Nutrition

Steroids 77 (2012) 323–331

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Steroids

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / s t e r o i d s

Review

Meal timing and composition influence ghrelin levels, appetite scores and weight loss maintenance in overweight and obese adults

Daniela Jakubowicz a,⇑, Oren Froy b, Julio Wainstein a, Mona Boaz c,d a Diabetes Unit, E. Wolfson Medical Center, Tel Aviv University, Holon 58100, Israel b Institute of Biochemistry, Food Science and Nutrition, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel c Epidemiology and Research Unit, E. Wolfson Medical Center, Holon 58100, Israel d School of Health Sciences, Department of Nutrition Sciences, Ariel University of Samaria, Israel

a r t i c l e i n f o a b s t r a c t

Article history: Received 13 October 2011 Accepted 22 November 2011 Available online 9 December 2011

Keywords: Meal timing Diet induced weight loss Weight regain Craving Ghrelin suppression

0039-128X/$ - see front matter � 2011 Elsevier Inc. A doi:10.1016/j.steroids.2011.12.006

⇑ Corresponding author. Tel.: +972 50 810 5552 (I fax: +972 3 502 8384.

E-mail addresses: [email protected] (D. Jak (O. Froy), [email protected] (J. Wains (M. Boaz).

Background: Although dietary restriction often results in initial weight loss, the majority of obese dieters fail to maintain their reduced weight. Diet-induced weight loss results in compensatory increase of hun- ger, craving and decreased ghrelin suppression that encourage weight regain. A high protein and carbo- hydrate breakfast may overcome these compensatory changes and prevent obesity relapse. Methods: In this study 193 obese (BMI 32.2 ± 1.0 kg/m2), sedentary non diabetic adult men and women (47 ± 7 years) were randomized to a low carbohydrate breakfast (LCb) or an isocaloric diet with high car- bohydrate and protein breakfast (HCPb). Anthropometric measures were assessed every 4 weeks. Fasting glucose, insulin, ghrelin, lipids, craving scores and breakfast meal challenge assessing hunger, satiety, insulin and ghrelin responses, were performed at baseline, after a Diet Intervention Period (Week 16) and after a Follow-up Period (Week 32). Results: At Week 16, groups exhibited similar weight loss: 15.1 ± 1.9 kg in LCb group vs. 13.5 ± 2.3 kg in HCPb group, p = 0.11. From Week 16 to Week 32, LCb group regained 11.6 ± 2.6 kg, while the HCPb group lost additional 6.9 ± 1.7 kg. Ghrelin levels were reduced after breakfast by 45.2% and 29.5% following the HCPb and LCb, respectively. Satiety was significantly improved and hunger and craving scores signifi- cantly reduced in the HCPb group vs. the LCb group. Conclusion: A high carbohydrate and protein breakfast may prevent weight regain by reducing diet- induced compensatory changes in hunger, cravings and ghrelin suppression. To achieve long-term weight loss, meal timing and macronutrient composition must counteract these compensatory mechanisms which encourage weight regain after weight loss.

� 2011 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324 2. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324

2.1. Study design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324 2.2. Participants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324 2.3. Diet Intervention Period (Week 0–Week 16). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 2.4. Follow-up Period (Week 16–Week 32) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 2.5. Anthropometric measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 2.6. Fasting blood assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 2.7. Breakfast meal challenge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 2.8. Blood analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 2.9. Appetite questionnaires . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 2.10. Craving scores questionnaire . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

ll rights reserved.

srael)/+1 3234107001 (USA);

ubowicz), [email protected] tein), [email protected]

324 D. Jakubowicz et al. / Steroids 77 (2012) 323–331

2.11. Sample size and study power . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 2.12. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

3. Results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

3.1. Patient dispensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 3.2. Weight loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 3.3. Fasting serum glucose, insulin and lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 3.4. Craving scores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 3.5. Cravings and weight change . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 3.6. Breakfast meal challenge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

3.6.1. Insulin response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 3.6.2. Ghrelin response. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 3.6.3. Hunger, satiety VAS scores. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

1. Introduction

Weight regain after weight loss represents one of the major obstacles in the therapeutic management of overweight and obes- ity, undoubtedly contributing to the epidemic of overweight which now exceeds 60% in United States adults and almost 20% of chil- dren [1–5]. Although dietary restriction often results in initial weight loss, the majority of obese dieters fail to maintain their reduced weight [5]. These diets are typified by short term [3– 6 months] success; however, most individuals cannot maintain such weight loss strategies over time [1,3,6–9].

Proposed predictors of weight regain after weight loss include increased subjective appetite scores, especially increased hunger and craving [6–12]. Energy and/or carbohydrate restricted weight loss diets have been shown to produce a carbohydrate withdrawal effect which further exacerbates hunger and carbohydrate crav- ings, ultimately resulting in weight regain [9,12–16]. The reward value of carbohydrates and the consequences of its withdrawal on hunger, cravings and satiety, are not addressed by many weight loss diets, including the more successful methods [17].

Most weight loss diets result in compensatory metabolic changes, including reduced energy expenditure [18,19], increased hunger [9,12,13,20,21] and craving scores [14–16], increased circu- lating ghrelin and decreased posprandial ghrelin suppression [21,22]. These alterations persist over time, even 1 year after initial weight reduction [21]; further, these changes promote weight re- gain after diet-induced weight loss. Long term strategies to coun- teract these changes and to facilitate maintenance of weight loss over time might include consideration of dietary macronutrient composition and meal timing.

Macronutrient composition of the diet has been shown to influ- ence hunger, satiety and cravings [16,23]. Several studies have shown that dietary protein is the most satiating of the macronutri- ents in conditions of both energy restriction and energy balance [24–27]. It has also been shown that the addition of carbohydrates to protein leads to additional reduction of hunger and increased satiety [28–30].

Meal timing also appears to influence its satiating properties. Specifically, protein consumed at breakfast (compared to lunch or dinner) leads to greater initial and sustained feelings of fullness, increased satiety and reduced levels of the appetite-regulating hor- mones such as ghrelin [31–35]. Moreover, the daily addition of a carbohydrate-rich snack (i.e. sweet) to breakfast has been shown to reduce the snack’s reward value decreasing cravings for sweets, breads, carbohydrates and fast food [36].

The present study was designed to address whether a change in diet macronutrient composition and meal timing impacts these metabolic outcomes (appetite and ghrelin levels) leading to long

term dietary adherence and prevention of weight regain. We stud- ied a population of overweight and obese adults and compared the effects of two isocaloric weight loss diets with different meal tim- ing and composition on appetite, craving scores, ghrelin levels, weight loss and maintenance during two consecutive periods: (1) Diet Intervention Period; and (2) Follow-up Period.

2. Materials and methods

2.1. Study design

The present study is a randomized, treatment controlled, open clinical trial comparing the effects of two isocaloric dietary interven- tions with different composition and meal timing on subjective appetite scores, craving, ghrelin suppression, weight loss and maintenance.

2.2. Participants

The study protocol initially included 193 obese/overweight sub- jects (115 women), recruited from outpatient clinics by means of personal interview and advertising. Inclusion criteria were adult (age 20–65 years); overweight or obese (body-mass index 25–37 kg/m2) non-diabetic [glucose <200 mg/dl 2 h after oral administration of 75 g glucose after an overnight fast]; with normal thyroid, liver and kidney function as assessed by standard blood tests. Exclusion criteria included individuals with diabetes or abnormal thyroid, liver or kidney function. Individuals who were presently dieting, using medications affecting body weight or who had experienced a change in weight >4.5 kg or a change in physical activity within the six months preceding study onset were excluded. Gastrointestinal problems possibly preventing dietary adherence; pregnancy or lactation; cancer or other characteristics [psychological or physical disabilities] deemed likely to interfere with participation in or compliance with the study were further exclusion criteria. Subjects taking antihypertensive or lipid-lower- ing medication were asked to maintain all medications and supple- ments at pre-study doses. Most subjects were sedentary at baseline and were asked to maintain their usual physical activity levels and to refrain from drinking >2 standard glasses of alcohol per week throughout the study.

The protocol and potential risks and benefits of the study were fully explained to each subject before he/she provided a written in- formed consent. All experimental procedures followed ethical standards of and were approved by the Institutional Review Board Helsinki Committee at the Wolfson Medical Center, Holon, Israel.

Table 1 Diet composition by treatment assignment and sex.

HCb Women LCb Women

Kcal gCh (%) gProt (%) gFat (%) Kcal gCh (%) gProt (%) gFat (%)

Breakfast 600 60 (40) 45 (30) 20 (30) 300 10 (13.3) 30 (40) 16 (48) Lunch 500 10 (8) 70 (56) 20 (36) 500 10 (8) 70 (56) 20 (36) Dinner 300 8 (10.7) 45 (60) 10 (30) 600 16 (10.6) 90 (60) 20 (30) Total 1400 78(19.6) 160 (48.6) 50 (32) 1400 36 (10.6) 190 (52) 56 (38)

HCb Men LCb Men

Breakfast 600 60 (40) 45 (30) 20 (30) 300 10 (13.3) 30 (40) 16 (48) Lunch 600 12 (8) 84 (56) 24 (36) 600 12 (8) 84 (56) 24 (36) Dinner 400 11 (10.7) 60 (60) 20 (30) 700 19 (10.6) 105 (60) 23 (30) Total 1600 83 (19.5) 189 (48.7) 64 (32) 1600 41 (10.7) 219 (52) 63 (38)

HCPb = high carbohydrate and protein breakfast diet. LCb = low carbohydrate breakfast diet; gCh (%) = grams of carbohydrate and %; gProt (%) = grams of protein and %; gFat (%) = grams of fat and %.

D. Jakubowicz et al. / Steroids 77 (2012) 323–331 325

2.3. Diet Intervention Period (Week 0–Week 16)

Subjects were assigned to one of two isocaloric weight loss diets which differed primarily in the composition of the breakfast meal:

a) Low carbohydrate diet (LCb): a low carbohydrate diet with a low calorie, and low carbohydrate breakfast; and

b) High carbohydrate- and protein-enriched breakfast diet (HCPb) with similar composition at lunch and at dinner to the low carbohydrate diet, but with a calorie-carbohy- drate-and protein-enriched breakfast. In this group, the breakfast also included a ‘‘dessert’’ on a daily basis. The ‘‘des- sert’’ was a sweet food selected from the following list: choc- olate, cookies, cake, ice cream, chocolate mousse or donuts.

Men were instructed to consume 1600 kcal while women were instructed to consume 1400 kcal daily. Composition of the diet interventions is presented in Table 1. In order to maintain daily en- ergy intake constant, the dinner in the HCPb was reduced from 600 to 300 kcals for women and from 700 to 400 kcals for men (Table 1). All subjects were counseled by a registered dietitian who in- structed subjects how to keep daily diet intake checklists for all foods consumed. The subjects’ body weights and dietary intake checklists were monitored every 4 weeks, and dietary adjustments were made as necessary.

2.4. Follow-up Period (Week 16–Week 32)

At the end of the Diet Intervention Period (Week 16), both groups entered the Follow-up Period (Week 16–Week 32). Partici- pants received individual counseling and written advice from a dietitian to continue the diets, including meal timing, followed during the Diet Intervention Period; however, they were to be self-supervised in terms of caloric restriction, and were free to eat as motivated by hunger or cravings. Nevertheless, the dietitian emphasized that the maintenance of weight loss is predicated on the participant’s ability to adhere to their previously assigned weight loss strategy over time. During the Follow-up Period, sub- jects continued visiting the clinic every 4 weeks, with the checklist for all foods consumed, for weighing and examinations, but with- out dietetic counseling. Food checklists were for post-hoc analyses

2.5. Anthropometric measurements

Subjects were weighed every 4 weeks during the study on a Detecto Physician Beam Scale (HOSPEQ, Inc., Miami, FL), before breakfast, wearing light clothes but no shoes. Waist circumference was measured using a tape measure at the umbilicus. Blood pres- sure was measured with the patient in a supine position using a

standard cuff and sphygmomanometer. The mean of three rested measures was recorded.

2.6. Fasting blood assays

All assays were performed after overnight fast on Week 0, Week 16 and Week 32, for measurement of lipids, glucose, insulin serum levels and ghrelin plasma levels.

2.7. Breakfast meal challenge

At three time points during the study, baseline (Week 0), Week 16 and Week 32, we conducted an acute meal challenge in which subjects consumed the breakfast prescribed by their assigned diet intervention. Specifically, subjects assigned to the HCPb diet re- ceived an enriched breakfast, as prescribed by the HCPb diet, while subjects assigned to the LCb diet received a low calorie, low carbohy- drate breakfast. The breakfast meals were consumed in their en- tirety within 15 min. On the day of the meal challenge, each subject reported to the laboratory at 07:00 after an overnight fast. After voiding, the subject was instructed to lie in a supine position on a bed. At 07:30, a catheter was placed in an antecubital vein of the non-dominant arm and kept in the patient for the next 240 min by saline drip. Thirty minutes after the catheter was in- serted, the fasting baseline blood sample was taken for measure- ment of insulin and ghrelin. Venous blood samples were collected before and 30, 60, 120, 180 and 240 min after breakfast to assess insulin and ghrelin responses. The appetite scores were concomi- tantly completed.

2.8. Blood analysis

Blood samples for measurement of glucose, insulin and lipid concentrations were collected in tubes with no additives and al- lowed to coagulate at room temperature for 30 min. Serum was isolated by centrifugation (Beckman, Fullerton, CA) at 600�g for 10 min at 4 �C and was frozen at �20 �C until analyzed. Serum glu- cose was determined by the glucose oxidase method (Beckman Glucose Analyzer, Fullerton, CA). Serum total cholesterol, HDL cho- lesterol, and triacylglycerols, were measured enzymatically using a Hitachi-Cobas Bio centrifugal analyzer (Roche) using standard enzymatic kits (Roche). Low-density lipoprotein cholesterol (LDL- C) was calculated according to the methods described [37]. Serum insulin was determined by a double antibody RIA [CIS Bio Interna- tional, Gif-Sur Yvette-Cedex, France), Sensitivity was 2.0 lU/ml and the intra- and inter-assay variability were 4.2% and 8.8%, respectively. Homeostasis model assessment (HOMA-R) index was calculated using the following formula: fasting serum insulin [mlU/ml] � fasting serum glucose (mmol/l)/22.5 [38].

326 D. Jakubowicz et al. / Steroids 77 (2012) 323–331

Blood samples for measurement of plasma ghrelin concentra- tions was collected in tubes containing EDTA and centrifuged at 3000 rpm at �4 �C for 15 min. The plasma was then separated and stored in microcentrifuge tubes at �80 �C for future analysis. Plasma total ghrelin was measured with an enzyme immunoassay kit (Phoenix Pharmaceuticals, Belmont, CA). The range of the kit was 0–261 pM/L. The assay sensitivity was 12 pM/L; the intra-as- say and inter-assay coefficients of variation for the assay control was 4%. All samples from a given subject were tested in duplicate and analyzed in the same assay. Total (insulin and ghrelin) and net [visual analog scores for appetite] areas under the curve during the 4-h breakfast meal tolerance test were calculated geometrically by using the trapezoidal rule.

2.9. Appetite questionnaires

Appetite scores for hunger and satiety were assessed using 100- mm visual analog scales (VAS), after acute meal challenge, at the same time points blood sampling was performed. Subjects were asked to make a single vertical mark on each scale somewhere between the 0 and 100 mm extremes (e.g., not at all hungry to very hungry) to indicate their feelings at that time point. Subjects did not discuss their ratings with each other and could not refer to their previous ratings when marking the scale. Reliability and validity of using VAS for assessing measures of appetite has been reported [39].

2.10. Craving scores questionnaire

Food cravings were assessed using the Food Craving Inventory (FCI), a 28-item questionnaire designed to measure the frequency of overall food cravings as well as cravings for specific types of foods [40]. Cravings for specific types of foods were measured by four independent subscales, each consisting of 4–8 items within

Fig. 1. Consort diagram. ⁄All randomized subjects are included in the analysis per intention to-treat principle. Missing data were imputed using last observation carried forward.

the food category: high fats [i.e., fried chicken, gravy, sausage, hot dogs, fried fish, corn bread, bacon, steaks]; sweets (i.e., cakes, cinnamon rolls, ice cream, cookies, chocolate, donuts, candy, brownies); carbohydrates/starches (i.e., sandwich bread, rice, bis- cuits, pasta, pancakes/waffles, rolls, cereal, baked potato]; and fast-food (i.e., pizza, French fries, hamburger, chips). Participants rated how often they experienced a craving for each of the foods using a 5-point Likert scale (1 = never, 5 = always/almost every day). In addition to the four independent subscales, an overall score was calculated by summing the subscales and represents the general food craving score. Craving scores were assessed 2 days prior to initiating the diet intervention; at Week 16 and Week 32 of the study.

2.11. Sample size and study power

A sample size of 130 participants (65 in each treatment group) provided 80% power to detect a true, between-group difference of 5 ± 10 kg at the end of follow-up. An additional 63 subjects were recruited to cover drop outs, which we predicted would reach almost 50% based on diet study drop-out rates in the literature.

2.12. Statistical analysis

All data are presented as the mean ± SEM. Statistical compari- sons of group differences were performed using one-way ANOVAs combined with Tukey’s post-hoc tests to compare the results between surgical groups (S-ADREC, ADREC and A-DEX) and cell treatment groups. Analysis of data was carried out using SPSS 11.0 statistical analysis software (SPSS Inc., Chicago, IL). For contin- uous variables, such as age, weight and biochemical measures, descriptive statistics were calculated and reported as mean ± stan- dard deviation. Normality of distribution of continuous variables was assessed using the Kolmogorov–Smirnov test (cut off at p = 0.01). Normally distributed continuous variables were com- pared by treatment assignment using the t-test for independent samples, while continuous variables with distributions signifi- cantly deviating from normal were compared by treatment assign- ment using the Mann Whitney U. Categorical variables, such as sex and treatment assignment, were described using frequency distri- butions and are presented as n (%). A model of each of the contin- uous outcomes: appetite scores, cravings scores, ghrelin and body weight was developed using general linear modeling (GLM) re- peated measures analyses. Treatment assignment and sex were in- cluded in all models as fixed factors and a sex-by-treatment interaction was assessed. Additionally, areas under the curve for biochemical measures, appetite and cravings scores over time were calculated using the trapezoidal rule and compared by treatment assignment using the t-test for independent samples. All tests fol- low the intention-to-treat principle and missing data were im- puted using last observation carried forward. All tests are two- tailed and considered significant at p < 0.05.

3. Results

3.1. Patient dispensation

Of the 193 subjects (BMI=32.3 ± 1.8 kg/m2) initially recruited and accepted for participation in the study, 96 (57 women and 39 men) were assigned to the HCPb group and 97 subjects (58 women and 39 men) were assigned to the LCb group. Patient dispensation is depicted in Fig. 1. As can be seen, a total of 144 participants completed the study, 74 (44 women) in HCPb group and 70 (42 women) in LCb group. Participants are compared by completion status in Table 2. In contrast to subjects who

Table 2 Characteristics of the study population by completion status.

HCPb group LCb group

Completed Withdrew Completed Withdrew

n = 74 n = 22 n = 70 n = 27 Follow-up time (weeks) 32 16.2 ± 10.4 32 15.5 ± 10.4 Age 46.7 ± 7.1 42.3 ± 7.3 47.5 ± 6.5 44 ± 8.3 Sex (females) 59.5 59.1 60 59.3 Weight week 0 (kg) 91.2 ± 9.8 93.5 ± 7.5 90.4 ± 9.2 93.3 ± 7.2 BMI week 0 (kg/m2) 32.2 ± 1.9 32.2 ± 2.0 32.3 ± 1.9 32.4 ± 1.5 Weight D Week 0–16 (kg) �13.6 ± 2.3 �1.4 ± 1.6 �15.3 ± 1.9 �2.1 ± 2.6 Hunger AUC240 min 19,391 ± 2355 19,343 ± 2328 35,628 ± 2497 35,374 ± 1761 Satiety AUC240 min 41,460 ± 3056 40,882 ± 3366 24,966 ± 2754 24,936 ± 1316

Craving Scores Week 0 Sweets 12.7 ± 1.6 14.0 ± 2.7 12.3 ± 2.3 13.9 ± 1.8 Fats 9.7 ± 1.1 11.6 ± 1.1 9.3 ± 1.6 11.1 ± 2.1 Carb/starches 12.5 ± 1.5 12.9 ± 1.5 12.5 ± 1.5 13.0 ± 1.6 Fast foods 13.1 ± 1.5 12.1 ± 1.5 13.5 ± 1.7 12.7 ± 1.3 General craving 48.0 ± 4.4 50.5 ± 5.2 47.6 ± 4.9 50.7 ± 3.2

Data are indicated as mean ± SD. Compared to participants who completed the study, those who withdrew (regardless of treatment assignment) were significantly younger (p = 0.001); had significant higher craving scores for sweets (p < 0.0001), fats (p < 0.0001), and general craving (p < 0.0001), but had significant lower scores for fast food craving (p = 0.001). Additionally, subjects who dropped out gained weight by Week 16, while completers had lost weight at Week 16 (p < 0.0001).

D. Jakubowicz et al. / Steroids 77 (2012) 323–331 327

completed the study, those who dropped out were significantly younger and had significantly higher general craving scores and craving scores for sweets and fats, and significantly lower craving scores for fast foods, regardless of treatment assignment. Addition- ally, subjects who withdrew had gained weight by Week 16, while those who completed the study had lost weight at this time point. Subjects who withdrew did not differ from completers in terms of sex or treatment assignment. All 193 subjects randomized to treat- ment are included in the analysis of results according to the inten- tion-to-treat principle and using last observation carried forward to impute values.

3.2. Weight loss

At baseline, body weight was similar by treatment group (Table 3). By the end of the Diet Intervention Period (Week 16), subjects in both treatment groups lost a significant amount of weight from baseline (Fig. 2). During the Follow-up Period, from Week 16 through Week 32, subjects in the HCPb group lost additional weight, while subjects in the LCb group regained weight. Thus, at the end of the Follow-up Period (Week 32), body weight was sig- nificantly different between the two groups and was significantly lower in the HCPb than LCb group (p < 0.0001) (Table 3).

3.3. Fasting serum glucose, insulin and lipids

Fasting concentrations of glucose, insulin and HOMA-IR decreased from baseline to Week 16 in both groups. From Week 16 to Week 32, these values further declined in the HCPb group. By contrast, these values increased from Week 16 to Week 32 in the LCb group. Values differed significantly between the groups at Week 32 (Table 3). At baseline, both groups were similar in total, HDL and LDL cholesterol and triglycerides (TG). By Week 16, TG values were significantly lower and HDL values significantly higher in the LCb group. At Week 32, total cholesterol, TG and LDL were all significantly lower, while HDL was significantly higher, in the HCPb vs. LCb group (Table 3).

3.4. Craving scores

At baseline, none of the food craving scores differed signifi- cantly by diet intervention group. At the end of the Diet Interven- tion Period (Week 16), all craving scores were significantly higher

in the LCb than in the HCPb group. By the end of the Follow-up Period (Week 32), all craving scores, including general cravings, sweets, high fats, carbohydrates/starches and fast foods, were significantly higher in the LCb than in the HCPb group (Table 3). The overall increase in craving scores in the LCb group was greatest for sweets, which was significantly greater than the increase in any other food category. Fat cravings were significantly greater than fast foods cravings in this group. The greatest reduction in cravings in the HCPb group was detected for sweets and fats. Other pair wise differences in cravings were not significant.

3.5. Cravings and weight change

Change in body weight during the Follow-up Period, Week 16 to Week 32, was significantly, positively associated with change in craving scores during the same phase. Specifically, in the Follow- up Period, weight change was associated with a change in cravings for sweets (r = 0.24, p = 0.004); carbohydrates and starches (r = 0.2, p = 0.02); fast foods (r = 0.25, p = 0.003); and general craving (r = 0.22, p = 0.007). An association between change in fats craving and change in body weight was not detected.

3.6. Breakfast meal challenge

3.6.1. Insulin response Insulin area under the curve [AUC] response to breakfast meal

challenge did not differ between diet intervention groups at the baseline. At Week 16, both groups exhibited a significant reduction of insulin-AUC from baseline. The HCPb group exhibited a further decrease at the end of Follow-up Period, while insulin AUC signifi- cantly increased in LCb group (Table 3). As shown in Table 3, at the Week 32 breakfast meal challenge, for insulin AUC was significantly, positively associated with body weight (r = 0.61, p < 0.0001).

3.6.2. Ghrelin response The nadir ghrelin value at baseline of the breakfast meal chal-

lenge was 301.2 ± 36.0 pg/ml in the HCPb group compared to 350.2 ± 26.4 pg/ml in the LCb group (p < 0.0001) (Table 3). Nadir ghrelin in response to HCPb breakfast was significantly decreased from baseline to Week 16 (p < 0.0001) and remained suppressed at Week 32 (Fig. 3). By contrast, in the LCb group, nadir ghrelin levels did not differ significantly between baseline and Week 16 (p = 0.06) and were significantly less decreased after the Follow-up

Table 3 Participant characteristic at baseline and after 16 and 32 weeks, n = 193 LCb group: n = 97; HCPb group: n = 96.

Group Baseline Week 16 Week 32

Weight (kg) HCPb 91.2 ± 9.8 77.6 ± 9.0 70.6 ± 8.7 LCb 90.4 ± 9.2 75.2 ± 8.1 86.9 ± 9.7 p-value 0.65 0.11 <0.001

BMI (kg/m2) HCPb 32.2 ± 1.9 27.4 ± 1.8 24.9 ± 1.9 LCb 32.3 ± 1.9 26.9 ± 1.7 30.9 ± 2.0 p-value 0.79 0.08 <0.001

Waist circumference (cm) HCPb 110.7 ± 3.1 103.3 ± 4.3 96.4 ± 5.3 LCb 110.4 ± 3.2 102.5 ± 4.3 108.7 ± 3.6 p-value 0.46 0.28 <0.001

FASTING VALUES Fasting glucose (mg/dl) HCPb 94.4 ± 7.0 86.2 ± 5.6 84.2 ± 4.6

LCb 94.6 ± 7.4 85.1 ± 6.7 95.5 ± 4.9 p-value 0.81 0.26 <0.001

Fasting insulin (lU/ml) HCPb 21.7 ± 3.6 12 6 ± 3.4 8.9 ± 3.9 LCb 21.7 ± 3.6 13.9 ± 4.8 23.69 ± 3.8 p-value 0.97 0.30 <0.001

HOMA-IR HCPb 5.0 ± 0.9 2.5 ± 0.5 1.6 ± 0.4 LCb 5.1 ± 0.9 2.4 ± 0.5 5.9 ± 0.9 p-value 0.89 0.19 <0.001

Total cholesterol (mg/dl) HCPb 211.8 ± 17.6 189.1 ± 10.6 179.2 ± 11.1 LCb 212.3 ± 19.8 188.6 ± 13.2 190.8 ± 18.2 p-value 0.87 0.81 <0.001

Triacylglycerol (mg/dl) HCPb 174.4 ± 17.6 140.8 ± 10.9 122.6 ± 9.7 LCb 174.5 ± 22.6 134.9 ± 7.9 174.5 ± 20.9 p-value 0.98 <0.001 <0.001

HDL cholesterol (mg/dl) HCPb 45.8 ± 5.3 48.8 ± 4.9 50.9 ± 4.9 LCb 47.4 ± 5.3 51.2 ± 5.0 48.0.2 ± 5.0 p-value N/A N/A N/A

LDL cholesterol (mg/dl) HCPb 157.2 ± 17.4 133.3 ± 10.8 122.2 ± 12.3 LCb 156.2 ± 20.6 130.7 ± 14.2 134.1 ± 19.5 p-value N/A N/A N/A

CRAVING SCORES Sweets HCPb 12.9 ± 1.9 9.7 ± 3.7 8.4 ± 4.3

LCb 12.78 ± 2.3 15.4 ± 1.8 17.1 ± 1.8 p-value 0.34 <0.001 <0.001

Fats HCPb 10.1 ± 1.8 9.2 ± 2.6 8.1 ± 2.9 LCb 9.8 ± 1.9 11.3 ± 1.7 12.3 ± 1.9 p-value 0.14 <0.001 <0.001

Carb/starch HCPb 12.6 ± 1.5 8.8 ± 3.8 8.2 ± 4.1 LCb 12.6 ± 1.6 15.7 ± 1.9 16.6 ± 1.9 p-value 0.85 <0.001 <0.001

Fast foods HCPb 12.8. ± 1.6 9.2 ± 3.6 8.5 ± 3.9 LCb 13.2 ± 1.6 15.9 ± 1.9 16.6 ± 2.0 p-value 0.15 <0.001 <0.001

General craving HCPb 48.6 ± 4.7 37.1 ± 12.9 33.2 ± 14.7 LCb 48.5 ± 4.8 58.4 ± 5.7 62.7 ± 6.1 p-value 0.57 <0.001 <0.001

BREAKFAST MEAL CHALLENGE AUC Ghrelin AUC240 min pg/ml � 240 min HCPb 219,431 ± 7479 204,325 ± 5579 201,115 ± 7295

LCb 275,432 ± 13,873 280,100 ± 11,735 282,968 ± 9526 p-value <0.001 <0.001 <0.001

Ghrelin nadir (pg/ml) HCPb 300.7 ± 35.9 243.3 ± 13.6 239.1 ± 23.4 LCb 350.5 ± 26.6 357.6 ± 17.1 363.9 ± 20.5 p-value <0.001 <0.001 <0.001

Insulin AUC 240 min lU/ml � 240 min HCPb 28,564 ± 3543 20,282 ± 3031 14,798 ± 4364 LCb 29,066 ± 3001 18,050 ± 3859 29,816 ± 5863 p-value 0.34 <0.001 <0.001

Hunger AUC240 min HCPb 19,346 ± 2310 19,301 ± 2475 19,890 ± 2204 LCb 35,499 ± 2436 40,651 ± 3264 40,639 ± 3110 p-value <0.001 <0.001 <0.001

Satiety AUC240 min HCPb 41,407 ± 3035 41,047 ± 3683 41,749 ± 2872 LCb 24,955 ± 2736 26,200 ± 6852 25,320 ± 2844 p-value <0.001 <0.001 <0.001

Data are indicated as mean ± SD. HCPb = energy-, carbohydrate- and protein-enriched breakfast diet; LCb = low carbohydrate breakfast diet. Conversion factors (metric units to SI units); glucose, mg/dl � 0.056 = mmol/l; insulin, lU/ml � 6.0 = pmol/L; ghrelin, pg/ml � 3.371 = pmol/L; total cholesterol, mg/dl � 0.0259 = mmol/l; triacylglycerol, mg/ dl � 0.0113 = mmol/l; HDL-cholesterol, mg/dl � 0.0259 = mmol/l.

328 D. Jakubowicz et al. / Steroids 77 (2012) 323–331

Period, (p = 0.03) in the LCb group. In the HCPb group after the Follow-up Period at Week 32, nadir ghrelin levels were significantly lower than at the end of the Follow-up Period in the LCb group (p < 0.0001) (Table 3). Nadir ghrelin, was significantly, inversely

correlated with body weight after Diet Intervention Period (r = �0.35, p < 0.0001) and after Follow-up Period (r = -0.42, p < 0.0001) in both groups. Additionally, nadir ghrelin was posi- tively correlated with all cravings scores at Week 16 and Week 32.

Fig. 2. Body weight by Diet Intervention Group. The p-value is for general linear model repeated measures comparisons. HCPb = energy-, carbohydrate- and pro- tein-enriched breakfast diet group, white squares: h LCb = low carbohydrate breakfast diet group, black squares: j.

D. Jakubowicz et al. / Steroids 77 (2012) 323–331 329

3.6.3. Hunger, satiety VAS scores At each breakfast challenge: baseline, Week 16 and Week 32,

hunger AUC was significantly lower, while satiety AUC was signif- icantly higher after the breakfast in the HCPb group than in LCb group (p < 0.0001) (Table 3). In the HCPb group, significant differ- ences in satiety and hunger scores were not detected from chal- lenge to challenge. By contrast, a significant increase in hunger was observed in the LCb group between baseline and after the Follow-up Period.

4. Discussion

In this study we observed that two isocaloric diets which dif- fered in meal timing and composition resulted in similar weight reduction at the end of the Diet Intervention Period. Weight regain after diet-induced weight loss was observed only in the LCb group, as has been reported in previous studies [4]. Subjects in the HCPb group were more successful in maintaining reduced weight; more- over, they continued losing weight during the Follow-up Period. Possible explanatory mechanisms for this between-group differ- ence in weight maintenance outcomes include the different influ- ence of both of the assigned diets on appetite, cravings and posprandial ghrelin levels.

Hunger and satiety response after the breakfast meal at baseline were consistent with previous reports [30,31,34]. Specifically, hunger scores were significantly lower and satiety scores signifi- cantly higher in the HCPb compared to the LCb group. By the end

Fig. 3. Ghrelin suppression after breakfast meal challenge at baseline, Week 16 and Week 32 by diet intervention group. The p-values are for GLM repeated measures comparison by group. HCPb = energy-, carbohydrate- and protein-enriched break- fast diet group, white squares: h LCb = low carbohydrate breakfast diet group, black squares: j.

of Diet Intervention Period, despite similar weight reduction in both groups, hunger scores increased significantly in the LCb group. This group reported significantly more hunger than subjects in the HCPb group. Contrastly, weight reduction was not associated with an increase in posprandial hunger in the HCPb group; further- more, HCPb subjects continued losing weight during the Follow-up Period and continued to report suppressed hunger throughout this period. This effect of an enriched breakfast on hunger and satiety persisted over time and was not less pronounced at Week 32 than after the baseline breakfast meal challenge, indicating a persistence of the treatment effect even in individuals habituated to a large breakfast [30]. These findings suggest that an enriched breakfast may represent a useful strategy to maintain weight loss and prevent weight regain over time.

All craving scores decreased in the HCPb group, especially for sweets and fats. By contrast, an overall increase in craving was ob- served in the LCb group, including general cravings and cravings for sweets, high fats, carbohydrates/starches and fast foods. The greatest between-group difference was craving for sweets, which were significantly higher in the LCb than in the HCPb group. Increased craving, particularly craving for sweets, was strongly associated with the regain of weight observed during the Follow- up Period in the LCb group. The weight reduction observed in the HCPb group during the Follow-up Period was correlated with decreased craving scores, especially for sweets and fats.

In many weight loss diets, energy is restricted concomitantly with the restricted intake of preferred foods, leading to an increase in the reinforcement value of the omitted or restricted food. This may be expressed as increased cravings for the desired food [14,41]. In contrast, repeated reinforcer presentation leads to a reduction of reinforcer efficacy and reduced motivation to obtain the desired food [36,42]. It is possible that the consumption of sweets at breakfast in the HCPb diet group [chocolate bar, choco- late mousse, cake, or donut] represents repeated reinforcement leading to reduced cravings.

Ghrelin suppression has been shown to be impaired in obese subjects, suggesting a defect in ghrelin-induced satiety mecha- nisms [43]. In this study, even before weight reduction, ghrelin levels were significantly more suppressed after HCPb than LCb breakfast, suggesting that breakfast composition might overcome the obesity related defect in ghrelin suppression. This between- group difference in ghrelin suppression is also consistent with previous reports showing greater ghrelin suppression after carbo- hydrate enriched vs. protein- or lipid-enriched meals [44,45].

Recent studies have shown that diet induced weight loss is associated with decreased posprandial ghrelin suppression, that persist over long time and that would be expected to facilitate re- gain of lost weight [21]. Despite similar weight loss in both groups at the end of the Diet Intervention Period, the association between- diet induced weight loss and decreased posprandial ghrelin sup- pression was seen only in the LCb group. By contrast, HCPb group subjects exhibited a significant increase in ghrelin suppression at Week 16. This suggests an improvement of ghrelin suppression after diet-induced weight loss which occurs selectively following a carbohydrate-enriched breakfast [46]. Moreover, despite addi- tional weight loss in the Follow-up Period in the HCPb group, nadir posprandial ghrelin remained suppressed. This implies that in the HCPb group, meal timing or diet composition or both, overcame or prevented the decrease of ghrelin suppression as has been shown in previous studies [21,22].

Cravings, especially for sweets and carbohydrates/starches, have been shown to be associated with ghrelin levels [47]. The strong association between nadir ghrelin levels and all craving scores categories observed in our study may represent an alterna- tive mechanism through which in the HCPb group the craving scores were significantly reduced.

330 D. Jakubowicz et al. / Steroids 77 (2012) 323–331

Findings of the present study must be considered in the frame- work of the study’s limitations. First, the between-group similarity in weight loss at Week 16 suggests similar within-group compli- ance, and the large between-group weight difference at Week 32 suggests that LCb subjects ceased dietary compliance while the subjects in the HCPb group maintained adherence even in the Fol- low-up Period. On the other hand, subjects in the HCPb group con- sumed added protein and carbohydrates in the morning, while the LCb group consumed a higher energy meal in the evening. This was necessary to ensure that the two diets remained isocaloric. Sub- jects in both groups lost weight until Week 16, indicating that both calorie-restricted diets resulted but in short term weight loss. The direct effects of meal timing (morning vs. evening consumption of carbohydrates) were not tested; however, this is the subject of our ongoing study.

In summary, increased hunger and craving scores coupled with decreased ghrelin suppression after diet induced weight loss in the LCb group was correlated with failed maintenance of weight reduction; on the contrary, progressive weight regain was ob- served during the Follow-up Period. This suggests that LCb subjects were not able to comply with this weight loss strategy over time. Subjects in the HCPb group continued losing weight during the Fol- low-up Period, implying that a carbohydrate- and protein-enriched diet may represent a strategy with which individuals can comply over the long term.

5. Conclusion

We found that the compensatory changes of appetite, craving and circulating as well as posprandial ghrelin that facilitate obesity relapse after diet-induced weight loss was prevented by addition of high carbohydrate, protein and calorie enriched breakfast. To achieve long term weight loss, the diet meal timing and macronu- trient composition has to counteract the compensatory mecha- nisms that encourage weight regain after weight loss.

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