EAL

NAP: Training (2014)

Citation:

Bartlett JK, Lourhelainen J, Iqbal Z, Cochran AJ, Gibala MJ, Gregson W, Close GL, Drust B, Morton JP. Reduced carbohydrate availability enhances exercise-induced p53 signaling in human skeletal muscle: Implications for mitochondrial biogenesis. Am J Physiol Regul Integ Comp Physiol. 2013; 304: R450-R458.

Study Design:

Randomized Crossover Trial

Class:

A - Click here for explanation of classification scheme.

Quality Rating:

POSITIVE: See Quality Criteria Checklist below.

Research Purpose:

To test that reduced carbohydrate (CHO) availability enhances p53 signaling and expression genes associated with regulation of mitochondrial biogenesis and substrate utilization in human skeletal muscle.

Inclusion Criteria:

Recreationally active male.

Exclusion Criteria:

History of neurological disease or musculoskeletal abnormality
Pharmacological treatment.

Description of Study Protocol:

Design

Randomized crossover, separated by at least seven days
HIIT running with high or low CHO availability.

Intervention

Low condition: Consumed diet low in CHO the day before HIIT, performed gycogen-depleting protocol in the evening prior to HIIT and restricted CHO intake before, during and after the HIIT protocol
High condition: Subjects reported to lab in the morning of main trial after consuming a high-CHO diet the day before, high-CHO breakfast on the morning of testing and were fed CHO immediately before, during and after exercise.

Statistical Analysis

Two-way repeated-measures general linear model where the within factors were time and condition. A comparison of the average physiological responses during exercise was analyzed using Student's T-test for paired samples.

Data Collection Summary:

Timing of Measurements
Muscle biopsies at pre-run, post-run and three hours after high-intensity interval (HIIT) running. Blood at pre-exercise; at 25 minutes and 43 minutes during exercise; immediately post-exercise; and one hour, two hours and three hours of recovery. Heart rate and perceived exertion (RPE) were monitored continuously during HIIT. CHO and lipid oxidation were collected.

Dependent Variables

Muscle glycogen, protein, anti-phospho ACC^Ser79, p53^Ser15, p38MAPK^{Tyr180/Thr182}, and GAPDH. total RNA, PGC-1a, PDK4, COXIV mRNA, cDNA: Muscle biopsy from the lateral portion of vastus lateralis
Plasma glucose, lactate, non-esterified fatty acid (NEFA), glycerol, and insulin: Blood via antecubital cannula.

Independent Variables

At 24 hours preceding the main experimental trial:
- Low-CHO diet of 3g per kg (6.8±0.71MJ):
  - CHO: 242±25g
  - Fat: 37±4g
  - Protein: 94±10g.
- High-CHO diet of 8g per kg CHO (14.5±1.5MJ):
  - CHO: 647±68g
  - Fat: 54±6g
  - Protein: 133±40g.
Day of testing: High group was given a high-CHO breakfast of 2g per kg (3.5±0.37MJ) with CHO, 166±17g; fat, 15±1.5g; protein, 24±2.5g two hours before HIIT. Ten minutes before exercise subjects in the High group are given a high-CHO beverage (Lucozade Sport). During the active recovery periods (19 minutes and 31 minutes), The high group was given 3ml per kg (14.5±1.5g CHO).
Post-exercise: Subjects in the High group drank 1.2g per kg body weight CHO drinks and snacks immediately after muscle biopsy and every hour until the three-hour biopsy
The Low group were glycogen-depleted and fasted on the day of testing, with only water ad libitum until after the three-hour biopsy. Evening prior to HIIT they performed an intermittent glycogen-depleting cycling protocol lasting 68±5 minutes. Within completion of the protocol, they consumed a low-CHO snack (less than 50g CHO).

Control Variables
A 50-minute, high-intensity interval running (HIIT): Six three-minute bouts at a velocity corresponding to 90% VO_2max interspersed with six three-minute recovery bouts at 50% VO_2max.Intermittent protocol started and finished with one seven-minute warm-up and cool-down at 70% VO_2max.

Description of Actual Data Sample:

Initial N: Eight males
Attrition (final N): Eight
Age: 25±5 years
Other relevant demographics: Vo_2max 55±6ml per kg per minute.

Anthropometrics

Body mass: 78±8kg
Height: 1.77±0.04m.

Location

Liverpool, UK.

Summary of Results:

Findings

No difference in average heart rate (P=0.38), VO₂ (P=0.38) and RPE (P=0.19) during exercise between High and Low groups. There was a significant difference in RER during exercise in High vs. Low (P=0.008). Total CHO oxidation was significantly greater in High compared with Low (P=0.008), while total lipid oxidation was greater in Low compared with High (P=0.008).
Exercise-induced decreases in muscle glycogen (P=0.02) were greater in High compared with Low (P=0.02), where total glycogen utilization during exercise was 142±34mmol and 30±12mmol per kg dw, respectively. Plasma glucose and serum insulin were higher in High vs. Low across all time points. Plasma NEFA and glycerol were elevated in Low vs. High (P<0.01).
Exercise in Low induced a threefold increase (P=0.04) immediately post-exercise in phosphorylation (P-) of ACC^Ser79 compared with no change in High. P-p53^Ser15 with Low demonstrating a 2.7-fold increase (P=0.04) at three hours post-exercise compared with no change in High. There was no difference in basal phosphorylation status of ACC and p53 between both diets (P>0.05). There was no change in P-p38MAPK^{Tyr180/Thr182} following exercise in either condition. No effects of exercise were observed for COXIV, Tfam, PDK4, CPT1 and SCO2 in either diet.
Both pre-exercise and post-exercise values for mRNA content of PGC-1a (P=0.05), COXIV (P=0.05), Tfam (P=0.02), PDK4 (P=0.016) and a trend for CPTI (P=0.09) were higher in LOW vs HIGH. SCO₂ mRNA displayed no significant effect of CHO availability (P=0.1). Exercise increased PGC-1a expression in both conditions (P=0.01) but the magnitude was not affected by CHO availability.

Author Conclusion:

Exercise-induced increase in p53 phosphorylation is enhanced in conditions of reduced CHO availability, which may be related upstream signaling through AMPK.

Funding Source:

Other:

not reported.

Reviewer Comments:

Quality Criteria Checklist: Primary Research
Relevance Questions
	1.	Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? (Not Applicable for some epidemiological studies)	Yes
	2.	Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about?	Yes
	3.	Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dieteticspractice?	Yes
	4.	Is the intervention or procedure feasible? (NA for some epidemiological studies)	Yes

Validity Questions
1.	Was the research question clearly stated?		Yes
	1.1.	Was (were) the specific intervention(s) or procedure(s) [independent variable(s)] identified?	Yes
	1.2.	Was (were) the outcome(s) [dependent variable(s)] clearly indicated?	Yes
	1.3.	Were the target population and setting specified?	Yes
2.	Was the selection of study subjects/patients free from bias?		Yes
	2.1.	Were inclusion/exclusion criteria specified (e.g., risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study?	Yes
	2.2.	Were criteria applied equally to all study groups?	Yes
	2.3.	Were health, demographics, and other characteristics of subjects described?	Yes
	2.4.	Were the subjects/patients a representative sample of the relevant population?	Yes
3.	Were study groups comparable?		Yes
	3.1.	Was the method of assigning subjects/patients to groups described and unbiased? (Method of randomization identified if RCT)	N/A
	3.2.	Were distribution of disease status, prognostic factors, and other factors (e.g., demographics) similar across study groups at baseline?	Yes
	3.3.	Were concurrent controls or comparisons used? (Concurrent preferred over historical control or comparison groups.)	Yes
	3.4.	If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were preexisting differences accounted for by using appropriate adjustments in statistical analysis?	N/A
	3.5.	If case control study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable.)	N/A
	3.6.	If diagnostic test, was there an independent blind comparison with an appropriate reference standard (e.g., "gold standard")?	N/A
4.	Was method of handling withdrawals described?		N/A
	4.1.	Were follow-up methods described and the same for all groups?	N/A
	4.2.	Was the number, characteristics of withdrawals (i.e., dropouts, lost to follow up, attrition rate) and/or response rate (cross-sectional studies) described for each group? (Follow up goal for a strong study is 80%.)	N/A
	4.3.	Were all enrolled subjects/patients (in the original sample) accounted for?	N/A
	4.4.	Were reasons for withdrawals similar across groups?	N/A
	4.5.	If diagnostic test, was decision to perform reference test not dependent on results of test under study?	N/A
5.	Was blinding used to prevent introduction of bias?		N/A
	5.1.	In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate?	N/A
	5.2.	Were data collectors blinded for outcomes assessment? (If outcome is measured using an objective test, such as a lab value, this criterion is assumed to be met.)	N/A
	5.3.	In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded?	N/A
	5.4.	In case control study, was case definition explicit and case ascertainment not influenced by exposure status?	N/A
	5.5.	In diagnostic study, were test results blinded to patient history and other test results?	N/A
6.	Were intervention/therapeutic regimens/exposure factor or procedure and any comparison(s) described in detail? Were interveningfactors described?		Yes
	6.1.	In RCT or other intervention trial, were protocols described for all regimens studied?	Yes
	6.2.	In observational study, were interventions, study settings, and clinicians/provider described?	N/A
	6.3.	Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect?	Yes
	6.4.	Was the amount of exposure and, if relevant, subject/patient compliance measured?	Yes
	6.5.	Were co-interventions (e.g., ancillary treatments, other therapies) described?	N/A
	6.6.	Were extra or unplanned treatments described?	N/A
	6.7.	Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups?	N/A
	6.8.	In diagnostic study, were details of test administration and replication sufficient?	N/A
7.	Were outcomes clearly defined and the measurements valid and reliable?		Yes
	7.1.	Were primary and secondary endpoints described and relevant to the question?	Yes
	7.2.	Were nutrition measures appropriate to question and outcomes of concern?	Yes
	7.3.	Was the period of follow-up long enough for important outcome(s) to occur?	Yes
	7.4.	Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures?	Yes
	7.5.	Was the measurement of effect at an appropriate level of precision?	Yes
	7.6.	Were other factors accounted for (measured) that could affect outcomes?	N/A
	7.7.	Were the measurements conducted consistently across groups?	Yes
8.	Was the statistical analysis appropriate for the study design and type of outcome indicators?		Yes
	8.1.	Were statistical analyses adequately described and the results reported appropriately?	Yes
	8.2.	Were correct statistical tests used and assumptions of test not violated?	Yes
	8.3.	Were statistics reported with levels of significance and/or confidence intervals?	Yes
	8.4.	Was "intent to treat" analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a dose-response analysis)?	N/A
	8.5.	Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (e.g., multivariate analyses)?	N/A
	8.6.	Was clinical significance as well as statistical significance reported?	Yes
	8.7.	If negative findings, was a power calculation reported to address type 2 error?	N/A
9.	Are conclusions supported by results with biases and limitations taken into consideration?		Yes
	9.1.	Is there a discussion of findings?	Yes
	9.2.	Are biases and study limitations identified and discussed?	Yes
10.	Is bias due to study's funding or sponsorship unlikely?		Yes
	10.1.	Were sources of funding and investigators' affiliations described?	No
	10.2.	Was the study free from apparent conflict of interest?	Yes