Effects of Weight Loss and Exercise on Fasting Ghrelin
The Nutrition and Exercise in Women (NEW) study was a 12-month randomized controlled trial to test the effects of dietary weight loss and/or exercise on circulating hormones and other outcomes. Study procedures were reviewed and approved by the Fred Hutchinson Cancer Research Center Institutional Review Board in Seattle, WA. All participants provided written informed consent.
Participants were overweight or obese (BMI ≥25 or BMI ≥30 kg/m (≥23 kg/m if Asian–American), postmenopausal women (50–75 y) from the greater Seattle area. They were recruited through media and mass mailings (Fig. 1). Exclusion criteria included: diagnosed diabetes; fasting blood glucose ≥126 mg/dL or use of diabetes medications; use of postmenopausal hormone therapy; history of invasive cancer within the past 10 years, excluding basal or squamous cell skin cancer; alcohol intake >2 drinks/day; current smoking; abnormal screening labs (e.g., haematocrit <32 or >48, white blood cell count <3·0 or >15·0, potassium <3·5 or >5·0, creatinine >2·0) or contraindication to the study interventions for any reason (e.g., abnormalities on screening physical, abnormal exercise tolerance test, uncontrolled hypertension, history of cardiac arrest or stroke, recent (within 6 months) myocardial infarction, pulmonary oedema, myocarditis, pericarditis, unstable angina, pulmonary embolism/deep vein thrombosis, orthostatic hypotension, moderate/severe asthma, uncontrolled arrhythmia, uncontrolled congestive heart failure, 3rd degree heart block, left bundle branch block, myocarditis, thrombophlebitis); participation in another structured weight loss program or use of weight loss medications; additional factors that might interfere with measurement of outcomes or with the success of the intervention (e.g., inability to attend facility-based sessions).
(Enlarge Image)
Figure 1.
Flow of participants through the NEW study.
Eligible women were randomized to: (i) dietary weight loss ('diet'; n = 118); (ii) moderate-to-vigorous intensity aerobic exercise ('exercise'; n = 117); 3) combined diet and exercise ('diet + exercise'; n = 117); or 4) control (no intervention) (n = 87). Computerized random assignment, using permuted blocks randomization to achieve a proportionally smaller control group, was stratified according to BMI (≥ or <30 kg/m) and race/ethnicity.
The dietary weight loss intervention was a modification of the Diabetes Prevention Program and Look AHEAD lifestyle behaviour change programs with goals of 1200–2000 kcal/day, <30% calories from fat, and 10% weight loss by 6 months with maintenance thereafter. Participants met individually with a dietitian at least twice, followed by weekly group meetings (5–10 women) for 6 months. Thereafter, they attended monthly group meetings in addition to biweekly phone or e-mail contact. Women completed daily food logs for at least 6 months or until they reached their 10% weight loss goal. Food logs, weekly weigh-ins and session attendance were tracked to promote dietary adherence.
Participants who did not meet their weight loss goal by 6 months were encouraged to continue weight loss efforts and were offered additional sessions; women who reached their goal were allowed to continue losing weight but were monitored to ensure that their BMI did not drop below 18·5 kg/m.
The exercise intervention progressed to 45 min of moderate-to-vigorous intensity exercise at a target heart rate of 70–85% observed maximum, 5 days/week by the 7th week. Participants attended 3 supervised sessions/week at the study facility and exercised 2 days/week at home. They recorded exercise mode, duration, peak heart rate and perceived exertion at each session. Activities of ≥4 metabolic equivalents (METs) were counted towards the prescribed target.
Women randomized to diet + exercise received separate sessions and were instructed not to discuss diet during supervised exercise. The control group was instructed not to change their diet or exercise habits for 12 months.
All study measures were obtained and analysed by trained personnel who were blinded to the participants' randomization status.
We collected demographic information, medical history and dietary intake (via a 120-item self-administered food frequency questionnaire) at baseline and 12 months. At both time points, participants wore pedometers (Accusplit, Silicon Valley, CA, USA) for seven consecutive days to determine an average daily step count. We assessed cardiorespiratory fitness (VO2max) using a maximal graded treadmill test according to a modified branching protocol.
BMI was calculated from weight and height, measured to the nearest 0·1 kg and 0·1 cm, respectively, with a balance beam scale and stadiometer. We measured waist circumference to the nearest 0·5 cm at the minimal waist. Body composition was measured on a DXA whole-body scanner (GE Lunar, Madison, WI, USA).
Fasting venous blood samples (50 ml) were collected during clinic visits prior to randomization and at 12 months. Participants ate no food and drank only water for 12 h prior to the blood draw, and were requested not to exercise for the preceding 24 h. Blood was processed within 1 h, centrifuged in refrigerated centrifuges kept between 4 and 8 degrees C. Samples were subsequently stored at −70 °C. The blood specimens, collected between February 2005 and July 2009, were frozen until June 2010 when the assays were performed; the samples had not been previously thawed.
Blood samples were analysed in batches with each participant's samples assayed simultaneously. The number of samples from each arm was approximately equal. Participant randomization dates were similar, and the sample order was random. Assays were performed at the University of Washington Northwest Lipid Research Laboratories.
We measured total, immune-reactive serum ghrelin by radioimmunoassay using the Millipore total human ghrelin assay (Millipore, Billerica, MA, USA) with limits of detection of 110 to 10 000 pg/ml. The intra- and inter-assay coefficients of variation (CV) were 11·8% and 15·9%, respectively.
Serum insulin was quantified by a 48-h, polyethylene glycol-accelerated, double antibody radioimmunoassay. The intra-assay CV was 4·5%. We used a Clinical Chemistry Autoanalyzer with the hexokinase method to quantify glucose. The intra- and inter-assay CVs were 1·1% and 3·5%, respectively. The homoeostasis assessment model (HOMA-IR = fasting insulin (mU/l) × fasting glucose (mmol/l)/22·5) was calculated as a surrogate measure of whole-body insulin resistance. Adiponectin was measured in serum samples using a radioimmunoassay (Linco Research, St. Charles, Missouri, USA) with I-labelled murine adiponectin and anti-adiponectin antibody; serum leptin was quantified using a Linco Research Human Leptin radioimmunoassay that utilizes I-labelled Human Leptin and the double antibody/PEG technique (Millipore). Intra- and inter-assay CVs for adiponectin were 8·4% and 9·8%, respectively; for leptin, they were 9·1% and 14·4%, respectively.
For the main analyses, all available data were used without imputation for missing values. Pearson correlation coefficients were calculated between total ghrelin and baseline anthropometric and blood measures. Descriptive data were presented as means (standard deviation, SD). Due to nonnormal distributions, blood parameters were log-transformed prior to further analysis, and these data are presented as geometric means with 95% confidence intervals (CI) unless otherwise indicated.
The mean change in fasting serum total ghrelin between baseline and 12 months in the diet, exercise and diet + exercise arms were computed and compared to the change among controls using the generalized estimating equation (GEE) modification of linear regression to account for intra-individual correlation overtime. The intervention effects were examined based on the assigned treatment at randomization, regardless of adherence or study retention (i.e., intent-to-treat). We used Bonferroni correction (two-sided alpha: 0·05/3 = 0·016) to adjust for multiple comparisons. The potential moderating effects of baseline ghrelin concentrations and BMI were also tested by including appropriate interaction terms in the models described above.
Subsequently, the effect of weight loss on total ghrelin was examined using a stratified analysis (<5%, 5–9·9% and ≥10% weight loss) performed within each intervention arm. These analyses were adjusted for age, race/ethnicity and baseline BMI.
Unstandardized bivariate regression coefficients were also calculated for the change in fasting total ghrelin associated with other metabolic parameters (leptin, adiponectin, glucose, insulin and HOMA-IR) in women randomized to diet, exercise or diet + exercise.
All statistical analyses were performed using SAS software version 9·1 (SAS Institute, Cary, NC, USA).
Methods
Design
The Nutrition and Exercise in Women (NEW) study was a 12-month randomized controlled trial to test the effects of dietary weight loss and/or exercise on circulating hormones and other outcomes. Study procedures were reviewed and approved by the Fred Hutchinson Cancer Research Center Institutional Review Board in Seattle, WA. All participants provided written informed consent.
Setting and Participants
Participants were overweight or obese (BMI ≥25 or BMI ≥30 kg/m (≥23 kg/m if Asian–American), postmenopausal women (50–75 y) from the greater Seattle area. They were recruited through media and mass mailings (Fig. 1). Exclusion criteria included: diagnosed diabetes; fasting blood glucose ≥126 mg/dL or use of diabetes medications; use of postmenopausal hormone therapy; history of invasive cancer within the past 10 years, excluding basal or squamous cell skin cancer; alcohol intake >2 drinks/day; current smoking; abnormal screening labs (e.g., haematocrit <32 or >48, white blood cell count <3·0 or >15·0, potassium <3·5 or >5·0, creatinine >2·0) or contraindication to the study interventions for any reason (e.g., abnormalities on screening physical, abnormal exercise tolerance test, uncontrolled hypertension, history of cardiac arrest or stroke, recent (within 6 months) myocardial infarction, pulmonary oedema, myocarditis, pericarditis, unstable angina, pulmonary embolism/deep vein thrombosis, orthostatic hypotension, moderate/severe asthma, uncontrolled arrhythmia, uncontrolled congestive heart failure, 3rd degree heart block, left bundle branch block, myocarditis, thrombophlebitis); participation in another structured weight loss program or use of weight loss medications; additional factors that might interfere with measurement of outcomes or with the success of the intervention (e.g., inability to attend facility-based sessions).
(Enlarge Image)
Figure 1.
Flow of participants through the NEW study.
Randomization and Interventions
Eligible women were randomized to: (i) dietary weight loss ('diet'; n = 118); (ii) moderate-to-vigorous intensity aerobic exercise ('exercise'; n = 117); 3) combined diet and exercise ('diet + exercise'; n = 117); or 4) control (no intervention) (n = 87). Computerized random assignment, using permuted blocks randomization to achieve a proportionally smaller control group, was stratified according to BMI (≥ or <30 kg/m) and race/ethnicity.
The dietary weight loss intervention was a modification of the Diabetes Prevention Program and Look AHEAD lifestyle behaviour change programs with goals of 1200–2000 kcal/day, <30% calories from fat, and 10% weight loss by 6 months with maintenance thereafter. Participants met individually with a dietitian at least twice, followed by weekly group meetings (5–10 women) for 6 months. Thereafter, they attended monthly group meetings in addition to biweekly phone or e-mail contact. Women completed daily food logs for at least 6 months or until they reached their 10% weight loss goal. Food logs, weekly weigh-ins and session attendance were tracked to promote dietary adherence.
Participants who did not meet their weight loss goal by 6 months were encouraged to continue weight loss efforts and were offered additional sessions; women who reached their goal were allowed to continue losing weight but were monitored to ensure that their BMI did not drop below 18·5 kg/m.
The exercise intervention progressed to 45 min of moderate-to-vigorous intensity exercise at a target heart rate of 70–85% observed maximum, 5 days/week by the 7th week. Participants attended 3 supervised sessions/week at the study facility and exercised 2 days/week at home. They recorded exercise mode, duration, peak heart rate and perceived exertion at each session. Activities of ≥4 metabolic equivalents (METs) were counted towards the prescribed target.
Women randomized to diet + exercise received separate sessions and were instructed not to discuss diet during supervised exercise. The control group was instructed not to change their diet or exercise habits for 12 months.
Outcomes and Follow-up
All study measures were obtained and analysed by trained personnel who were blinded to the participants' randomization status.
We collected demographic information, medical history and dietary intake (via a 120-item self-administered food frequency questionnaire) at baseline and 12 months. At both time points, participants wore pedometers (Accusplit, Silicon Valley, CA, USA) for seven consecutive days to determine an average daily step count. We assessed cardiorespiratory fitness (VO2max) using a maximal graded treadmill test according to a modified branching protocol.
BMI was calculated from weight and height, measured to the nearest 0·1 kg and 0·1 cm, respectively, with a balance beam scale and stadiometer. We measured waist circumference to the nearest 0·5 cm at the minimal waist. Body composition was measured on a DXA whole-body scanner (GE Lunar, Madison, WI, USA).
Fasting venous blood samples (50 ml) were collected during clinic visits prior to randomization and at 12 months. Participants ate no food and drank only water for 12 h prior to the blood draw, and were requested not to exercise for the preceding 24 h. Blood was processed within 1 h, centrifuged in refrigerated centrifuges kept between 4 and 8 degrees C. Samples were subsequently stored at −70 °C. The blood specimens, collected between February 2005 and July 2009, were frozen until June 2010 when the assays were performed; the samples had not been previously thawed.
Blood Analysis
Blood samples were analysed in batches with each participant's samples assayed simultaneously. The number of samples from each arm was approximately equal. Participant randomization dates were similar, and the sample order was random. Assays were performed at the University of Washington Northwest Lipid Research Laboratories.
We measured total, immune-reactive serum ghrelin by radioimmunoassay using the Millipore total human ghrelin assay (Millipore, Billerica, MA, USA) with limits of detection of 110 to 10 000 pg/ml. The intra- and inter-assay coefficients of variation (CV) were 11·8% and 15·9%, respectively.
Serum insulin was quantified by a 48-h, polyethylene glycol-accelerated, double antibody radioimmunoassay. The intra-assay CV was 4·5%. We used a Clinical Chemistry Autoanalyzer with the hexokinase method to quantify glucose. The intra- and inter-assay CVs were 1·1% and 3·5%, respectively. The homoeostasis assessment model (HOMA-IR = fasting insulin (mU/l) × fasting glucose (mmol/l)/22·5) was calculated as a surrogate measure of whole-body insulin resistance. Adiponectin was measured in serum samples using a radioimmunoassay (Linco Research, St. Charles, Missouri, USA) with I-labelled murine adiponectin and anti-adiponectin antibody; serum leptin was quantified using a Linco Research Human Leptin radioimmunoassay that utilizes I-labelled Human Leptin and the double antibody/PEG technique (Millipore). Intra- and inter-assay CVs for adiponectin were 8·4% and 9·8%, respectively; for leptin, they were 9·1% and 14·4%, respectively.
Statistical Analysis
For the main analyses, all available data were used without imputation for missing values. Pearson correlation coefficients were calculated between total ghrelin and baseline anthropometric and blood measures. Descriptive data were presented as means (standard deviation, SD). Due to nonnormal distributions, blood parameters were log-transformed prior to further analysis, and these data are presented as geometric means with 95% confidence intervals (CI) unless otherwise indicated.
The mean change in fasting serum total ghrelin between baseline and 12 months in the diet, exercise and diet + exercise arms were computed and compared to the change among controls using the generalized estimating equation (GEE) modification of linear regression to account for intra-individual correlation overtime. The intervention effects were examined based on the assigned treatment at randomization, regardless of adherence or study retention (i.e., intent-to-treat). We used Bonferroni correction (two-sided alpha: 0·05/3 = 0·016) to adjust for multiple comparisons. The potential moderating effects of baseline ghrelin concentrations and BMI were also tested by including appropriate interaction terms in the models described above.
Subsequently, the effect of weight loss on total ghrelin was examined using a stratified analysis (<5%, 5–9·9% and ≥10% weight loss) performed within each intervention arm. These analyses were adjusted for age, race/ethnicity and baseline BMI.
Unstandardized bivariate regression coefficients were also calculated for the change in fasting total ghrelin associated with other metabolic parameters (leptin, adiponectin, glucose, insulin and HOMA-IR) in women randomized to diet, exercise or diet + exercise.
All statistical analyses were performed using SAS software version 9·1 (SAS Institute, Cary, NC, USA).
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