Calorie Restriction and Gastric Bypass Effects on FGF21
Calorie Restriction and Gastric Bypass Effects on FGF21
The research design and methods have been described elsewhere. In short, we included obese females eligible for dietary or surgical treatment. The subjects had normal fasting glucose (NGT) or T2DM (treated with oral medication only, Table S1 http://onlinelibrary.wiley.com/store/10.1111/cen.12496/asset/supinfo/cen12496-sup-0001-suppmat.doc?v=1&s=34462a94371903d4bb1a9315c6123ef09a5fa9e1) according to WHO standards. Age-matched, healthy females with normal BMI served as a control group for preintervention comparisons. The protocol (ClinicalTrials.gov: NTC01167959) was approved by the medical ethics committee of the Leiden University Medical Center, and all subjects provided written informed consent before participation.
Subjects were studied (after ≥10 h fasting overnight) within a month before (median 2·5 weeks before), 3 weeks after, and 3 months after intervention (Figure S1 http://onlinelibrary.wiley.com/store/10.1111/cen.12496/asset/supinfo/cen12496-sup-0001-suppmat.doc?v=1&s=34462a94371903d4bb1a9315c6123ef09a5fa9e1). Fasting and multiple postprandial (after 266 ml Nutridrink®, 400 kcal; 49 calorie% carbohydrate, 35 calorie% lipids, 16 calorie% protein) blood samples were taken before and 3 weeks after intervention. Blood was collected in a SST® Gel and Clot Activator tube (Becton and Dickinson Breda, Netherlands) and a vacutainer on EDTA with added Aprotinin or Dipeptidyl-Peptidase-IV (DPP-IV) inhibitor as appropriate.
Logistic reasons precluded the study of participants the day prior to operation; however, we ensured that no changes in weight and diet had occurred between the first test occasion and the actual intervention. None of the subjects followed a preoperative very-low-calorie diet, and subjects in the VLCD group maintained normal dietary habits prior to start of the intervention.
Standard operating procedures were followed for GB and RYGB and patients were prescribed a staged meal plan after surgery. T2DM subjects undergoing dietary intervention (VLCD) were prescribed commercially available Prodimed® (Prodimed Benelux BV, Valkenswaard, The Netherlands), a high-protein, low-calorie meal replacement plan.
All samples were analysed after one freeze-thaw cycle. Serum cholesterol, high-density-lipoprotein (HDL), triglycerides (TG), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and C-Reactive Protein (CRP) were measured on a Modular Analytics P-800 system (Roche Diagnostics, Mannheim, Germany). Low-density-lipoprotein (LDL) cholesterol was calculated according to the Friedewald equation. Commercial kits were employed for the assay of nonesterified fatty acids (Wako Richmond, USA) and total bile salts (Diazyme Poway, USA). Serum FGF19 and FGF21 levels were measured with in-house developed ELISAs as described elsewhere. The interassay coefficients of variation of the FGF19 and FGF21 ELISA were 8·3% and 6·7%, respectively. The lower limit of detection of both ELISAs is 0·01 ng/ml, and the assays are linear up to 0·30 and 0·50 ng/ml for FGF19 and FGF21, respetively.
Data were analysed using spss 17.0. (Armonk, NY, USA) Data are presented as means ± SEM. Non-normally distributed data were log transformed. Differences between subject groups (NGT vs T2DM) and lean controls at baseline were compared by one-way anova. The effect of the meal challenge, at baseline and 3 weeks after intervention was analysed by repeated measures anova. Between-group treatment effects at 3 weeks and 3 months as compared to baseline were compared with a mixed-effects model with the patient groups and diabetes as fixed effects and the subject-specific deviances modelled with random intercepts. Bonferoni correction was applied to correct for multiple comparisons. A P < 0·05 was considered statistically significant.
Subjects and Methods
Subjects and Study Design
The research design and methods have been described elsewhere. In short, we included obese females eligible for dietary or surgical treatment. The subjects had normal fasting glucose (NGT) or T2DM (treated with oral medication only, Table S1 http://onlinelibrary.wiley.com/store/10.1111/cen.12496/asset/supinfo/cen12496-sup-0001-suppmat.doc?v=1&s=34462a94371903d4bb1a9315c6123ef09a5fa9e1) according to WHO standards. Age-matched, healthy females with normal BMI served as a control group for preintervention comparisons. The protocol (ClinicalTrials.gov: NTC01167959) was approved by the medical ethics committee of the Leiden University Medical Center, and all subjects provided written informed consent before participation.
Subjects were studied (after ≥10 h fasting overnight) within a month before (median 2·5 weeks before), 3 weeks after, and 3 months after intervention (Figure S1 http://onlinelibrary.wiley.com/store/10.1111/cen.12496/asset/supinfo/cen12496-sup-0001-suppmat.doc?v=1&s=34462a94371903d4bb1a9315c6123ef09a5fa9e1). Fasting and multiple postprandial (after 266 ml Nutridrink®, 400 kcal; 49 calorie% carbohydrate, 35 calorie% lipids, 16 calorie% protein) blood samples were taken before and 3 weeks after intervention. Blood was collected in a SST® Gel and Clot Activator tube (Becton and Dickinson Breda, Netherlands) and a vacutainer on EDTA with added Aprotinin or Dipeptidyl-Peptidase-IV (DPP-IV) inhibitor as appropriate.
Logistic reasons precluded the study of participants the day prior to operation; however, we ensured that no changes in weight and diet had occurred between the first test occasion and the actual intervention. None of the subjects followed a preoperative very-low-calorie diet, and subjects in the VLCD group maintained normal dietary habits prior to start of the intervention.
Interventions
Standard operating procedures were followed for GB and RYGB and patients were prescribed a staged meal plan after surgery. T2DM subjects undergoing dietary intervention (VLCD) were prescribed commercially available Prodimed® (Prodimed Benelux BV, Valkenswaard, The Netherlands), a high-protein, low-calorie meal replacement plan.
Assays
All samples were analysed after one freeze-thaw cycle. Serum cholesterol, high-density-lipoprotein (HDL), triglycerides (TG), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and C-Reactive Protein (CRP) were measured on a Modular Analytics P-800 system (Roche Diagnostics, Mannheim, Germany). Low-density-lipoprotein (LDL) cholesterol was calculated according to the Friedewald equation. Commercial kits were employed for the assay of nonesterified fatty acids (Wako Richmond, USA) and total bile salts (Diazyme Poway, USA). Serum FGF19 and FGF21 levels were measured with in-house developed ELISAs as described elsewhere. The interassay coefficients of variation of the FGF19 and FGF21 ELISA were 8·3% and 6·7%, respectively. The lower limit of detection of both ELISAs is 0·01 ng/ml, and the assays are linear up to 0·30 and 0·50 ng/ml for FGF19 and FGF21, respetively.
Statistical Analysis
Data were analysed using spss 17.0. (Armonk, NY, USA) Data are presented as means ± SEM. Non-normally distributed data were log transformed. Differences between subject groups (NGT vs T2DM) and lean controls at baseline were compared by one-way anova. The effect of the meal challenge, at baseline and 3 weeks after intervention was analysed by repeated measures anova. Between-group treatment effects at 3 weeks and 3 months as compared to baseline were compared with a mixed-effects model with the patient groups and diabetes as fixed effects and the subject-specific deviances modelled with random intercepts. Bonferoni correction was applied to correct for multiple comparisons. A P < 0·05 was considered statistically significant.