HORMONAL RESPONSES OF WOMEN AFTER WEIGHT LOSS FROM PHYSICAL TRAINING AND A LOW CARBOHYDRATE DIETING Respostas hormonais de mulheres após redução de peso por treinamento físico e dieta com restrição de carboidratos Bárbara Lúcia Fonseca CHAGAS1*; Ana Carolina Santos Barbosa MACHADO1; Marina de Macedo Rodrigues LEITE1; Marzo Edir da Silva GRIGOLETTO1; Ivina Elaine dos Santos ROCHA2 ; Liliane Viana PIRES3; Raquel Simões MENDES-NETTO1,3 1Postgraduate Program in Physical Education, Department of Physical Education, Federal University of Sergipe, Ave. Marechal Rondon, 49100-000, São Cristóvão, Sergipe, Brazil. 2 Laboratory Analysis in the Clinical Teaching Hospital of Federal University of Sergipe. Brazil. Av. Cláudio Batista, 49060-108, New City, Aracaju, Sergipe, Brazil 3Postgraduate Program in Nutrition Sciences, Department of Nutrition, Federal University of Sergipe, Ave. Marechal Rondon, 49100-000, São Cristóvão, Sergipe, Brazil. * Corresponding author: Raquel Simões Mendes Netto (email: raquel@academico.ufs.br). (Recebido em 21 de setembro de 2020; aceito em 19 de julho de 2021) ________________________________________________________________________________ ABSTRACT Aim. The present study aimed to analyze the effect of a 12-week low-carbohydrate diet and regular intermittent training on weight loss and hormone levels. Methodology. Participants in the study were overweight and obese adult women who were randomized to two low-calorie dietary groups: those receiving a diet with reduced carbohydrate levels and those receiving a diet with adequate carbohydrate levels. Anthropometric measures and hormone levels of the participants were assessed at the beginning and at the end of the 12-week program. Two-way analysis of variance was performed to compare the groups, and significance was set as p<0.01. Results. Participants of both groups exhibited weight loss and improvements in body composition with training and hypocaloric diets, regardless of the diet’s CHO content. However, reduced levels of the hormones T4, free T4, and testosterone were found in the L-CHO but not the A-CHO group. Conclusion. Weight loss through hypocaloric diets in combination with training resulted in hormonal changes favorable to insulin and catabolic control. The low-carbohydrate diet, in contrast, resulted in undesirable changes in the homeostasis of thyroid and anabolic hormones. Keywords: Obesity, Weight Loss, Diet; Carbohydrate, Hormones, Training. *Brazilian Clinical Trials Registry, n. RBR-5n9g5f. RESUMO Objetivo. O presente estudo teve como objetivo analisar o efeito da perda de peso baseado na redução de carboidratos na dieta, sobre respostas hormonais após seguimento dietético e prática regular de treinamento intermitente em doze semanas. Metodologia. Participaram do estudo mulheres adultas com excesso de peso as quais foram randomizadas em dois diferentes grupos de dieta, com redução de carboidratos e com adequados teores deste nutriente, ambos grupos submetidos a treinamento intermitente. As participantes foram avaliadas no início e ao final de 12 semanas do programa. Foi realizado teste ANOVA two-way para comparação entre os grupos. Utilizou-se o valor significativo de p<0,001. Resultados. Ambos os grupos apresentaram semelhanças na redução de peso e melhoria na composição corporal após as 12 semanas de realização do programa com treinamento físico e dietas hipocaloricas, independente do tipo de dieta. Porém os marcadores de T4, T4 livre e testosterona apenas demonstraram diminuição ao longo do tempo para o grupo R-CHO. Conclusão. A redução de peso através de dietas hipocaloricas em combinação com exercício físico resulta em alterações favoravéis para insulina e controle do catabolismo. Por outro lado a dieta restrita em carboidratos promoveu alterações indesejáveis na homeostase de hormônios tireoidianos e anabólicos. Palavras-Chave: Obesidade; Redução de peso; Carboidratos; Dieta; Hormônios; Treinamento. Introduction Lifestyle changes, such as increased physical activity and better eating habits, are essential for the prevention and treatment of excessive weight gain of the population1-4. Recently, altering the nutrient content in weight loss diet plans has been receiving increased attention5,6 in this context, low-carbohydrate (L-CHO) diets have been widely adopted as a strategy to promote weight loss7,8,9. In a meta-analysis10, the researchers analyzed studies that included diets with CHO levels ranging from 4% to 40% of the total energy intake (TEI) and those using a CHO intake restricted to 20g/day; they found that these diets were associated with improvements in the lipid and insulin profiles of the participants. These findings are similar to those of other studies that proposed weight loss based on caloric restriction; these studies reported reduce in the metabolic stress and increase in the levels of appetite-regulating hormones, thyroid hormones, and sex hormones11-13. However, when CHO restriction is too severe (<20g/day or <4% of the Total Energetic Intake - TEI), a lowered enthusiasm for physical activity, decreased lean mass, subsequent weight gain, and lower adherence to dietary follow-up have been reported14,15. Regular intermittent training can also contribute to weight loss and favorably affect the resting metabolic rate and anabolic hormonal responses in individuals participating in weight reduction programs16-19. Therefore, it is important to clarify the effect of L-CHO diets in combination with regular training on hormone levels, taking the metabolic consequences resulting from this type of intervention into account. In the present study, we analyzed the effects of dietary CHO reduction associated with intermittent training on weight loss and hormonal responses. Methods Population The present study was a randomized controlled clinical trial designed to assess changes in weight, body composition, and hormone levels of women participating in a 12-week weight loss program. Women aged 18–59 years with a body mass index (BMI) between 25 kg/m² and 34.9 kg/m²who were linked to an academic institution and who were defined as being sedentary or having a low activity level based on their International Physical Activity Questionnaire20 (IPAQ) scores were included in this study. Participants who self-reported the continuous use of anorectic drugs or undergoing hormone therapy and those who were under medical/nutritional monitoring for weight loss were ineligible. The study followed the recommendations of the Declaration of Helsinki and was approved by the research ethics committee of the university at which the study was conducted. We obtained registration in the Brazilian Clinical Trials Registry, n. RBR-5n9g5f. Study design All participants followed a weight loss program that involved semi-supervised tranning sessions three times/week and a monthly nutrition consultation. Study participants were randomly allocated into two groups. The first group received a dietary plan containing reduced CHO levels (L-CHO group), while the dietary plan given to the second group included adequate CHO levels (A-CHO group) (Figure 1). Both diets were low-calorie diets. During the monthly individualized nutrition consultation, dietary (24-hour dietary recall) and anthropometric (body weight and abdominal circumference) assessments were performed. The subjects answered a questionnaire asking questions on their adherence to the diet and a 24-hour dietary recall. The participants were also asked to complete at least three food records/month. Subjects analyzed in the group with adequacy of the dietary carbohydrate content (A-CHO) (n=8) Excluded from analyses: Men (n=8) Subjects analyzed in the group with reduction in the dietary carbohydrate content ( L-CHO) (n=11) Excluded from analyses: • Men (n=4) Eligible (n=106) Initiated intermittent training (n=24) Initiated other type of training (n=32) Concluded the 12-week intermittent training program (n=16) Loss to follow-up: Dropout/abandonment (n=8) Follow-up Allocation Inclusion Analysis Group with adequacy of the dietary carbohydrate content A-CHO ( n=56) Group with reduction in the dietary carbohydrate content L-CHO (n=50) Initiated intermittent training (n=25) Initiated other type of training (n=25) Concluded the 12-week intermittent training program (n=15) Loss to follow-up: Dropout/abandonment (n=10) Excluded Physical limitations (n=2) Abandonment (n=5) Did not meet the inclusion criteria (n=248) Demonstration of interest in inning the program (n=361) Figure 1. Flowchart of the experimental design. Weight loss program The calorie restriction protocol of the diets was based on the target of a reduction of 5% to 10% of the study participants’ body weight within 12 weeks. After analyzing the Estimated Energy Requirement (EER) of the individuals21, the calorie restriction required for each participant was individually calculated based on a 500-kcal deficit for individuals classified as overweight (BMI 25– 29.99 kg/m2), and a 1,000-kcal deficit for those classified as obese (BMI>30.0 kg/m2).Two types of hypocaloric diets that differed by their CHO content were administered. The diet adopted by the L- CHO group contained a CHO restriction that allowed a daily intake of around 100 g/day, which represents the minimum content of this nutrient according to the Dietary Reference Intake21 (DRI). In contrast, individuals in the A-CHO group were given a diet with a CHO content of around 250 g/day. The physical tranning was performed 3 times/week in sessions lasting approximately 60 minutes. Two of the three sessions were supervised by coach and guidance was offered on the third day of training that was otherwise performed by the participants without direct supervision. Each training session was conducted in three stages. The first stage was a standardized dynamic warm-up routine. The second stage included neuromuscular stimuli and was subdivided into neuromuscular sessions I and II. The neuromuscular I session was characterized by pushing, pulling, and, squatting exercises in a circuit training routine that was designed for achieving greater power, speed, agility, and coordination. The neuromuscular II session comprised strength exercises. For the neuromuscular sessions, five-minute circuits comprising five different exercises lasting one minute each were performed twice. The rest intervals between the exercises decreased along the 12-week duration of the program, with increased training intensity. The third stage included cardiorespiratory exercises using cognitive stimuli and lasted for five minutes. Body composition and hormone markers During the initial evaluation and at the end of the 12-week period, the participants’ weight was measured with a 100g-capacity precision digital scale (LIDER®, P150C, Ribeirão Preto, São Paulo, Brazil). Waist and hip circumferences were evaluated with a non-elastic tape measure (SANNY®, American Medical do Brazil Ltda., São Bernardo do Campo, São Paulo, Brazil), and body composition was measured through electrical bioimpedance (Biodynamics®, 310, Corporation, EUA 310). The participants underwent blood tests both, pre- and post-intervention. A 12-mL aliquot of blood was extracted by venipuncture of an antecubital vein after a 12-hour fast. Hormone analyses of insulin, cortisol, T3, T4, thyroid-stimulating hormone (TSH), and testosterone were performed using the Immunoassay Analyzer (Abbott Architect i1000SR Analyzer, USA). Statistical analysis Descriptive statistics, Delta Variation, and standard errors were used for data comparison between groups and as a function of time. The Shapiro-Wilk test was performed to determine the normality of the data. Two-way analysis of variance (ANOVA) and when F-ratio was significant, Bonferroni’s post hoc test was applied to identify the differences between the groups (G: L- CHO vs A-CHO), time (T: Pre - vs Post intervention), and time vs group interaction (G x T). Data were analyzed with the statistical software Statistical Package for the Social Sciences SPSS, version 20 for Windows, Chicago, USA. For all statistical analyses, p<0.01 was considered significant. The effect size (ES) was calculated by the post-intervention mean minus the pre- intervention mean, divided by the mean pre- and post-intervention standard deviations. To classify the magnitude of the differences, the clinical effect22 was considered small when the ES ranged from 0.20 to 0.49, medium when the ES ranged from 0.50 to 0.79, and large when the ES was >0.80 Results A total of 19 adult women with overweight and obesity (11 and 8 in the L-CHO and A-CHO groups, respectively) were included in the study. Both group shad similar characteristics (L-CHO vs. A-CHO: age,32 ± 7.7 years vs.28.5 ± 9.3 years; body weight, 79.4 ± 9.1 kg vs.76.7 ± 10.5 kg; and BMI, 30.4 ± 2.3vs. 28.8± 2.3 kg/m2). Moreover, both groups showed significant reductions in anthropometric measures and body composition after the 12-week intervention; no differences were seen between the two groups (Table 1). A large clinical effect was observed for most variables except lean mass and Estimated Energy Requirement (EER), which showed small effects. Table 2 depicts the variations in hormone levels after 12 weeks of tranning and dietary follow- up. We found a significant reduction in plasma T3, insulin, and cortisol levels in both groups; these changes were classified as large clinical effects (ES>0.8). The plasma levels of T4, free T4, and testosterone showed significant reductions over time in the L-CHO but not the A-CHO group; however, these effects were not significant in the G × T interaction analysis (p>0.05). TSH levels did not show any changes in the G, T, and G×T analyses. Table 1. Variations in anthropometric measures and body composition in overweight and obese women according to dietary plan Variation, mean (SE) Variables L-CHO (n = 11) A-CHO (n = 8) ANOVA effect F p Weight (kg) Pre-intervention 79.35 (2.92) 76.67 (3.42) G 50.41 <0.001 Post-intervention 73.35 (2.78)* 71.73 (3.26)* T 0.25 0.621 Δ (Δ%) -6.04 (-7.51) -4.94 (-6.42) G×T 0.142 0.712 ES -0.54 -0.66 BMI (kg/m2) Pre-intervention 30.38 (0.70) 28.80 (0.82) G 1.644 0.214 Post-intervention 28.07 (0.69)* 26.97 (0.81)* T 56.30 <0.001 Δ (Δ%) -2.32 (-7.51) -1.83 (-6.42) G × T 1.037 0.323 ES -0.94 -0.73 Abdominal circumference (cm) Pre-intervention 97.03 (1.96) 97.35 (2.30) G 0.004 0.951 Post-intervention 90.16 (2.10)* 89.47 (2.47)* T 90.12 <0.001 Δ (Δ%) -6.87 (-7.13) -7.87 (-8.04) G × T 0.45 0.835 ES -0.95 -1.0 Fat (%) Pre-intervention 36.97 (0.95) 34.31 (1.11) G 1.99 0.172 Post-intervention 33.03 (0.87)* 31.83 (1.02)* T 87.75 <0.001 Δ (Δ%) -3.94 (-10.57) -2.47 (-7.18) G × T 0.796 0.385 ES -0.9 -1.02 Fat (kg) Pre-intervention 29.46 (1.56) 26.55 (1.83) G 0.90 0.356 Post-intervention 24.34 (1.38)* 23.05 (1.62)* T 69.57 <0.001 Δ (Δ%) -5.12 (-17.21) -3.5 (-13.04) G × T 0.369 0.552 ES -0.78 -0.91 Lean mass (kg) Pre-intervention 50.78 (1.66) 49.01 (1.89) G 0.67 0.423 Post-intervention 49.37 (1.55)* 47.15 (1.76)* T 35.45 <0.001 Δ (Δ%) -1.41 (-2.75) -1.86 (-3.71) G × T 0.888 0.365 ES 0.31 -0.39 EER (kcal) Pre-intervention 1544.77 (50.7) 1490.14 (57.5) G 0.67 0.422 Post- intervention 1501.00 (47.4)* 1434.0(53.8)* T 37.68 <0.001 Δ (Δ%) -28.36(-1.76) -43.62 (-2.91) G × T 0.869 0.362 ES 0.31 -0.39 * Intragroup difference over time (pre- × post-intervention) A-CHO, adequate carbohydrate diet; ANOVA, analysis of variance; BMI, body mass index; EER, Estimated Energy Requirement, G, group; G × T, group × time interaction; ES, effect size; L-CHO, low-carbohydrate diet; SE, standard error; T, time Table 2. Variation in hormone levels in overweight and obese women according to the type of diet. Variation mean (SE) Variables L-CHO (n = 11) A-CHO (n = 8) ANOVA effect F p T3 Pre-intervention 1.25 (0.086) 1.30 (0.10) G 0.551 0.468 Post-intervention 0.95 (0.07)* 1.08 (0.08)* T 48.38 <0.001 Δ (Δ%) -0.30 (-22.3) -0.23 (-17.7) G × T 1.215 0.286 ES -0.75 -1.08 T4 Pre-intervention 6.81 (0.66) 7.09 (0.70) G 0.29 0.596 Post-intervention 6.16 (0.65)* 6.91 (0.69) T 5.45 <0.05 Δ (Δ%) -1.4 (-14.19) -0.19 (-2.95) G × T 0.61 0.444 ES -0.12 -0.37 Free T4 Pre-intervention 1.01 (0.04) 0.96 (0.04) G 0.03 0.862 Post-intervention 0.92 (0.03)* 0.96 (0.03) T 4.72 <0.05 Δ (Δ%) -0.08 (-7.79) -0.005 (-0.3) G × T 0.46 0.501 ES -0.37 -0.52 TSH Pre-intervention 2.24 (0.40) 1.63 (0.46) G 0.13 0.722 Post-intervention 1.62 (0.29) 1.88 (0.34) T 0.54 0.475 Δ (Δ%) -0.62 (-11.3) 0.25 (9.3) G × T 0.33 0.574 ES -0.19 -0.27 Insulin Pre-intervention 8.23 (0.84) 9.21 (1.03) G 0.085 0.772 Post-intervention 6.35 (0.74) 5.96 (0.91)* T 11.49 <0.001 Δ (Δ%) -3.91 (-27.9) -5.57 (-32.9) G × T 0.10 0.743 ES -0.87 -0.97 Cortisol Pre-intervention 12.53 (1.20) 10.32 (1.61) G 1.34 0.272 Post-intervention 9.15 (0.94)* 7.54 (1.26)* T 17.58 <0.001 Δ (Δ%) -5.3 (-31.17) -4.65 (-25.7) G × T 1.04 0.322 ES -0.92 -0.93 Testosterone Pre-intervention 0.48 (0.06) 0.37 (0.06) G 0.61 0.445 Post-intervention 0.41 (0.05)* 0.38 (0.06) T 2.68 0.124 Δ (Δ%) -0.06 (-12.48) 0.01 (3.36) G × T 0.12 0.733 ES -0.04 -0.29 * Intragroup difference over time (pre- × post-intervention) A-CHO, adequate carbohydrate diet; ANOVA, analysis of variance, G, group; G × T, group × time interaction; ES, effect size; L-CHO, low-carbohydrate diet; SE, standard error; T, time; TSH, thyroid- stimulating hormone Discussion The results of this study showed that participants of both groups exhibited weight loss and improvements in body composition with intermittent tranning and hypocaloric diets, regardless of the diet’s CHO content. However, reduced levels of the hormones T4, free T4, and testosterone were found in the L-CHO but not the A-CHO group. A moderate weight reduction can to be sufficient to affect the homeostasis of thyroid hormones13. In the present study, overweight and obese individuals who underwent individualized diets planned to create a deficit of 500–1000 kcal/day with the goal of achieving a 5–10% weight loss, exhibited decreased T3 levels. On the other hand, only the L-CHO group showed decreased T4 and free T4 levels after the 12-week intervention. A study with obese japanese patients23 reported a negative correlation between free T4 levels and weight reduction in 29 obese female premenopausal patients; higher free T4 levels were associated with successful weight loss after a six-month intervention. Therefore, a decrease in free T4 might reflect a reduction in the basal metabolic rate, which in the long run might impair the weight loss process24-27 and might result in subsequent weight gain. The total weight and fat mass loss seen in both groups in this study might have caused the reduction in cortisol and insulin levels28. However, dietary CHO reduction was sufficient to compromise testosterone homeostasis, despite that other studies relate the benefits of intermittent tranning routine in anabolic profile29-31. The main strengths of this study include: 1) the use of a CHO reduction diet that complied with the minimum limits proposed by standard nutritional guidelines, 2) individualized dietary planning, and 3) follow-up performed by health professionals to improve program adherence. However, this study also has a limitation, where we could not include an experimental group with very low CHO (<20g/d) diet in this study. This would have required greater monitoring of patients which was not possible in our study design. Conclusion This study suggests that weight loss through hypocaloric diets in combination with intermittent tranning promotes hormonal changes favorable to insulin and catabolic control. CHO reduction resulted in undesirable changes in the homeostasis of thyroid and anabolic hormones. The diet and exercise strategies adopted in this study are easily reproducible and low-cost. This facilitates the development of weight loss programs, for example in primary healthcare settings. 1. Kaila B, Raman M. Obesity: A review of pathogenesis and management strategies. Can J Gastroenterol. 2008; 22(1): 61-68. [acesso em 04 janeiro de 2018]. Disponível: https://doi.org/10.1155/2008/609039 2. Poobalan AS, Aucott LS, Precious E, Crombie IK, Smith WC. Weight loss interventions in young people (18 to 25 year olds): a systematic review. Obes Rev. 2010; 11(8): 580- 92. [Acesso em 02 julho de 2018]. Disponível: https://doi.org/10.1111/j.1467- 789X.2009.00673.x 3. Makris A, Foster GD. 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