Disrupted Control of Xenin and Ghrelin in Night Workers
This cross-sectional study was conducted at the Laboratory of Investigation in Metabolism and Diabetes (LIMED), at the State University of Campinas (UNICAMP), after approval by the Institutional Ethics Review Board. The study included two groups according to fixed shift work: night workers (n = 12) and day workers (n = 12). Night workers worked from 7 pm to 7 am. Day workers had conventional hours for work (business hours) as well as for meals and sleep and served as control. Volunteers were invited among cleaning service employees at the hospital of UNICAMP, and provided written informed consent before participation. Inclusion criteria were: female gender; aged 20–40 years; overweight [body mass index (BMI) 25–29·9 kg/m]; stable weight (variation < 5%) within the last three months; sedentarism; having worked in the same fixed shift in the last two years. Exclusion criteria were: known or suspected pregnancy; diabetes; smoking; use of lipid-lowering drugs or systemic corticosteroids within the last three months; diagnosed or suspected psychiatric disorder or use of psychotropic drugs.
All evaluations were taken in the morning after a sleep night and 12-h overnight fast, out of the menstrual period.
Anthropometrics included height, weight, BMI [weight divided by height squared (kg/m)] and waist circumference, above right upper iliac crest. Body composition (body fat and lean masses) was estimated by electric bioimpedance (Biodynamics Model 310, Biodynamics Corp., Seattle, WA).
The subjects completed a 24-h food record, kept on three alternate days, one being a weekend day. Food intake was described in household portions and by meals. The same dietician provided a standard food diary, filling instructions and entered the record data in the software Dietpro 5·1i (A. S. Sistemas, Viçosa, MG, Brazil) for analysis of the mean three-day total energy intake and macronutrients composition (lipids, proteins, carbohydrates).
Pittsburgh Sleep Quality Index (PSQI), adapted and validated for Brazil, was applied. This self-administered questionnaire assesses quality of sleep during the previous month and contains nineteen self-rated questions yielding seven components: subjective sleep quality; sleep latency; sleep duration; sleep efficiency; sleep disturbances; use of sleep medications and daytime dysfunction. Each component is scored from 0 to 3, yielding a global PSQI score between 0 and 21, with higher scores indicating lower quality of sleep. A global PSQI score >5 indicates that a person is a "poor sleeper", having severe difficulties in at least two areas or moderate difficulties in more than three areas.
Subjects were submitted to standard meal tolerance test (MTT), based on a mixed meal containing 515 kcal (41·8% fat, 40·7% carbohydrate, 17·5% protein). The test was performed in the morning after a sleep night and 12-h overnight fast for both groups. The meal was eaten within 10 min. Meal start was considered time zero. Blood samples were drawn at times −60, −30, 0, 15, 30, 45, 60, 90, 120, 150 and 180 min for analysis of glucose, insulin, ghrelin, xenin, PYY 3–36, oxyntomodulin and GLP-1. The area under the curve (AUC) of each parameter was calculated by the trapezoidal method. The incremental AUC (AUCi) was calculated as total AUC minus the area under the basal value.
Glucose was assayed by glucose oxidase method. Blood samples were collected in tubes with EDTA.K3 plus aprotinin for ghrelin, PYY3–36, oxyntomodulin and xenin, and in tubes with EDTA.K3 plus Sigma diprotin A for GLP-1. Serum samples were stored in a freezer at −80°C for posterior analysis of insulin (ELISA, Bayer Corp., Tarrytown, NY); total ghrelin, PYY 3–36, oxyntomodulin (ELISA, Phoenix Pharmaceuticals, Inc., Burlingame, CA); xenin (xenin-25) (RIA, Phoenix Pharmaceuticals, Inc., Burlingame, CA); GLP-1 (Millipore Corp., St.Charles, MO); adiponectin, leptin, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and ultra-sensitive C-reactive protein (usCRP) (ELISA, R&D Systems Inc., Minneapolis, MN). Total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides were immediately analysed using standard methods.
Insulin sensitivity (IS) was estimated by two methods: Homeostatic Model Assessment (HOMA-IR) [glucose (mmol/l) Insulin (μU/ml)/22,5], for which higher values represent lower IS, and Stumvoll Index (ISIStumvoll), calculated from BMI and MTT values of glucose and insulin: [0·22−(0·0032 BMI)−(0·0000645 2-h insulin (pmol/l)]−[0·0037 90-min glucose (mmol/l)]. ISIStumvoll corresponds to the metabolic clearance rate of glucose; higher values represent higher IS.
SPSS v16·0 (SPSS Inc., Chicago, IL) was used for statistical analysis. Data are presented as mean and standard deviation (mean ± SD). Comparisons between groups were obtained by Mann–Whitney test and, for MTT curves, also by the general linear model for repeated measures. Statistical significance was assumed if P < 0·05.
Methods
This cross-sectional study was conducted at the Laboratory of Investigation in Metabolism and Diabetes (LIMED), at the State University of Campinas (UNICAMP), after approval by the Institutional Ethics Review Board. The study included two groups according to fixed shift work: night workers (n = 12) and day workers (n = 12). Night workers worked from 7 pm to 7 am. Day workers had conventional hours for work (business hours) as well as for meals and sleep and served as control. Volunteers were invited among cleaning service employees at the hospital of UNICAMP, and provided written informed consent before participation. Inclusion criteria were: female gender; aged 20–40 years; overweight [body mass index (BMI) 25–29·9 kg/m]; stable weight (variation < 5%) within the last three months; sedentarism; having worked in the same fixed shift in the last two years. Exclusion criteria were: known or suspected pregnancy; diabetes; smoking; use of lipid-lowering drugs or systemic corticosteroids within the last three months; diagnosed or suspected psychiatric disorder or use of psychotropic drugs.
All evaluations were taken in the morning after a sleep night and 12-h overnight fast, out of the menstrual period.
Anthropometrics and Body Composition
Anthropometrics included height, weight, BMI [weight divided by height squared (kg/m)] and waist circumference, above right upper iliac crest. Body composition (body fat and lean masses) was estimated by electric bioimpedance (Biodynamics Model 310, Biodynamics Corp., Seattle, WA).
Diet Composition
The subjects completed a 24-h food record, kept on three alternate days, one being a weekend day. Food intake was described in household portions and by meals. The same dietician provided a standard food diary, filling instructions and entered the record data in the software Dietpro 5·1i (A. S. Sistemas, Viçosa, MG, Brazil) for analysis of the mean three-day total energy intake and macronutrients composition (lipids, proteins, carbohydrates).
Sleep Quality
Pittsburgh Sleep Quality Index (PSQI), adapted and validated for Brazil, was applied. This self-administered questionnaire assesses quality of sleep during the previous month and contains nineteen self-rated questions yielding seven components: subjective sleep quality; sleep latency; sleep duration; sleep efficiency; sleep disturbances; use of sleep medications and daytime dysfunction. Each component is scored from 0 to 3, yielding a global PSQI score between 0 and 21, with higher scores indicating lower quality of sleep. A global PSQI score >5 indicates that a person is a "poor sleeper", having severe difficulties in at least two areas or moderate difficulties in more than three areas.
Meal Tolerance Test
Subjects were submitted to standard meal tolerance test (MTT), based on a mixed meal containing 515 kcal (41·8% fat, 40·7% carbohydrate, 17·5% protein). The test was performed in the morning after a sleep night and 12-h overnight fast for both groups. The meal was eaten within 10 min. Meal start was considered time zero. Blood samples were drawn at times −60, −30, 0, 15, 30, 45, 60, 90, 120, 150 and 180 min for analysis of glucose, insulin, ghrelin, xenin, PYY 3–36, oxyntomodulin and GLP-1. The area under the curve (AUC) of each parameter was calculated by the trapezoidal method. The incremental AUC (AUCi) was calculated as total AUC minus the area under the basal value.
Blood Analysis
Glucose was assayed by glucose oxidase method. Blood samples were collected in tubes with EDTA.K3 plus aprotinin for ghrelin, PYY3–36, oxyntomodulin and xenin, and in tubes with EDTA.K3 plus Sigma diprotin A for GLP-1. Serum samples were stored in a freezer at −80°C for posterior analysis of insulin (ELISA, Bayer Corp., Tarrytown, NY); total ghrelin, PYY 3–36, oxyntomodulin (ELISA, Phoenix Pharmaceuticals, Inc., Burlingame, CA); xenin (xenin-25) (RIA, Phoenix Pharmaceuticals, Inc., Burlingame, CA); GLP-1 (Millipore Corp., St.Charles, MO); adiponectin, leptin, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and ultra-sensitive C-reactive protein (usCRP) (ELISA, R&D Systems Inc., Minneapolis, MN). Total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides were immediately analysed using standard methods.
Insulin Sensitivity
Insulin sensitivity (IS) was estimated by two methods: Homeostatic Model Assessment (HOMA-IR) [glucose (mmol/l) Insulin (μU/ml)/22,5], for which higher values represent lower IS, and Stumvoll Index (ISIStumvoll), calculated from BMI and MTT values of glucose and insulin: [0·22−(0·0032 BMI)−(0·0000645 2-h insulin (pmol/l)]−[0·0037 90-min glucose (mmol/l)]. ISIStumvoll corresponds to the metabolic clearance rate of glucose; higher values represent higher IS.
Statistics
SPSS v16·0 (SPSS Inc., Chicago, IL) was used for statistical analysis. Data are presented as mean and standard deviation (mean ± SD). Comparisons between groups were obtained by Mann–Whitney test and, for MTT curves, also by the general linear model for repeated measures. Statistical significance was assumed if P < 0·05.
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