Testosterone After a Glucose and Protein Drink in Obese Boys
Overweight and obese but otherwise healthy adolescent boys age 8–17 were recruited as part of a larger study of girls and boys across the weight spectrum exploring childhood and adolescent obesity (HISTORY: High Impact Strategies Towards Overweight Reduction in Youth) and the appetite hormone response related to clinical pubertal status. Recruitment occurred via flyers posted in the hospital, and a print ad in the local newspaper. All eligible overweight and obese boys were included in the analysis. Obesity was defined using the Center for Disease Control BMI charts where overweight is categorized as 85th–95th percentile, and obesity is categorized as ≥95th percentile. Subjects were excluded if they had a history of prematurity, chronic illness or were taking any medications known to affect glucose homeostasis, appetite or pubertal development. The results presented in this paper are based on a secondary analysis of a previous study to examine appetite hormones in response to a mixed glucose–whey drink. The study was approved by the University of Toronto Research and Ethics Board for Humans and The Hospital for Sick Children Research Ethics Board.
After a 10–12 h-fast, participants arrived at 08:00 and were administered a motivation-to-eat VAS scale, which measures dimensions of subjective appetite and has been previously validated for use in boys after glucose preloads. The scale was composed of four questions: (i) How strong is your desire to eat? ("very weak" to "very strong"), (ii) How hungry do you feel? ("not hungry at all" to "as hungry as I've ever felt"), (iii) How full do you feel? ("not full at all" to "very full") and (iv) How much food do you think you can eat? (prospective food consumption, PFC) ("nothing at all" to "a large amount"). Participants were instructed to read each question and place an "x" along the 100-mm line depending on their current feelings. An intravenous catheter was placed for blood sampling.
Participants were then administered a mixed beverage containing 30 g of glucose monohydrate (Grain Process Enterprises, Toronto, ON, Canada) and 30 g of whey protein isolate (plain whey–protein isolate; Interactive Nutrition International Inc., Ottawa, ON, Canada) plus aspartame-sweetened, orange-flavoured crystals (1·1 g, Sugar Free Kool-Aid, Kraft Canada Inc., Don Mills, ON, Canada) to standardize flavour. The combination of a standardized glucose load and protein has been shown to generate a greater insulin increase and ghrelin decrease in comparison with a glucose load only potentially through protein-stimulated GLP-1 secretion. Thus, this combination was chosen to promote a greater change in hormone flux. The beverage was consumed within 5 min, followed by 50 ml of water to minimize aftertaste.
Blood samples were taken of testosterone, LH, FSH GLP-1 and ghrelin at fasting and at 15, 30, and 60 min after completely ingesting the mixed beverage. Fifteen and 30 min were chosen to capture the rapid postprandial rise (GLP-1) and fall (ghrelin) of these peptides in the circulation. The 60-min time point was chosen as a previous study in adults has indicated postglucose load nadir testosterone values at this time.
In a separate visit conducted within 4 weeks of the visit described above, body composition was analysed via BOD POD (Life Measurement Incorporated, Concord, CA, USA) air-displacement plethysmography (ADP). A standard, calibrated scale and wall-mounted stadiometer were used to measure weight and height, and body mass index (BMI) was calculated as weight (kg)/height (m). Three trials of these measurements were completed, and the mean was taken for analyses. Puberty was assessed through a validated Tanner staging questionnaire and via examination by a paediatric endocrinologist. A multiple-sampled (0. 30, 60, 90 and 120 min) oral glucose tolerance test (7 1·75 g/kg to a max of 75 g glucose) was performed to assess insulin sensitivity. Insulin sensitivity was calculated by the Matsuda Model whole body insulin sensitivity index (WBISI) where WBISI = (10 000/square root of [fasting glucose × fasting insulin] × [mean glucose × mean insulin during OGTT]). Assessment of normal glucose tolerance, impaired fasting or 2-h glucose and type 2 diabetes was completed by measurement of the 0- and 120-min glucose levels using the Canadian Diabetes Association Clinical Practice Guidelines.
Glucose was analysed using Glu Microslides, Vitros 950 chemical system (Ortho Clinical Diagnostics). Insulin was measured using a chemiluminescence immunoassay (Siemens Immulite 2500 platform; range 15–2165 pmol/l, intra- and interassay coefficient of variation [CV] <7·6%). Human active GLP-1 (intra-CV: <8%; inter-CV: <5%; #EGLP-35K), total and acylated ghrelin (intra-CV: <2%; inter-CV: <8%; #EZGRT-89K) were measured with ELISA kits (Millipore, Billerica, MA, USA). Samples were stored at −80 °C until analysis, and all samples were run in duplicate.
FSH was assayed to ensure that none of the males had evidence of primary testicular insufficiency (indicated by an elevated FSH) using a chemiluminescent assay platform (Abbott ARCHITECT ci8200, Saint-Laurent, Québec, Canada) used for clinical and research purposes at the Hospital for Sick Children. The same platform was also used to measure testosterone and LH (testosterone sensitivity 0·3 nmol/l and cross-reactivity of 0·01–2·11% for various metabolites; LH sensitivity of 0·07 IU/l and cross-reactivity of 0·84% for TSH with no cross-reaction to FSH or hCG).
Sample size was assessed using PS Power and Sample Size Calculations (version 3.0.43, 2009, Vanderbilt University). The mean of three adult studies showed decreases of testosterone of 24% following an OGTT. Based on this estimated relative decrease, and using mean and standard deviation values from studies measuring testosterone in boys from T1 to T5 puberty, 13 boys were required to achieve power of 0·8 and an α = 0·05.
A two-way repeated measured anova was used to analyse the effects of puberty and time and their interactions on testosterone before and after the glucose and protein beverage. A Tukey's test post hoc analysis was then performed. Pearson's correlation was performed to evaluate testosterone (baseline) and change in testosterone to measures of adiposity, insulin sensitivity (WBISI), and change in LH, ghrelin and GLP-1. All analyses were performed with Statistical Product and Service Solutions software, version 17.0 (SPSS Inc., Chicago, IL, USA). Effects were considered significant at P < 0·05, and data are presented as mean ± (SEM).
Materials and Methods
Subjects
Overweight and obese but otherwise healthy adolescent boys age 8–17 were recruited as part of a larger study of girls and boys across the weight spectrum exploring childhood and adolescent obesity (HISTORY: High Impact Strategies Towards Overweight Reduction in Youth) and the appetite hormone response related to clinical pubertal status. Recruitment occurred via flyers posted in the hospital, and a print ad in the local newspaper. All eligible overweight and obese boys were included in the analysis. Obesity was defined using the Center for Disease Control BMI charts where overweight is categorized as 85th–95th percentile, and obesity is categorized as ≥95th percentile. Subjects were excluded if they had a history of prematurity, chronic illness or were taking any medications known to affect glucose homeostasis, appetite or pubertal development. The results presented in this paper are based on a secondary analysis of a previous study to examine appetite hormones in response to a mixed glucose–whey drink. The study was approved by the University of Toronto Research and Ethics Board for Humans and The Hospital for Sick Children Research Ethics Board.
Protocol
After a 10–12 h-fast, participants arrived at 08:00 and were administered a motivation-to-eat VAS scale, which measures dimensions of subjective appetite and has been previously validated for use in boys after glucose preloads. The scale was composed of four questions: (i) How strong is your desire to eat? ("very weak" to "very strong"), (ii) How hungry do you feel? ("not hungry at all" to "as hungry as I've ever felt"), (iii) How full do you feel? ("not full at all" to "very full") and (iv) How much food do you think you can eat? (prospective food consumption, PFC) ("nothing at all" to "a large amount"). Participants were instructed to read each question and place an "x" along the 100-mm line depending on their current feelings. An intravenous catheter was placed for blood sampling.
Participants were then administered a mixed beverage containing 30 g of glucose monohydrate (Grain Process Enterprises, Toronto, ON, Canada) and 30 g of whey protein isolate (plain whey–protein isolate; Interactive Nutrition International Inc., Ottawa, ON, Canada) plus aspartame-sweetened, orange-flavoured crystals (1·1 g, Sugar Free Kool-Aid, Kraft Canada Inc., Don Mills, ON, Canada) to standardize flavour. The combination of a standardized glucose load and protein has been shown to generate a greater insulin increase and ghrelin decrease in comparison with a glucose load only potentially through protein-stimulated GLP-1 secretion. Thus, this combination was chosen to promote a greater change in hormone flux. The beverage was consumed within 5 min, followed by 50 ml of water to minimize aftertaste.
Blood samples were taken of testosterone, LH, FSH GLP-1 and ghrelin at fasting and at 15, 30, and 60 min after completely ingesting the mixed beverage. Fifteen and 30 min were chosen to capture the rapid postprandial rise (GLP-1) and fall (ghrelin) of these peptides in the circulation. The 60-min time point was chosen as a previous study in adults has indicated postglucose load nadir testosterone values at this time.
In a separate visit conducted within 4 weeks of the visit described above, body composition was analysed via BOD POD (Life Measurement Incorporated, Concord, CA, USA) air-displacement plethysmography (ADP). A standard, calibrated scale and wall-mounted stadiometer were used to measure weight and height, and body mass index (BMI) was calculated as weight (kg)/height (m). Three trials of these measurements were completed, and the mean was taken for analyses. Puberty was assessed through a validated Tanner staging questionnaire and via examination by a paediatric endocrinologist. A multiple-sampled (0. 30, 60, 90 and 120 min) oral glucose tolerance test (7 1·75 g/kg to a max of 75 g glucose) was performed to assess insulin sensitivity. Insulin sensitivity was calculated by the Matsuda Model whole body insulin sensitivity index (WBISI) where WBISI = (10 000/square root of [fasting glucose × fasting insulin] × [mean glucose × mean insulin during OGTT]). Assessment of normal glucose tolerance, impaired fasting or 2-h glucose and type 2 diabetes was completed by measurement of the 0- and 120-min glucose levels using the Canadian Diabetes Association Clinical Practice Guidelines.
Biochemical Assays
Glucose was analysed using Glu Microslides, Vitros 950 chemical system (Ortho Clinical Diagnostics). Insulin was measured using a chemiluminescence immunoassay (Siemens Immulite 2500 platform; range 15–2165 pmol/l, intra- and interassay coefficient of variation [CV] <7·6%). Human active GLP-1 (intra-CV: <8%; inter-CV: <5%; #EGLP-35K), total and acylated ghrelin (intra-CV: <2%; inter-CV: <8%; #EZGRT-89K) were measured with ELISA kits (Millipore, Billerica, MA, USA). Samples were stored at −80 °C until analysis, and all samples were run in duplicate.
FSH was assayed to ensure that none of the males had evidence of primary testicular insufficiency (indicated by an elevated FSH) using a chemiluminescent assay platform (Abbott ARCHITECT ci8200, Saint-Laurent, Québec, Canada) used for clinical and research purposes at the Hospital for Sick Children. The same platform was also used to measure testosterone and LH (testosterone sensitivity 0·3 nmol/l and cross-reactivity of 0·01–2·11% for various metabolites; LH sensitivity of 0·07 IU/l and cross-reactivity of 0·84% for TSH with no cross-reaction to FSH or hCG).
Statistical Analyses
Sample size was assessed using PS Power and Sample Size Calculations (version 3.0.43, 2009, Vanderbilt University). The mean of three adult studies showed decreases of testosterone of 24% following an OGTT. Based on this estimated relative decrease, and using mean and standard deviation values from studies measuring testosterone in boys from T1 to T5 puberty, 13 boys were required to achieve power of 0·8 and an α = 0·05.
A two-way repeated measured anova was used to analyse the effects of puberty and time and their interactions on testosterone before and after the glucose and protein beverage. A Tukey's test post hoc analysis was then performed. Pearson's correlation was performed to evaluate testosterone (baseline) and change in testosterone to measures of adiposity, insulin sensitivity (WBISI), and change in LH, ghrelin and GLP-1. All analyses were performed with Statistical Product and Service Solutions software, version 17.0 (SPSS Inc., Chicago, IL, USA). Effects were considered significant at P < 0·05, and data are presented as mean ± (SEM).
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