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Iranian Journal of Health and Physical Activity (2011) 2 (2), 1-8

Six Consecutive Days of Anaerobic Interval Exercise Trainingand Its Hormonal Effects in Young Females

Mahnaz Manshouri 1, Abbass Ghanbari-Niaki 2*

1 Physical Education Center, Isfahan University of Technology, Isfahan, Iran2 Exercise Biochemistry Division, Faculty of Physical Education and Sports Sciences, University of Mazandaran, Baboulsar, Iran

Received 15 May2011 Accepted 11August 2011

Abstract

Introduction : Ghrelin and Obestatin are orexigenic/anorexigenic peptides secreted from the gastrointestinaltract. Obestatin, a 23-amino acid peptide, has been recently discovered and isolated from rat stomach.

Purpose: The aim of this study was to investigate how an anaerobic interval exercise would affect plasmaObestatin and hormonal responses .

Material and Methods : Twenty-five young female college students were assigned into either a controlgroup (19.5 0.27 yr, 163.250.62 cm, 58.811.96kg, 22.050.67 BMI, 39.072.8 ml/kg.min, n= 10) or a

Running-based anaerobic sprint test (RAST) group (200.4yr, 161.880.78cm, 54.221.80kg, 20.660.58BMI, 42.631.23 ml/kg.min, n=15). Individuals in the training group performed 5 sets of Running-basedAnaerobic Sprint Tests (RAST), each set consisting 635 meter sprints. Intervals between each 35 meter sprintand between sets of 6 sprints were, 10 seconds and 5 minutes respectively. Blood samples from overnight fastand luteal phase individuals from both groups were obtained pre-, and at specified intervals, post- 6consecutive days of RAST exercise training. Plasma levels of Obestatin, Glucose, Growth hormone, Insulin,Cortisol, Testosterone, and DHEA-S were determined.

Results: No significant changes in plasma Obestatin levels were observed (P

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Hormonal Effects of Anaerobic Training2

levels has been reported [20-24], the effects of sixconsecutive days of a running-based anaerobicsprint test (RAST) on plasma Obestatin, glucoseand hormonal responses has not been investigated.Following our earlier work [25], we report here,results of a study designed to examine the effectsof anaerobic interval exercise on Glucose, Insulin,Dihydroepiandrosterone sulfate (DHEA-S), andTestosterone (T) in young female college students.Thus, this study was conducted to investigate theeffect of a highly energy demand activity with aconsiderable energy deficiency situation on plasmaObestatin and hormonal response in young femalecollege students.

Material and MethodsSubjects and research design

The study was approved by the ethic committeeof the School of Medical Sciences of TarbiatModares University and was conducted inaccordance with the policy statement of theDeclaration of the Iranian Ministry of health.

Twenty-five young female college students wereassigned into either a control group (19.5 0.27 yr,163.250.62 cm, 58.811.96kg, 22.050.67 BMI,39.072.8 ml/kg.min, n= 10) or a Running-basedanaerobic sprint test (RAST) group (200.4yr,161.880.78cm, 54.221.80kg, 20.660.58 BMI,42.631.23 ml/kg.min, n=15) groups. Subjectswere asked to go through a medical examinationand were examined for cardiac, respiratory, renal,and metabolic diseases.They were not using any

steroids.

Exercise testing proceduresBefore the main trial, participants were taken to

gym two times, to perform anaerobic intervalexercise (RAST test) and to become familiar withthe procedure. The subjects were allowed to have asmuch time as they needed to recover from eachattempt. On the third visit, the subjects completed a

practice session to ensure that each participant wasable to complete the entire exercise session. Thetraining sessions were held in the morning, between08.30 to 11.00 AM, in order to avoid the effects of

circadian rhythm. The program consisted of a5-minute warm up, 5 sets of RAST-test (6 35meters every 10 seconds) with 5-minute rest period

between sets, and a 5-min cool down period. Theduration of the whole program was 40 minutes. All

participants were in luteal phase. All exercises wereconducted after an overnight fast (at least 12h,allowed to drink water only). The subjects wereinstructed to follow a normal lifestyle , maintaindaily habits, avoid the consumption of anymedication, and refrain from exercise 3 days prior

to the experiment session .

Blood collectionBlood samples were collected into heparinzed

Vacutainer tubes (Becton Dickinson, NJ, USA)from antecubital vein 24hrs before and 24hrs aftertraining program while subjects were overnight fastfor at least 12hrs. Plasma was separated bycentrifugation within 15min of collection andaliquots were kept frozen at -80C for subsequentanalysis (within 2-3 weeks)

Hormonal Elisa assaysThe samples were analyzed for Obestatin, GH,

lnsulin, DHEA-S, and Tstosterone. PlasmaObestatin was measured using a kit (EIA, PeninsulaLaboratories LLC, CA, USA, Sensitivity 0.002ng/mL). GH was determined by ELISA kit(Diagnostic Biochem Canada Inc, London, Ontario,Canada, Sensitivity 2 ng/mL). Plasma Insulin wasdetermined by ELISA kit (Mercodia, AB, Uppsala,Sweden, Sensitivity 1mIU/L). Plasma Testosteroneand DHEAS were determined by Enzyme ImmunoAssay Method (EIA, Diagnostic Biochem CanadaInc, London, Ontario, Canada, Sensitivity 0.022ng/mL and 0.005g/dL, respectively). TheIntraassay coefficients of variation were 8.3%,5.95, 4.1%, 7.5% and 7.85 % for Obestatin, GH,insulin, testosterone and DHEA-S, respectively.Hemoglobin and Hematocrit were also determinedusing automated hematology analyzer (Sysmax

K-4500, Toa Medical Electric Co., Kobe Japan).Plasma Glucose was measured by Glucose Oxidasemethod. Lactate was obtained by kit (Randox, LOT086904 UK BT294 QX) and lactate dehydroge-nase (LDH) was determined automatically (RA1000 auto Analyzer) by kit (Pars Azmoun, Tehran,Iran). The changes of plasma volume werecalculated based on hemoglobin and Hematocritestimation [26].

Statistical analysisAll results are expressed as the mean SEM.

Pre- and post-exercise results were compared using

Mann-Whitney non-parametric two-tailed t-test forwithin and between group comparisons. All thestatistical analyss were performed using GraphPad5.0, and differences were accepted at P

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3 Iranian Journal of Health and Physical Activity

RAST groups regarding age, height, weight, and pre-physical conditioning. There was a significantdifference in post-training exercise values betweenRAST and control groups. (Table 1 and Figure 1).

No changes in plasma volume were noted in either

groups, due to fasting, luteal phase, and exerciseroutine, which reflects no association betweenobserved results and the hydration state of studysubjects (data not shown).

Table.1: physical characteristics of the participants (mean SEM).

VARIABLES Control Group RAST Test Group

Age (yr) 20.00 0.40 19.56 0.27

Height (cm 161.88 0.78 163.25 0.62

Weight (kg)54.22 1.80 58.81 1.96

BMI (kg/m 2) 20.66 0.58 22.05 0.67

VO 2max (ml/kg/min-1)

42.63 1.37 39.09 2.81

Obestatin

1 2 7-0.8-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8Group 1Group 2

P>0.05

P>0.05

P>0.05

Days After Last Exerc ise (Day 6)

D i f f e r e n c e

f r o m

D a y

0

Figure 1 : Plasma Obestatin concentrations in control and exercise groups 24h before and 24h after anaerobic intervalexercise training. Data was expressed as mean SEM.

Hormonal changes as a function of six consecutive days RAST

No significant difference was observed in

pre- and post-RAST plasma Obestatin levels,within and/or between the study groups. (Table 2and Figure 1A). However, a marginal 24h increasein plasma Obestatin levels was observed in theRAST-group, followed by a decreasing trend 2 and7 days post-exercise. (Table 3). Although theseresults were not statistically significant, weobserved a suggestive trend which should be furtherinvestigated useing a larger group of test subjectswhere statistical power is ensured. A statisticallysignificant decrease was noted in the

RAST-training group, both inGH and Testosterone plasma levels, P=0.032 and P=0.043 respectively.(Table2). No significant differences were noted in

plasma insulin, Cortisol, and DHEAS levels withinor between study groups (Table 2). Furtherhormones analysis, indicated a statisticallysignificant increase (P=0.03) in the ratio between-adrenergic hormone DHEAS and the anabolichormone Testosterone in the RAST-group (Figure2B).

Relationship between hormonal changes and metabolic response

Lactate level was higher in training group

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compared to the control group on days 1, 2, and 7 post-RAST exercises (Table 3). This difference,however, is not considered statistically significant,and is hypothesized to be a consequence of fasting

prior to blood collection. Furthermore, lactatedehydrogenase (LDH) was significantly lower inthe plasma of the exercise group (P

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Figure 2: Plasma GH and Testosterone concentrations in control groups 24h before and 24h after anaerobic intervalexercise training. Data were expressed as mean SEM. * Pre vs. post (P

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loss, which is then followed by plasma volumeexpansion. This expansion may be 6% to 25%greater than baseline, occurs within 3 hours follow-ing acute exercise and persists for 3 to 5 daysfollowing the cessation of training documented[28,29].Although the effects of acute and chronic(aerobic /anaerobic, endurance and resistance)exercise training on plasma GH concentrations inmale and female subjects are well documented[31-38], our findings indicate that plasma GHconcentration significantly decreased following theanaerobic training. We are not aware of any studiesthat have examined the effects of prospective sixconsecutive days of anaerobic exercise training onthe release of GH at rest. According to Kanaley etal. [39] treadmill runnings for 30min at threedifferent times of day (07.00, 19.00, 24.00h)resulted in a transient suppression in GH release.

No change in GH levels following heavy- resis-tance training has been observed by Kreamer et al[40]. Chwalbinska-Moneta et al. [41] reported thatresting plasma GH level was insignificantly lowerfollowing one week of interval training andsignificantly decreased at the end of first and thirdweeks of aerobic exercise training at 75% ofVO2max. Fry et al. [42] demonstrated a reductionin post-exercise concentrations of lactate andgrowth hormone after 1 week of high-volumeweight-lifting training. The same results were alsoreported by Kraemer et al. [43] after 3 consecutivedays of heavy resistance exercise. A suppression ofGH may reflect autonegative feedback of GH on its

own secretion, as suggested by Lanzi &Tannenbaum [44]. A change in resting plasmalactate post-anaerobic training could be an effectivefactor in lowering GH concentration at rest. In the

present study lower GH levels were accompaniedwith a reduction in plasma lactate and LDHconcentrations at rest. A stimulatory effect oflactate on GH secretion was reported by Sutton etal. [45] and other investigators [46, 41]. However, alower GH concentration in the present study can be

justified by an increased post-exercise training plasma volume.

Testosterone is an endogenous anabolic hormone

which is believed to be involved in muscle tissuegrowth and remodeling. The concentration of plasma in male subjects is higher than females [35,37]. The effect of acute and chronic exercise andtraining (endurance and resistance type) on plasmatestosterone in male and female subjects welldocumented [32, 35, 37, 47]. Slowinska-Lisowska& Majad [48] reported a reduction in total and freeTestosterone concentrations after a 400m sprint inmale elite athletes. According to Fry et al. [49]during the 24h of monitoring hormonal response of

14 subjects _with varying fitness levels of _ to anintensive anaerobic exercise, a reduction in plasmaTestosterone has been observed. According toKraemer et al. [43] total serum Testosteronesignificantly increased immediately after heavyresistance exercise and declined to below restingvalues on day 3 in placebo taking condition, and onday 2, and 3 during supplementation. Flynn et al.[50] observed a significant reduction during10-day

periods of increased training volume (200% ofindividual normal training) in 11 well-traineddistance runners.

It has been suggested that calorie intake in theform of carbohydrate and protein, has an acuteattenuating effect on circulating Testosterone con-centrations [51, 52, 43]. In our study we did notrecord the subjects food consumption and theywere overnight fast during the blood sampling. Thereduction in plasma Testosterone in the presentstudy might be due to the increased plasma volume.A decline in post-exercise training Testosteronelevel was accompanied with a modest increase(1.72mU/L) in plasma Insulin level in trainedgroup.

The Dehydroepiandrosterone sulfate (DHEAS)has a critical role in the maintenance of bonedensity and overall health. Previous studies haveshown changes in plasma DHEAS level in responsto short-term resistance and endurance exercise [35,37, 47]. In the present study, our results indicatethat plasma DHEAS concentration remainedunchanged in response to six consecutive days of a

running-based anaerobic sprint test (RAST).According to Aizawa et al. [53] plasma DHEASconcentration significantly increased in femaleswho performed a resistance training program for 8weeks. Flynn et al. [50] has shown a significantreduction in DHEAS level during 10 days ofincreased training volume. These diversities could

be the result of differences in procedures andsubjects characteristics such as gender (female vs.male) age (young vs. old), type of exercise

performed (anaerobic vs. resistance and aerobic),duration of exercise (six days vs. 3, 10 days or8weeks), blood sampling time (immediately/less

than 12h vs.24h), and nutritional status (overnightfasting vs. fed-condition). To summarize, our dataindicates that six consecutive days of anaerobicinterval exercise training, in our experimentalconditions increased plasma volume and decreasedresting plasma GH and Testosterone as signs ofearly metabolic adaptations to high intensityexercise. According to the present study, it could besuggested that any exercise-induced changes in

plasma Obestatin levels might be mashed byshort-term fasting and elevated plasma volume. It

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appears that plasma Obestatin does not contributeto anaerobic exercise training-induced reduction of Growth hormone. Further studies should beconducted in order to investigate plasma Obestatinand Ghrelin communal response to d ifferentconditions regarding mode of exercise, its intensityand duration and nutritional interventions.

Acknowledgments

This work was supported by Mrs. MahnazManshouri. We wish to thank Professor TaghiManshouri, Dr Gholam-Ali Naderi for their kindhelp and sincere cooperation. We also thank allsubjects for their participation in this study.

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