63 Journal of Exercise Physiologyonline June 2015 Volume 18 Number 3 Editor-in-Chief Official Research Journal of Tommy the American Boone, PhD, Society MBA of Review Board Exercise Physiologists Todd Astorino, PhD Julien Baker, ISSN 1097-9751 PhD Steve Brock, PhD Lance Dalleck, PhD Eric Goulet, PhD Robert Gotshall, PhD Alexander Hutchison, PhD M. Knight-Maloney, PhD Len Kravitz, PhD James Laskin, PhD Yit Aun Lim, PhD Lonnie Lowery, PhD Derek Marks, PhD Cristine Mermier, PhD Robert Robergs, PhD Chantal Vella, PhD Dale Wagner, PhD Frank Wyatt, PhD Ben Zhou, PhD Official Research Journal of the American Society of Exercise Physiologists ISSN 1097-9751 JEPonline The Comparative Effects of Two Different Carbohydrate Gels on Post-Exercise Glucose and Plasma Free-Fatty Acids of Long Distance Runners Beatriz G. Ribeiro1, Roberto Carlos-Burini2, Tiago C. Leite1, Anderson P. Morales1,3, Felipe Sampaio-Jorge1,3, Gabriela M. O. Coelho1 1Laboratory Research and Innovation in Sports Sciences, Federal University of Rio de Janeiro/Macaé, RJ, Brazil, 2Department of Public Health, Botucatu Medical School - University State São Paulo, São Paulo, Brazil, ³Laboratory of Chemistry and Biomolecules, Higher Institutes of Education of CENSA, Campos dos Goytacazes, RJ, Brazil ABSTRACT Ribeiro BG, Carlos-Burini R, Leite TC, Morales AP, SampaioJorge F, Coelho GMO. The Comparative Effects of Two Different Carbohydrate Gels on Post-Exercise Glucose and Plasma Free-Fatty Acids of Long Distance Runners. JEPonline 2015;18(3):63-73. The purpose of this study was to determine the post-exercise glucose and free fatty acid (FFA) responses to two different carbohydrate gel (CHO gel) supplementations. Body composition and dietary intake of 8 long distance runners were assessed prior to a 90-min run at 70% VO2 max on a motorized treadmill. Blood samples were collected preand post-exercise when they received 1 g·CHO·kg–1 orally, either as maltodextrin-glucose-fructose (MGF) or maltodextrin-glucose (MG) or placebo (P). In both MGF and MG trials, blood glucose levels increased similarly during the first 30 min of recovery in response to the post-exercise ingested CHO supplement. In the P trial, blood glucose levels remained unchanged during recovery. The area under the curve was higher after the consumption of MG than MGF during recovery. Plasma FFA levels decreased in both CHO supplement trials and remained suppressed during recovery when compared to the P trial (P<0.05). We conclude that supplement CHO gel MG trial induced different patterns of plasma glucose postprandial time 60 and 90 min after exercise. Plasma FFA levels showed similar responses after exercise, regardless of supplemental CHO (MG and MGF trials). Furthermore, the decrease on plasma glucose levels occurred earlier with MGF loading rather than MG alone. Key Words: Carbohydrate Gel Supplementation, Athletes 64 INTRODUCTION Post-exercise recovery represents an important challenge to modern athletes. Many athletes undertake strenuous training programs involving one or more prolonged high-intensity exercise sessions each day, typically with 6 to 24 hrs for recovery between sessions. This usually leads to near depletion of body glycogen stores. Maximal glycogen storage is desirable for both athletic competition and quality training. Muscle glycogen resynthesis after prolonged exercise has been well investigated in relation to the amount, and timing of ingested carbohydrates (CHO) (5,21,24,30). Ingestion of high CHO foods after exercise can increase liver and muscle glycogen storage. The optimum restoration speed of muscular glycogen for athletes can be achieved when 1.0 to 1.85 g·CHO·kg-1·BW·hr-1 is offered right after exercise (4). However, the efficacy of a particular CHO in promoting glycogen resynthesis depends on the CHO composition (6), and also on the plasma glucose response to the CHO load (10). Previous studies have shown that ingestion of a high glycemic index (GI) CHO is more effective in promoting muscle glycogen resynthesis than the ingestion of a lower GI CHO (3,29). On the contrary, Donaldson, Perry and Rose (9) reported in a review study that the GI is not as important as the interaction between CHO and fat oxidation, which may play a role in increasing endurance exercise performance. Fructose seems to promote only a very modest increase in muscle glycogen, while the consumption of fructose or sucrose provides a significant increase in liver glycogen storage during recovery period (5,8). Little is known about the influence of co-ingestion of glucose and fructose supplementation (e.g., pre-exercise, during exercise, and post-exercise) on the plasma residence time of glucose and FFA post-exercise. The metabolic fate of fructose supplementation includes greater FFA blood concentration, glucose, and glycogen synthesis (12). On the other hand, glucose supplementation decreases post-exercise FFA concentration by the inhibition of lipolysis in adipocyte leading to a lower rate of FFA oxidation (28). The purpose of this study was to determine the post-exercise glucose and FFA responses to two different CHO gels, maltodextrin-glucose-fructose or maltodextrin-glucose, during a 2-hr recovery from 90 min of submaximal motorized treadmill running. METHODS Subjects Eight long distance runners, volunteered to participate in this study. The mean age (30 4.7 yrs), height, body mass, lean body mass, maximum oxygen uptake (VO 2 max) and maximum heart rate (HR max) were determined (Table 1). All subjects were considered healthy, non-smokers. They were not taking any medication, and did not have evidence of cardiovascular or metabolic diseases. The experimental procedures and possible study risks were verbally explained. Afterwards, each subject signed an informed consent form. The study protocol was approved by the ethics committee of the Federal University of Rio de Janeiro. 65 Table 1. Subjects Characteristics. Variables Height (cm) 174 8.1 Weight (kg) 62.8 6.9 Body fat (%) 5.4 1.0 LBM (kg) 59.3 6.4 VO2 Max (mL·kg-1·min-1) 63.3 4.3 Heart Rate (beats·min-1) 183.8 9.3 Training Frequency (times·wk-1) 6.0 0.4 Training Duration (hrs·wk-1) 9.8 2.3 Values (Mean ± SD) for 8 subjects; LBM = Lean Body Mass; VO2 max = Maximal Oxygen Uptake Pre-Experiment Measurements Daily energy intake and dietary composition of habitual and 24-hr pre-test meals of long distance runners were assessed using a 3-day food record. Dietary data (Table 2) were analyzed for energy and macronutrient intake by the Evaluation System Nutritional Center for Health Informatics Paulista School of Medicine, version 2.0, Brazil. One week before the start of experimental trials, the subjects were familiarized with running on the treadmill and experimental procedures. During the familiarization session, each subject was given instructions for the use (i.e., proper running form) of the motorized treadmill (Inbrasport Master®). The subjects undertook one preliminary test in order to determine VO 2 max using an uphill incremental treadmill running test to exhaustion (17). Expired air samples were collected using the Cardio2 CPX/D Med Graphics Cardiopulmonary Exercise System and analyzed for VO2, carbon dioxide (VCO2) production, and respiratory exchange ratio (RER). The VO 2 value obtained during the last min of the VO2 max test was taken as the VO2 max value of the individual. One week before the first experimental trial, the subjects undertook a 60-min treadmill run at 70% VO2 max in order to confirm the relative exercise intensity and to fully familiarize themselves with prolonged treadmill running and the measurements used during the experimental trials. Testing Sessions The subjects arrived at the laboratory at 7 a.m. on three occasions, each separated by 1 wk. They were instructed to abstain from caffeine, alcohol, tobacco, and from heavy exercise 24 hrs prior to each main trial. Daily energy intake and composition of each subject’s diet were assessed using a 24hr recall (Table 2). Before body mass (BM) was obtained, each subject was asked to empty his bladder. Subsequently, a catheter (BD AngiocathTM, 18GAX1.88in, Bencton Dickinson, Brazil) was inserted in an ante-cubital vein. A 10 mL resting venous blood sample was obtained. 66 In order to standardize the effect of dehydration on muscle metabolism during exercise, the subjects consumed 4 mL·kg-1·BM-1 of cool water immediately before the warm-up and 2 mL·kg-1·BM-1 of water every 20 min during the treadmill running test. The warm-up consisted of a 5-min run at an exercise intensity equivalent to 60% of VO2 max, which was followed by a 90-min run at 70% of VO2 max. A second venous blood sample was collected immediately after the run was completed. Then, the subject’s dry post-exercise nude body mass was obtained. The subjects recovered for 2 hrs. Immediately after the treadmill running test the subjects ingested a CHO gel that provided 1 g·kg-1· BW·hr-1 of carbohydrate or placebo gel. The subjects ingested a CHO gel, over 2 min (t = −2 to 0 min), of either: (a) MGF: 35% maltodextrin, 50% glucose and 15% fructose; (b) MG: 50% maltodextrin and 50% glucose: and (c) P: artificial sweetener. The artificially sweetened placebo mixture consisted of sucralose® and was indistinguishable from the CHO-gel mixture. The CHO-gel was prepared according to procedures described by Pierucci, Ribeiro, and Soares (25). The order of the experiments was randomized and administered in a blind cross-over design. The subjects remained in the laboratory and venous blood samples were taken every 30 min during a 2-hr resting recovery period to determine glucose and FAA blood levels. Blood Sample Collection and Analysis Each venous blood sample was collected into an EDTA tube except for a 3 mL aliquot, which was placed into a Sodium Flouride di-sodic EDTA for subsequent plasma glucose colorimetric determination. Plasma was obtained by centrifugation at 2.500 rev·min-1 at 4ºC for 20 min. The resultant plasma was stored at -20ºC until the analyses were performed. Plasma was analyzed for glucose using a commercially available kit (GOD-POD-Kit Celm, Brazil), and for FFA with the colorimetric method described by Novak (20). Blood (0.5 mL) was immediately analyzed for hemoglobin concentrations (Kit Cellmlise II, CC-530 Celm, Brazil), and hematocrit using micro capillary method. The hemoglobin and hematocrit values were used to calculate changes in plasma volume (VP) (1). All analyses were made in duplicate. Statistical Analyses Descriptive statistics was used for all measured outcomes. The data are expressed as mean ± SD. Differences between treatment groups were determined by two-way ANOVA of which one factor is subject and the other is group. If a significant difference was indicated (P<0.05), the Tukey’s post hoc test was used. RESULTS Dietary Assessment The average total energy intake and dietary composition of 24 hrs before the trial was similar to the daily habitual energy intake (Table 2). Total CHO intake was 9.9 ± 3.3 g·kg-1, 9.8 ± 3.7 g·kg-1, and 10.2 ± 3.5 g·kg-1 in the MGF, MG, and P trials, respectively. Dietary intake did not differ among the trials. 67 Table 2. Dietary Composition of Habitual and 24 Hrs Pre-Test Meals of Long Distance Runners. Nutritional Analysis Dietary 24 Hrs 24 Hrs 24 Hrs Component Before Before Before Habitual MGF MG P 4234.0 1565 3984.2 1125 CHO (g) 633.3 209.9 624.0 193.8 615.2 237.9 641.0 222.7 CHO (%) 59.8 5.7 62.5 7.3 56.9 6.1 59.6 6.5 Protein (g) 154.7 48.2 157.2 50.1 186.3 108.4 175.5 54.2 Protein (%) 14.9 2.8 16.0 2.75 15.9 2.31 16.7 2.7 Fat (g) 125.2 73.1 100.7 41.5 141.1 81.2 119.6 65.4 Fat (%) 25.2 7.4 22.4 6.2 27.1 6.4 23.7 5.9 9.9 2.4 9.9 3.0 9.8 3.8 10.2 3.6 Energy (kcal) CHO (g·kg·BM-1·d-1) 4458.4 2017 4345.8 1569 Values (Mean ± SD) for 8 Subjects. Maltodextrin-Glucose-Fructose (MGF), Maltodextrin-Glucose (MG), and Placebo (P); CHO = Carbohydrates; BM = Body Mass Fluid Ingestion, Volume Plasma Changes, and Body Mass The mean water ingested was 848.7 ± 140.9 mL in the MGF trial, 926.2 ± 224.1 mL in the MG trial, and 1000.0 ± 173.9 mL in the P trial during motorized treadmill. The consumption did not differ among trials. Percentage changes in plasma volume were similar between the trials during motorized treadmill (MGF: + 3 ± 4%; MG: +2 ± 4%; P: +5 ± 6%). Changes in body mass were similar among the three trials during the treadmill running test (MGF: 1.8 ± 0.4 kg; MG: 1.7 ± 0.3 kg; P: 1.7 ± 0.5 kg). Blood Glucose In both the MGF and the MG trials, the subjects’ blood glucose levels increased during the first 30 min of the recovery period in response to the CHO gel ingested after the treadmill running test (Figure 1). In the P trial, the blood glucose levels remained unchanged during recovery period. In contrast, in the MG trial, the blood glucose levels remained high until the 60 min of recovery period and was higher (P<0.05) compared to P and at 90 min compared to MGF trial (Figure 1). The area under the curve of plasma glucose levels was geometrically calculated by applying the trapezoid rule to provide a measure of plasma glucose response to oral CHO gel load. The area under the curve was higher after consumption of MG (87.12 mmol·L -1) than MGF (72.15 mmol·L-1) during the recovery period. 68 Figure 1. Plasma Glucose Concentration Before (Pre) and After (Post) 90-Min Treadmill Run at 70% VO2 max and During Recovery After Ingestion or Not of 1 g·CHO·kg–1 Orally. Values are means SE (error bars) for 8 subjects. Maltodextrin-Glucose-Fructose (MGF), Maltodextrin-Glucose (MG), and Placebo (P). *Represents a significant difference between P and MGF and MG trials (P<0.05). **Represents a significant difference between P and MG trial (P<0.05). ***Represents a significant difference between MGF and MG trials (P<0.05). Plasma FFA Plasma FFA levels were higher after exercise compared to the resting values (P<0.05) (Figure 2). FFA blood concentrations levels decreased in both CH gel trials (MGF and MG) and remained suppressed until 90 min of recovery period when compared to P trial (P<0.05). Furthermore, the blood FFA levels remained high in the P trial during recovery period. Figure 2. Plasma FFA Concentration Before (Pre) and After (Post) 90-Min Treadmill Run at 70% VO2 Max and During Recovery After Ingestion or Not of 1 g·CHO·kg–1 Orally. Means SE (error bars). Values are means SD for 8 subjects. Maltodextrin-Glucose-Fructose (MGF), Maltodextrin-Glucose (MG), and Placebo (P). *Represents a significant difference between P in post exercise time point and are both trials (MGF and MG), P<0.05. 69 DISCUSSION Accepted standards on carbohydrate ingestion studies that promote glucose and FFA responses have always been performed with intravenous infusions of glucose and fructose or with oral ingestion of glucose polymers, sucrose, glucose or fructose isolated taken in liquid or solid states (5,24,30). This study focused on the effects of oral CHO gel supplement intake on plasma glucose and FFA during a 2-hr recovery period. The CHO gel consisted of maltodextrin - glucose with or without fructose because athletes use these supplements regularly, especially when the sport includes aerobic resistance training. The main finding in this study is that different CHO gel induced different postprandial plasma glucose patterns. The maltodextrin-glucose-fructose (MGF) gel resulted in a smaller area of the glucose curve after 60 and 90 min of recovery from exercise compared to the maltodextrin-glucose (MG) gel. The results also showed that the MG gel stimulated a higher glycemic response than the MGF gel without changing the plasma FFA concentrations. Some studies have demonstrated that energy supplements containing fructose and sucrose resulted in lower glycemic response when compared to those containing either glucose or its polymers (5,24,30). Elevation of glucose levels, as well as its duration depends on carbohydrate absorption speed especially glucose absorption speed, which varies according to several factors such as gastric emptying, hydrolysis speed, and the diffusion of hydrolyzed products in the small intestine (4). It is well known that fructose by itself has a low rate of intestinal absorption, but the mixture of glucose and fructose is well absorbed through additional transport mechanisms (32). This mechanism is explained by the different membrane transport, as facilitated passive transport independent of sodium via GLUT5 for fructose and facilitated diffusion via SGLT1 for glucose (33). It is believed that glucose and fructose are not competing for the same carrier, indicating a better efficiency in absorption and oxidation (13). Currell and Jeukendrup (7) confirmed that a mixture of glucose and fructose (1.2 and 0.6 g·min-1, respectively), resulted in significantly better cycling performance in a time-trial compared with a beverage containing only glucose. In the present study, the addition of fructose to glucose and maltodextrin in CHO gel did not reduce the 30-min postprandial glucose response (Figure 1). On the other hand, the lower AUC for the MGF could be explained by the difference in CHO composition of the gels. The fructose metabolism is predominantly hepatic with slight impact of insulin levels and/or glucose plasma levels in healthy subjects (16). Petersen and colleagues (22) detected that fructose infusion tripled hepatic glycogen synthesis in healthy subjects during the euglycemic hyper insulin clamp. The mechanism is not fully understood but it is known that fructose-1-phosphate (a metabolite formed in the liver by fructokinase that phosphorylates quickly fructose, using a molecule of ATP), has the ability to dissociate glucokinase from glucokinase regulatory protein (GKRP). The glucokinase dissociation from GKRP allows the glucokinase translocation from nucleus to the cytosol where it can phosphorylate glucose, thus increasing liver glycogen resynthesis (19). This result is well established through negative correlation in a high level of intracellular glucose-6-phosphate (via glucose substrate) and a low concentration of ATPs (via substrate fructose) G6-P/ATP with the activation state of enzyme glycogen synthase (GS) in hepatocytes from normal and diabetic animals incubated with a mixture of 20 mM glucose and 3 mM fructose (6). This is a likely explanation for the decreased blood glucose found in MGF compared to MG (Figure 1), within 90 min of recovery. 70 However, Casey et al. (5) found that the consumption of 1 g·kg-1 of weight of glucose post-exercise promoted higher increase of plasma glucose than equal amounts of sucrose. Besides glycemia differences, storage of glycogen found in the liver and the muscle were similar to both groups. Differences in CHO administration route (oral x parenteral) and in techniques of liver glycogen evaluation could explain the diversity of results found in different studies. Casey et al. (5) pointed out that, 1 g·kg-1 BM glucose or sucrose is sufficient to start post-exercise liver glycogen resynthesis. Therefore, the intake of energy supplements containing fructose between intense workouts would provide energy substrates for liver glycogen restoring and physical exercise continuation. The behavior of plasma glucose and FFA varies after exercise based upon the intensity and duration of the physical effort. In accordance with previous studies (2,14,15) the present study (after the 90min run at 70% of VO2 max) showed a significant increase of FFA levels in the placebo group, which was maintained for up to 2 hrs after exercise (Figure 2). Borsheim, Knardahl, and Hostmark (2) demonstrated that the plasma concentration of non-esterified fatty acids remained significantly elevated and constant even 3 hrs after the end of a long duration exercise. The effect is attributed to adrenergic stimulation of hormone-sensitive adipocyte lipase. The lack of this plasma FFA response was seen, in both groups supplemented with carbohydrates, at plasma glucose of 30 min (Figures 1 and 2). This response was probably due to reduced lypolysis, re-esterification of FFA into TG, increase or an elevation in the oxidative tissue uptake or a combination of the three possibilities. The reduced lipolysis was a consequence of higher plasma insulin triggered by higher plasma glucose levels. Insulin diminishes cyclic AMP concentrations in adipocyte; consequently, blocking the hormone sensitive lipase stimuli (18,27). Under these conditions, a high level of insulin and a lower concentration of urinary catecholamines were found in 20 men supplemented with a mixture of 50 g glucose and 15 g fructose in the recovery period after aerobic exercise on a bicycle ergometer (11). It is believed that such addition of supplemental fructose results in less sensitivity to high levels of blood insulin. It has been suggested that an overload formation of lipid metabolites (Glycerol-3Phosphate - precursor in the synthesis of triacylglycerol) derived from fructose, could interfere with the insulin signaling (31), accompanied by an inflammatory response mediated gene activated NF-κB (nuclear factor κB) and involvement of inflammatory cytokines, such as muscle TNF-α (tumor necrosis factor-α) (26). CONCLUSIONS In conclusion, this study showed that both trials with supplementation after exercise are effective in increasing plasma glucose during recovery. However, the presence of fructose attenuated plasma glucose levels after 60 and 90 min of recovery without changes in plasma FFA levels. Therefore, to restore the glycogen as soon as possible, we recommend not eating fructose during the early recovery period because attenuated plasma glucose levels will decrease the rate of glycogen synthesis. 71 ACKNOWLEDGMENTS The authors would like to thank the IMMT/Macaé, FESPORTUR/Macaé and FAPERJ. 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