IGF-1 administration muscle glycogen storage has been previously reportedincreased muscle GLUT4 expression above control level, in humans and animals (6, 7, 12). This improvementglycogen increase was not observed. Our data suggest is thought, to some extent, associated with the in-that IGF-1 can partially reproduce exercise training effect creased GLUT4 protein expression in exercised muscleon improving glycemic control. (6, 17, 27). The underlying mechanism for the ex- ercise-induced increase in GLUT4 expression hasKey Words: insulin-like growth factor, insulin resistance, not been fully understood. Available data suggest diabetes, glycogen, aging 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, JhongchengRd., Shihlin District, Taipei City 111, Taiwan, Republic of China. Fax: +886-2-28753383, E-mail: firstname.lastname@example.org*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 112
Exercise and Muscle Fiber Conversion 113like growth factor 1) level (5, 24, 28), and this signal ercise, according to Hou et al. (11). This recovery pe-has been suggested to be associated with exercise riod included a 12-h fast prior to the glucose intubationtraining-dependent improvement in muscle prop- for OGTT. Blood samples were withdrawn from theerties (5, 19). In this study, we compared the effect tail at 0 (fasted sample), 15, and 45 min after the oralof IGF-1 administration and exercise training on glucose load (1 g/kg BW) for blood glucose and se-GLUT4 protein expression in rat muscles, as well as rum insulin measurements, according to the procedureits association to glycogen storage and whole-body given in Cortez et al. (8). A glucose analyzer (Lifescan,glucose tolerance. Milpitas, CA, USA) was used for glucose concentra- tion determination; the glucose oxidase method wasMaterials and Methods used. The consistency of the analyzer was tested using real blood samples twice before use. SerumAnimal Care and Experimental Design insulin levels were measured using enzyme-linked immunosorbent assay (ELISA) with anti-insulinForty male Sprague-Dawley rats from the National monoclonal antibody.Animal Laboratory of the NSC (National ScienceCouncil, Taipei, Taiwan, ROC) weighing 200 g each GLUT4 Proteinwere housed 3 per cage and were provided normal ratchow (PMI Nutrition International, Brentwood, MO, Three days after the OGTT, and 18 h after the last ex-USA) and water ad libitum. The temperature of the ercise bout with an immediate glucose intubation (1animal room was maintained at 23°C, with a 12-h g/kg BW), muscles were surgically removed forlight-dark cycle. After 1 week of familiarization, the analysis of glycogen, and GLUT4 protein levels, andrats were weight-matched and divided into 4 groups: citrate synthase activity assay. Muscle samples forcontrol (C, n = 10), exercise (E, n = 10), IGF-1 (I, n = GLUT4 protein were homogenized in ice-cold HES10), and exercise + IGF-1 (EI, n = 10). Exercise train- (20 mM N-2-hydroxyethylpiperazine-N’-2-ethane-ing protocol consisted of 90 min of swimming for sulfonic acid, 1 mM EDTA, and 250 mM sucrose, pHthe first week and gradually increased to 180 × 2 min 7.4) buffer (1: 20) with a Polytron homogenizer(1-h rest in between) for the third week. Swimming (Kinematica, Littau, Switzerland). Sample homo-training started at 9 am every morning with a break on genates and standards were diluted 1: 1 with LaemmliSunday. The temperature of the water, 25 cm in depth sample buffer (125 mM Tris, 20% glycerol, 2% SDS,in a plastic barrel, was maintained at 34° ± 1°C; 3 rats and 0.008% bromophenol blue, pH 6.8). The Westernwere placed in each barrel at the same time. For IGF- blotting procedure for GLUT4 analysis was followed1 administration, recombinant human IGF-1 was pro- the previously described method (11). Muscle homo-duced from eukaryotic cells (Leinco Technologies, genates containing 75 µg (red gastrocnemius andSt. Louis, MO, USA). IGF1 was dissolved in saline plantaris muscles) of protein were subjected to SDS-and was injected (ip) 2 µg/kg 1 h after exercise polyacrylamide gel electrophoresis (PAGE) andtraining (15). At the end of the third week, rats were electrophoretically transferred to a PVDF membrane.anesthetized at a time when they had recovered 18 h Two heart homogenates containing 15 and 30 µg ofafter the cessation of the last exercise bout, and red protein were loaded in parallel with the muscle sam-and white portion of the quadriceps muscles were ex- ples. GLUT4 antiserum (Chemicon, Temecula, CA,cised and examined for GLUT4 protein level, glycogen USA) was used for immunoblotting (directly againstcontent, and citrate synthase activity. Oral glucose the carboxyl-terminus of the GLUT4 protein) in a di-tolerance test (OGTT) was performed 3 days before lution of 1: 5000. GLUT4 protein was visualized us-muscle sample excision; and the muscle tissue analysis ing an ECL Western blot detection kit (Amersham,for glycogen, GLUT4 protein, and citrate synthase Arlington Heights, IL, USA) on x-ray film accordingwas performed 3 days after the OGTT to avoid the to the manufacturer’s instructions.potential interference effect of OGTT procedure onthe muscle glycogen result (IGF-1 was continuously GLUT4 mRNAgiven during the next 3 days after OGTT). The samevolume of saline was daily injected as a placebo into For RNA extraction, muscle tissues were homogenizedcontrol animals. In the EI group, IGF-1 was dissolved in guanidium isothiocyanate-beta-mercaptoethanolin saline and injected (2 µg/kg) 1 h after exercise buffer with a Polytron. Total RNA was isolated fromtraining. frozen tissue samples. For Northern blotting analysis, equal amounts of total RNA (20 µg) were denaturedOral Glucose Tolerance Test (OGTT) by heating at 60°C for 10 min and separated on 1% agarose-formaldehyde gels. Ethidium bromide stain-OGTT was performed 18 h after the last bout of ex- ing of the formaldehyde gel and the transferred blots
114 Tzeng, Lai, Hou, Chen, Lee, Huang, Chen, Liu, Liou, Kao and Kuowere used for determining the quality of the RNA Asample. Treatment groups were always analyzed in 2500parallel. GLUT-4 mRNA level was determined byhybridization with DIG-labeled anti-sense GLUT-4 Area under Curve of GlucosecRNA. GLUT4 mRNA was quantified on the blots 2000using densitometric analysis with NIH image software. *Both 28S ribosomal RNA and beta-actin mRNA were 1500 *used as an internal standard on each blot. The amount *of GLUT-4 mRNA present in each sample was deter-mined by comparing the intensity of the treatment 1000band with control band on each membrane.Glycogen 500About 50 mg of muscle sample was dissolved in 1 N 0KOH at 70°C for 30 min. Dissolved homogenate was C E I IEneutralized by glacial acetic acid and incubated over-night in acetate buffer (0.3 M sodium acetate, pH 4.8) Bcontaining amyloglucosidase. The reaction mixture 50was neutralized with 1 N NaOH. Samples were then 45analyzed by measuring glucosyl units using the Trinder 40 Area under Curve of Insulinreaction (Sigma, St. Louis, MO, USA). 35Citrate Synthase Activity 30 25Citrate synthase (CS) activity was determined in theplantaris and red gastrocnemius muscles as originally 20described by Srere (25). Briefly, samples were homo- 15genized in HES buffer in a 1: 40 dilution. The super- 10natant was assayed spectrophotometrically usingDTNB. Assays were performed at 37°C in a spectro- 5photometer (Beckman, Fullerton, CA, USA) equipped 0with a thermoelectric flow cell and a 1-cm light path. C E I IE Fig. 1. Area under curve of glucose (A) and insulin (B) of oralStatistical Analysis glucose tolerance. C: control; E: exercise-training; I: IGF-1 administration; IE: IGF-1 administration + exer-A two-way analysis of variance among the experi- cise training. *Significance against C group, P < 0.05.mental groups was performed for all variables. Fisher’sprotected least significance test, which holds the val-ue of type I errors to 0.05 for each test, was used to Data for muscle glycogen content are showed indistinguish significant differences between pairs of Fig. 2. Exercise training significantly elevated glyco-groups. P < 0.05 was considered statistically signifi- gen content in RQ and WQ (P < 0.05), whether IGF-cant. All values are expressed as the means ± SE. 1 was treated or not. IGF-1 administration alone did not cause significant difference in glycogen contentResults from control for both muscles. For both RQ and WQ, no significant difference in glycogen content was ob-OGTT was performed under overnight fasted con- served between the E and IE groups.dition. Fig. 1 displays the mean value for area under All GLUT4 proteins given are relative to meancurves (AUC) of glucose (Fig. 1A) and insulin (Fig. control level. Data for muscle GLUT4 protein is dis-1B) during OGTT, as indicators for the whole body played in Fig. 3 (3A for RQ; 3B for WQ). Both ex-glucose tolerance and insulin sensitivity. The AUC ercise training and IGF-1 administration elevatedof glucose for the E, I, and IE groups was significantly GLUT4 protein levels in RQ and WQ (P < 0.05).lower than that in the C group (P < 0.05). No signifi- Muscles for the IE group also showed greater GLUT4cant difference was found among the E, I, and IE protein level than those for the C group (P < 0.05).groups. The AUC of insulin level was not different For both muscles, IGF-1 administration, with exerciseamong all groups. or not, significantly elevated GLUT4 protein level
Exercise and Muscle Fiber Conversion 115 A A 60 3.0 50 * * 2.5 *# *# RQ GLUT4 Protein (% control) RQ Glycogen (mmol/g) 40 2.0 * 30 1.5 20 1.0 10 0.5 0 C E I IE 0.0 C E I IE B B 45 1.8 40 * 1.6 *# * *# WQ GLUT4 Protein (% control) 35 1.4 WQ Glycogen (mmol/g) * 30 1.2 25 1.0 20 0.8 15 0.6 10 0.4 5 0.2 0 0.0 C E I IE C E I IEFig. 2. Glycogen. RQ: red portion of quadriceps muscle; Fig. 3. GLUT4 protein. RQ: red portion of quadriceps muscle; WQ: white portion of quadriceps muscle. *Significance WQ: white portion of quadriceps muscle. *Significance against C group, P < 0.05. against C group, P < 0.05. #Significance against E group, P < 0.05.above control and exercise-trained levels (P < 0.05). Result for muscle CS activity as a mitochondriaThere is no difference in GLUT4 protein level of RQ marker is shown in Fig. 5 (5A for RQ; 5B for WQ). Theand WQ between the I and IE groups. activity of CS in the E, I, and IE groups was signifi- All GLUT4 mRNA given are relative to mean cantly greater than that in both RQ and WQ (P < 0.05).control level. Data for muscle GLUT4 mRNA are There is no difference among the E, I, and IE groups.displayed in Fig. 4 (4A for RQ; 4B for WQ). Both ex-ercise training and IGF-1 administration elevated DiscussionGLUT4 mRNA levels in RQ and WQ (P < 0.05).Muscles for the IE group also showed greater GLUT4 Previous studies on animal muscle have shown thatmRNA level than those of the C group (P < 0.05). For following weight overload there is an increase inboth muscles, the IGF-1 treated group, with exercise the expression of IGF-1 mRNA in muscle (21, 22).or not, displays greater GLUT4 protein level than Recently, Bamman et al. (2) reported a 62% increasethose in the control and exercise-trained groups (P < in IGF-1 mRNA concentration in human muscle 48 h0.05). For RQ and WQ, no significant difference in after a single bout of exercise. It was unknown howGLUT4 protein level was observed between the I much, or in what aspect, the training effect is mediatedand IE groups. by exercise-induced IGF-1 production. Thus we hy-
116 Tzeng, Lai, Hou, Chen, Lee, Huang, Chen, Liu, Liou, Kao and Kuo A A 4.0 60 *# * * *# * 3.5 50 RQ GLUT4 mRNA (% control) RQ CS Activity (µmol/g/min) 3.0 40 2.5 * 2.0 30 1.5 20 1.0 10 0.5 0.0 0 C E I IE C E I IE B 3.0 B 30 2.5 *# WQ GLUT4 mRNA (% control) *# 25 * WQ CS Activity (µmol/g/min) * * 2.0 20 * 1.5 15 1.0 10 0.5 5 0.0 0 C E I IE C E I IEFig. 4. GLUT4 mRNA. RQ: red portion of quadriceps muscle; Fig. 5. Citrate synthase activity. RQ: red portion of quadriceps WQ: white portion of quadriceps muscle. *Significance muscle; WQ: white portion of quadriceps muscle. *Sig- against C group, P < 0.05. #Significance against E group, nificance against C group, P < 0.05. P < 0.05.pothesized that, without exercise training, chronic GLUT4 protein expression, regardless of treatmentIGF-1 administration can simulate the exercise training of IGF-1 or exercise training. This result suggestseffect on GLUT4 protein expression, glycogen storage, that exercise training-induced GLUT4 elevation couldand thus affecting the whole-body glucose tolerance. be mediated by IGF-1 signaling pathway. Alterna-This hypothesis was partially demonstrated by the tively, a cross-talk between exercise-derived signalcurrent study. If exercise and IGF-1 mediate different and IGF-1 signaling pathways to enhance GLUT4mechanism to enhance GLUT4 protein expression, protein expression was reciprocally inhibited to eachthen additive effect should be observed. Here we other (14).found IGF-1 significantly elevated GLUT4 protein Although the increased insulin AUC in the IGF-and mRNA level in both IGF-1 treated and exercise- 1 injected rats did not reach statistical significance,trained muscle, and no additive effect was found. we could not rule out the possibility that reduced glu- Both white and red skeletal muscles had similar cose AUC was affected by greater insulin secretion.training adaptation in GLUT4 increases, suggesting It has been reported that IGF-1 administration canthat both muscle groups were recruited by our exercise stimulate beta-cell proliferation (16). To furthertraining protocol. Cortez has previously reported that clarify whether IGF-1 effect is completely mediatedexercise training-induced GLUT-4 gene and protein by GLUT4 protein expression requires data concernedexpression occurred only in the recruited muscle (8). insulin-stimulated glucose uptake and GLUT-4 proteinIt is noteworthy that the improvement in the whole- translocation in skeletal muscle. IGF-1 can also in-body glucose tolerance is associated with greater crease the angiogenesis in skeletal muscle which may
Exercise and Muscle Fiber Conversion 117also play a role for the observed improvement in an improvement in insulin sensitivity. This resultglucose tolerance (18). suggests that the elevation of muscle oxidative The generally observed post-exercise glycogen capacity with both treatments may also take part insupercompensation (greater than pre-exercise glyco- improving glucose tolerance and insulin sensitivity.gen storage) is partly associated with increased GLUT4 In addition, this result also implies that the generallyprotein expression. The causal relationship was dem- observed exercise training effect can be partly simu-onstrated in the muscle with greater GLUT4 protein lated by IGF-1 treatment. The fact that no additiveexpression by germ-line manipulation that leads to a effect by exercise-training and IGF-1 also suggestsgreater insulin-stimulated glycogen storage (26). The that both stimulations share common signalingpresent study that increased glycogen storage by ex- mechanism for inducing mitochondria biogenesis.ercise training was occurred in parallel with an in- Previous study has shown that the beneficial ef-crease in GLUT4 protein following a 16-h recovery fect of exercise training on insulin sensitivity is at-also supports this idea. However, in the IGF-1 treated tenuated in elderly and high body mass indexgroup glycogen level was not elevated even though individuals (20), the population generally displayswe saw a greater GLUT4 protein level. This result lower IGF-1 level (1, 23). Apparently, IGF-1 signalingimplicates that, during the post-exercise recovery system is one anabolic signal that can be stimulatedphase, metabolic need for glycogen utilization was by exercise (24, 28). In this study, the evidencealso elevated by IGF-1 administration. IGF-1 is gen- implicates that IGF-1 is relevant for upregulatingerally known as an enhancer for metabolism in skeletal GLUT4 protein expression and citrate synthasemuscle due to greater energy requirement for muscle activity. Apparently, the benefit of IGF-1 adminis-growth or protein turnover (10). Therefore, the lower tration could not simulate all the benefits of exerciseglycogen storage in IGF-1 treated muscle compared training. Greater glycogen storage was only observedto the trained muscle could be due to greater glyco- in exercise-trained muscle, which highlights thegen turnover for the enhanced rate of synthesis for unique effect of exercise over the chronic IGF-1protein. administration. Glycogen is an important anaerobic The fact that glycogen supercompensation phe- substrate relevant for the body to encounter metabolicnomenon was only observed in the exercise-trained stress, and thus is important for survival under variousmuscle, also suggests that motor unit recruitment is challenge in normal life.essential for the training effect. It is known that the We found that IGF-1 administration can partlyglucose transport across the plasma membrane for mimic the effect of exercise training in enhancingproviding substrate for glycogen synthesis is relied GLUT4 protein expression, citrate synthase activity,on the number of GLUT4 protein presence on plasma and glucose tolerance. However, training-inducedmembrane, which can be regulated by insulin (12). glycogen supercompensation was absent with IGF-1Brozinick et al. (4) has shown that exercise training treatment alone, suggesting the importance of musclecan increase GLUT4 protein concentration in the contraction component in the full benefit of exerciseproximity adjacent to plasma membrane, as observed training.48-h post exercise. Therefore, increased distributionof GLUT4 protein to plasma membrane by exercise Acknowledgmentstraining could lead to a faster rate of glucose transportfor glycogen synthesis after meal, which might in turn The present work was partly sponsored by Ministry ofexplain why greater glycogen level did not occur in Education.IGF-1 treated muscle without exercise. The ability to maintain glucose homeostasis Referencesafter carbohydrate ingestion relies on the efficientglucose metabolism in skeletal muscle, resulting from 1. Alderete, T.L., Byrd-Williams, C.E., Toledo-Corral, C.M., Conti,parallel expression of the proteins controlling glucose D.V., Weigensberg, M.J. and Goran, M.I. 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