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J Appl Physiol 97: 2148-2153, 2004. First published June 25, 2004; doi:10.1152/japplphysiol.00517.2004
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Effects of 7 wk of endurance training on human skeletal muscle metabolism during submaximal exercise

Paul J. LeBlanc,1 Krista R. Howarth,2 Martin J. Gibala,2 and George J. F. Heigenhauser1

1Department of Medicine, McMaster University, Hamilton, Ontario L8N 3Z5; and 2Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada L8S 4K1

Submitted 13 May 2004 ; accepted in final form 24 June 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
This is the first study to examine the effects of endurance training on the activation state of glycogen phosphorylase (Phos) and pyruvate dehydrogenase (PDH) in human skeletal muscle during exercise. We hypothesized that 7 wk of endurance training (Tr) would result in a posttransformationally regulated decrease in flux through Phos and an attenuated activation of PDH during exercise due to alterations in key allosteric modulators of these important enzymes. Eight healthy men (22 ± 1 yr) cycled to exhaustion at the same absolute workload (206 ± 5 W; ~80% of initial maximal oxygen uptake) before and after Tr. Muscle biopsies (vastus lateralis) were obtained at rest and after 5 and 15 min of exercise. Fifteen minutes of exercise post-Tr resulted in an attenuated activation of PDH (pre-Tr: 3.75 ± 0.48 vs. post-Tr: 2.65 ± 0.38 mmol·min–1·kg wet wt–1), possibly due in part to lower pyruvate content (pre-Tr: 0.94 ± 0.14 vs. post-Tr: 0.46 ± 0.03 mmol/kg dry wt). The decreased pyruvate availability during exercise post-Tr may be due to a decreased muscle glycogenolytic rate (pre-Tr: 13.22 ± 1.01 vs. post-Tr: 7.36 ± 1.26 mmol·min–1·kg dry wt–1). Decreased glycogenolysis was likely mediated, in part, by posttransformational regulation of Phos, as evidenced by smaller net increases in calculated muscle free ADP (pre-Tr: 111 ± 16 vs. post-Tr: 84 ± 10 µmol/kg dry wt) and Pi (pre-Tr: 57.1 ± 7.9 vs. post-Tr: 28.6 ± 5.6 mmol/kg dry wt). We have demonstrated for the first time that several signals act to coordinately regulate Phos and PDH, and thus carbohydrate metabolism, in human skeletal muscle after 7 wk of endurance training.

energy status; pyruvate; pyruvate dehydrogenase

CARBOHYDRATES AND FAT ARE the major metabolic fuels for ATP production in skeletal muscle at rest and during exercise (see Ref. 12 for review). At workloads above 65–70% of maximal oxygen uptake (O2 max), carbohydrates, primarily in the form of muscle glycogen, are the dominant fuel source (32, 34). Several weeks of repeated bouts of exercise (endurance training) alter skeletal muscle metabolism, with a resulting decreased carbohydrate metabolism during submaximal exercise. Endurance training has been shown to increase sensitivity of mitochondrial respiration to the energy status of the cell (9) and decrease flux through glycogenolysis (17, 20) and glycolysis (17) during submaximal exercise, with a net glycogen-sparing effect.

Two important enzymes of glycogen metabolism are glycogen phosphorylase (Phos), the flux-generating enzyme of glycogenolysis, and pyruvate dehydrogenase (PDH), the rate-determining enzyme that regulates the entry of carbohydrate-derived acetyl units into oxidative metabolism. Both Phos and PDH play important roles in dictating the rate of carbohydrate-derived acetyl units for ATP production. A previous study has demonstrated that metabolic signals related to contraction and energy status of the cell result in altered carbohydrate metabolism through coordinated regulation of Phos and PDH in healthy active subjects at varying exercise power outputs (18). Thus the response of these two enzymes to endurance training may provide insight into training-induced regulation of skeletal muscle substrate utilization during submaximal exercise.

Thus the aim of the present study was to investigate the adaptive regulation of skeletal muscle Phos and PDH activation during submaximal exercise before and after 7 wk of endurance training (Tr). Surprisingly, no study has examined the effects of endurance training on the activation state of Phos and PDH during submaximal exercise. We hypothesized that Tr would result in a posttransformationally regulated decreased flux through Phos and an attenuated activation of PDH during exercise, due to endurance training-induced alterations in key allosteric modulators of Phos and PDH.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Subjects.   Eight healthy, active men were recruited to participate in the study (age 22 ± 1 yr; height 179 ± 2 cm; weight 91.6 ± 4.3 kg). None of the subjects was specifically trained in cycling exercise. Verbal and written explanation of the experimental protocol and its attendant risks were provided, and informed consent was obtained from each subject. The study was approved by the Hamilton Health Sciences/Faculty of Health Sciences Research Ethics Board in accordance with the Declaration of Helsinki.

Preexperimental protocol.   Individuals completed an initial incremental maximal exercise test on an electromagnetically braked cycle ergometer (Lode Excalibur, Quinton Instruments, Seattle, WA) to determine maximal work capacity and O2 max using a metabolic measurement system (Moxus Modular VO2 System, AEI Technologies, Pittsburgh, PA).

Training protocol.   Individuals were trained on a cycle ergometer at a workload that elicited 75% of their O2 max for 1 h per day, 5 days a week, for a total of 7 wk. For the first week, the 1 h of training was broken up into four 15-min intervals with 5 min of rest in between. For the second and third weeks, the 1 h of training was broken up into three 20-min intervals with 5 and 2.5 min of rest in between, respectively. For the third to eighth week, the 1 h of training was broken up into three 20-min intervals with 1 min of rest in between. On the fourth week, individual subjects completed an incremental maximal exercise test to determine changes in maximal work capacity and O2 max so that adjustments could be made to the workload to maintain a training intensity equivalent to 75% of each subject's O2 max.

Experimental protocol.   Each subject participated in two experimental trials, before and after training. For a given subject, the two experimental trials were performed at the same time of day. Individuals served as their own control. Before the beginning of the exercise protocol, a venous catheter was inserted in an anticubital vein for blood sampling and was maintained patent with saline. One thigh was prepared for the extraction of needle biopsy samples from the vastus lateralis as described by Bergström (2). Three incisions were made through the skin to the deep fascia under local anesthesia (2% lidocaine without epinephrine). Subjects cycled at the same absolute workload (206 ± 5 W), eliciting 80% of their peak power output pre-Tr. Muscle biopsies were obtained at rest and after 5 and 15 min of exercise. Blood samples were taken at rest and after 10 min of exercise.

Nutritional control.   Individuals were asked to refrain from caffeine, alcohol, and exercise for 48 h before each trial. Individuals also consumed the same diet 24 h before each trial, confirmed by subsequent nutritional analyses of dietary records (Nutritionist 5, version 1.7, First Data Bank, San Bruno, CA) to confirm total energy intake and proportion of energy from carbohydrate, fat, and protein. The dietary analysis revealed no significant difference between pretraining (2,733 ± 175 kcal; 53 ± 4% carbohydrate, 30 ± 3% fat, and 17 ± 2% protein) and posttraining (2,768 ± 301 kcal; 53 ± 3% carbohydrate, 29 ± 3% fat, 18 ± 2% protein) diets. Three hours before each experimental protocol, subjects consumed a standardized meal that contained 711 kcal, derived from 87% carbohydrate, 3% fat, and 10% protein.

Blood sampling and analysis.   Venous blood samples (~7 ml) were collected in heparinized syringes (Sarstedt, Nümbrecht, Germany) and placed on ice. Blood was centrifuged at 15,900 g for 2 min to isolate plasma. One portion of plasma was incubated with 5 M NaCl (4:1 vol/vol plasma-NaCl) at 56°C for 30 min and then frozen for later analysis of free fatty acids (Wako NEFA kit, Wako Chemicals). A second portion of plasma was deproteinized with 500 mM perchloric acid (PCA; 1:2 vol/vol plasma-PCA) and then frozen for later analysis for glucose and lactate (1).

Muscle analysis.   Muscle biopsies were immediately frozen by plunging the needles into liquid N2. Small pieces were chipped from each biopsy (under liquid N2) for determination of the activity of PDH in the active form (PDHa; Ref. 30) and citrate synthase (CS; Ref. 16). The remainder of the sample was freeze-dried, dissected free of blood and connective tissue, and powdered. One aliquot was analyzed for total (a+b) and active Phos (Phos a+b and Phos a respectively; Ref. 36), and the maximal velocity and mole fraction of Phos a+b and Phos a were calculated from the measured activities as described by Chasiotis et al. (6). Phos measurements were made only on exercise samples because accurate resting samples are obtainable only if the biopsy is held from liquid N2 freezing for 30 s, because of Ca2+ release during muscle sampling, which artificially increases the transformation of Phos b to a (31). Thus it was deemed unethical to obtain extra samples for resting Phos, and previous studies have reported values of ~10% for the mole fraction of Phos a at rest in human skeletal muscle (31). A second aliquot was extracted in 500 mM PCA and 1 mM EDTA, neutralized to pH 7.0 with 2.2 M KHCO3, and analyzed for acetyl-CoA, free CoA, acetylcarnitine, free carnitine (5), ATP, pyruvate, lactate, phosphocreatine, creatine, glucose, glucose-6-phosphate (G-6-P), glucose-1-phosphate (G-1-P), fructose-6-phosphate (F-6-P), and glycerol-3-phosphate (Gly-3-P; Ref. 1). The final aliquot was used to measure muscle glycogen (14). All muscle metabolites, along with Phos, PDH, and CS enzyme measurements, were normalized to the highest total creatine content for a given individual to correct for non-muscle contamination.

Calculations.   The content of free ADP (ADPf) and free AMP (AMPf) were calculated from the near-equilibrium reactions of creatine kinase and adenlylate kinase, respectively (9); however, these calculations do not provide information as to the exact location of these metabolites within the cell. The skeletal muscle content of free inorganic phosphate (Pi) was calculated as the difference between resting and exercise PCr content, minus the accumulation of G-6-P, F-6-P, and Gly-3-P, plus the assumed resting content of 10.8 mmol/kg dry wt (9).

Previous studies have shown that in vivo flux through PDH is similar to its in vitro level of activity at varying exercise power outputs (11, 18, 28, 29). Thus pyruvate oxidation, estimated by PDHa flux, was calculated from PDHa activity as measured in wet tissue and converted to dry tissue using the wet-to-dry muscle ratio of 4:1 at rest and 4.5:1 during exercise (27).

Pyruvate production was calculated from the sum of the measured rate of muscle lactate and pyruvate accumulation, plus the rate of blood lactate accumulation (distribution volume of blood lactate was assumed to be 0.64·body weight), plus the flux of pyruvate through PDHa. The calculation of blood lactate accumulation does not account for lactate oxidation by other tissues and would result in a slight underestimation of lactate accumulation.

Statistical analysis.   Student's paired t-test was used to determine differences between pre- and post-Tr values of cardiorespiratory measurements, CS and Phos enzyme activity, and blood metabolites. All other data were analyzed by using a two-factor, repeated-measures ANOVA. Significant main effects and interactions were subsequently analyzed by using a Tukey's post hoc test. Assumptions for normality and independence were verified by generating appropriate residual plots. Data transformations (log, square root, and inverse square root) were used when appropriate to meet the above assumptions. The level of significance for all analyses was set at P ≤ 0.05. All data are presented as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Cardiorespiratory measurements and muscle enzymes. O2 max increased 6% after 7 wk of aerobic training (pre-Tr, 3.90 ± 0.15 l/min; post-Tr, 4.13 ± 0.13 l/min; P < 0.05).

Training resulted in increased resting muscle CS maximal activity from 26.8 ± 1.4 to 34.1 ± 3.4 mmol·min–1·kg wet wt–1 (P < 0.05). The percent mole fraction of Phos a was unaffected by Tr after 5 min (pre-Tr, 43.0 ± 5.4%; post-Tr, 39.5 ± 2.2%) and 15 min (pre-Tr, 40.4 ± 3.7%; post-Tr, 46.8 ± 2.4%) of exercise. PDHa increased during exercise compared with rest in both conditions, with no further increase after 5 min of exercise (Fig. 1). After 15 min of exercise, PDHa was lower in post-Tr compared with pre-Tr.



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  		Fig. 1. Active form of muscle pyruvate dehydrogenase (PDHa) at rest and during exercise before (Pre) and after 7 wk of aerobic training (Post). Values are means ± SE. ww, Wet weight. *P ≤ 0.05 vs. rest. {dagger}P ≤ 0.05 vs. pretraining.

 
Muscle metabolites.   Resting glycogen content was higher post-Tr compared with pre-Tr at all time points (Table 1). At the end of 15 min of exercise, glycogen content decreased by 49 and 17% during pre-Tr and post-Tr, respectively. Glycogenolytic rate was lower post-Tr compared with pre-Tr (Fig. 2). At rest, all glycolytic intermediates were similar between conditions (Table 1). G-1-P was not detectable. After 5 min of exercise, glucose content was elevated compared with rest, but there was no difference between conditions. After 5 min of exercise, content of G-6-P, F-6-P, and Gly-3-P were lower post-Tr compared with pre-Tr. With the exception of G-6-P, all glycolytic intermediates were lower after the last 10 min of exercise post-Tr compared with pre-Tr.


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  		Table 1. Muscle contents of glycogen, glucose, and glycolytic intermediates at rest and during exercise before and after 7 wk of aerobic training

 


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  		Fig. 2. Estimated rates of muscle glycogen use during exercise before and after 7 wk of aerobic training. Values are means ± SE. Numbers on the x-axis are time (in min). dw, Dry weight. {dagger}Significantly different from pretraining, P ≤ 0.05.

 
Resting pyruvate and lactate content were similar between both conditions (Table 1). During exercise, both pyruvate and lactate content increased from rest. After 5 min of exercise, pyruvate content was similar between conditions; however, after 15 min of exercise, pyruvate content was lower post-Tr compared with pre-Tr. After both 5 and 15 min of exercise, lactate content was lower post-Tr compared with pre-Tr.

ATP content was unchanged during exercise in both conditions (Table 2). PCr content decreased during pre-Tr after 5 min of exercise, with no further change during the last 10 min of exercise. In comparison, PCr content decreased during post-Tr only after 15 min of exercise and remained higher during exercise compared with pre-Tr. Calculated ADPf, AMPf, and Pi content increased in pre-Tr after 5 and 15 min of exercise and were higher than post-Tr at both time points. In contrast, ADPf and AMPf content were unaffected by exercise post-Tr, and free Pi was higher at 15 min of exercise compared with 5 min and rest.


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  		Table 2. Muscle contents of high-energy phosphates at rest and during exercise before and after 7 wk of aerobic training

 
With the exception of acetylcarnitine content being lower post-Tr compared with pre-Tr after 5 min of exercise, muscle acetyl-CoA, free CoA, and free carnitine content at rest and during exercise were unaffected by training (Table 3).


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  		Table 3. Muscle contents of CoA, carnitine, and their acetylated forms at rest and during exercise before and after 7 wk of aerobic training

 
Blood metabolites.   Plasma glucose concentration was similar between conditions at rest (pre-Tr, 4.4 ± 0.3 mM; post-Tr, 4.7 ± 0.4 mM) and during exercise (pre-Tr, 4.0 ± 0.3 mM; post-Tr, 4.1 ± 0.1 mM). Similarily, plasma free fatty acid concentration was similar at rest (pre-Tr, 0.19 ± 0.02 mM; post-Tr, 0.13 ± 0.03 mM) and during exercise (pre-Tr, 0.18 ± 0.04 mM; post-Tr, 0.13 ± 0.02 mM). Plasma lactate concentration was similar between conditions at rest (pre-Tr, 0.7 ± 0.2 mM; post-Tr, 0.8 ± 0.1 mM). During exercise, plasma lactate concentration was higher pre-Tr (5.1 ± 0.4 mM) compared with post-Tr (3.1 ± 0.3 mM).

Pyruvate production and oxidation and lactate accumulation.   In the first 5 min of exercise, less pyruvate was produced and less lactate accumulated post-Tr compared with pre-Tr, with no change in pyruvate oxidation (Fig. 3). During the subsequent 10 min of exercise, there was less pyruvate produced and oxidized and less lactate accumulation post-Tr compared with pre-Tr.



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  		Fig. 3. Comparison of the fate of pyruvate produced during exercise before and after 7 wk of aerobic training. *Significantly different from 0–5 min, P ≤ 0.05. {dagger}Significantly different from pretraining, P ≤ 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
This is the first study to examine the effects of endurance training on the activation state of Phos and PDH in human skeletal muscle at rest and during submaximal exercise. The two major findings from the present study were that 7 wk of endurance training resulted in 1) an attenuated exercise-induced activation of PDH, possibly mediated by decreased pyruvate availability and improved energy status of the cell, and 2) a decreased flux through Phos, regulated posttransformationally by allosteric modulators linked to the energy status of the cell, that contributed to endurance training-induced glycogen-sparing during submaximal exercise.

Effects of training on pyruvate metabolism.   The activation of PDH is controlled by the relative activities of PDH kinase (PDK) and PDH phosphatase (PDP), which inhibit and activate PDH, respectively. Increased ratios of acetyl-CoA/CoA, ATP/ADP, and NADH/NAD+ stimulate PDK and inhibit PDP. In addition, pyruvate inhibits PDK (4), whereas Ca2+, H+, and insulin activate PDP (see Refs. 8, 35 for review). The increased Ca2+ concentration at the onset of exercise stimulates PDP and crudely activates PDH, whereas the other allosteric modulators fine tune PDH activation.

This is the first study to demonstrate an attenuated activation of PDH post-Tr. A previous study reported no effect on PDH activation during exercise (27); however, training was only for 2 wk. In the present study, the apparent mechanisms regulating the attenuated exercise-induced activation of PDH post-Tr may be mediated by two allosteric modulators. PDK activity is ATP dependent, and ADP competitively inhibits this reaction (24). The decreased ADPf during exercise post-Tr would release inhibition on PDK and attenuating PDH activation. Also, reduced glycogenolysis and glycolysis during exercise post-Tr was associated with attenuated pyruvate production and skeletal muscle pyruvate content, which is consistent with previous studies (13, 25). Attenuated pyruvate content may have released inhibition on PDK and reduced activation of PDH after 15 min of exercise. The inhibition constant (Ki) of pyruvate for PDK has been reported in cardiac muscle to be in the range of 0.08–2 mM (see Ref. 35 for review). If it is assumed that the fluid volume of muscle is 75% of the total volume, muscle pyruvate concentration after 15 min of exercise decreased from 0.31 mM pre-Tr to 0.15 mM post-Tr. Although the Ki of pyruvate in human skeletal muscle has yet to be confirmed, the possibility remains that it may be similar in magnitude to the pyruvate concentrations we observed pretraining in the present study. In addition, the sensitivity of PDK to pyruvate is positively correlated with ADP (26). It is important to note that the pyruvate and ADPf concentrations reported here are of the whole muscle cell, and further research is needed to ascertain the subcellular distribution of these modulators because the inner mitochondrial concentration would influence the activity of PDK.

PDH control can also be accomplished through stable alterations to PDK. A recent study conducted in our laboratory demonstrated that 8 wk of endurance training resulted in a twofold increase in PDK activity, mainly due to increased protein expression of the PDK-2 isoform (23). Although not measured, it can be assumed that there was a similar increased PDK activity and PDK-2 isoform expression in the present study due to similar training protocols. These results present a twofold effect on the attenuated activation of PDH during submaximal exercise post-Tr. First, an increased PDK activity post-Tr would enhance phosphorylation and resultant lower activity of the PDH complex during exercise. Second, endurance training-induced increase in PDK-2 isoform protein expression would enhance post-Tr skeletal muscle PDK sensitivity to pyruvate as PDK-2 is the most sensitive to inhibition by dichloroacetate, a pyruvate analog (3). Thus the training-induced increases in PDK activity and PDK-2 protein may play an important role in PDH regulation, and in turn carbohydrate metabolism, during exercise post-Tr.

Effects of training on glycogenolysis.   In the present study, glycogenolytic rate and resulting pyruvate production during exercise were lower in post-Tr compared with pre-Tr. This is consistent with previous studies, demonstrating a conservation of glycogen in endurance-trained human skeletal muscle (13, 15, 19, 20, 22, 25).

Muscle glycogenolytic flux is regulated by Phos, which is subject to both covalent and allosteric regulation. During exercise, covalent transformation of Phos from the less active (Phos b) to the more active (Phos a) form is regulated mainly by Ca2+ (6). The changes in glycogenolytic flux through Phos during exercise demonstrated in the present study that post-Tr took place in the absence of any change in total Phos activity or the percent mole fraction of Phos a. Previous studies have demonstrated that exercise-induced covalent transformation of Phos b to a is always well in excess of glycogenolytic flux at submaximal exercises (7, 18). As a result, the decreased glycogenolytic rate post-Tr compared with pre-Tr is more likely due to posttransformational allosteric regulators.

Posttransformationally, Phos b is stimulated by AMPf and free IMP, and inhibited by ATP and G-6-P, whereas Phos a is stimulated by AMPf and ADPf (10, 21, 33). In addition, the availability of one of the substrates, Pi, is critical in increasing the catalytic rate of Phos (6). After 5 and 15 min of exercise post-Tr, the endurance training-induced increased mitochondrial sensitivity to ADPf resulted in a 46–70 and 24–47% attenuated increase of AMPf and ADPf, respectively. This, in turn, decreased the reliance on PCr, resulting in 50–63% reduced availability of Pi. Attenuated skeletal muscle content of ADPf, AMPf, and Pi would decrease Phos activity. Tighter metabolic control has previously been postulated to be linked to endurance training-induced glycogen sparing in human skeletal muscle via Phos (13, 25), and this study demonstrates that this regulation of Phos occurs posttransformationally.

Endurance training-induced improvement in the energy status of the cell would also affect other key enzymes of carbohydrate metabolism, namely phosphofructokinase (PFK). PFK is an important rate-determining step in glycolysis and is subject to allosteric modulation by metabolites linked to energy status. During exercise post-Tr, attenuated skeletal muscle content of ADPf, AMPf, and Pi would inhibit PFK, resulting in coordinated glycogenolytic and glycolytic flux through PFK and Phos.

Conclusion.   The results from the present study demonstrated that 7 wk of endurance training influences skeletal muscle carbohydrate metabolism during submaximal exercise. This was evident by a decreased flux through Phos and attenuated exercise-induced activation of PDH. It appears that several signals act to coordinate glycogen utilization posttraining. Improved energy status of the cell (ADPf, AMPf, Pi) decreased glycogenolytic rate via Phos allosteric posttransformational regulation. Attenuated pyruvate production from glycogenolysis potentially released a pyruvate-mediated inhibition of PDK, resulting in a decreased activation of PDH.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was supported by operating grants from the Canadian Institute of Health Research (G. J. F. Heigenhauser) and the National Sciences and Engineering Research Council (NSERC) (M. J. Gibala). P. J. LeBlanc was supported by an Ontario Graduate Scholarship and K. R. Howarth by a NSERC Postgraduate Scholarship.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank the subjects for time and effort.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. J. F. Heigenhauser, McMaster University Health Science Centre, Hamilton, ON, Canada L8N 3Z5 (E-mail: heigeng{at}mcmaster.ca)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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