Adipose Tissue Biology: Association With IR in Children
Adipose Tissue Biology: Association With IR in Children
Subcutaneous AT samples were obtained from 171 Caucasian children (0–18 years) undergoing elective orthopedic surgery (n = 98), herniotomy/orchidopexy (n = 54), or other surgeries (n = 19). Obtained tissue samples weighed 0.04 to 16.4 g. Children were free of severe diseases and medication potentially influencing AT biology. The following exclusion criteria were applied: diabetes, generalized inflammation, malignant disease, genetic syndromes, or permanently immobilized children. Written informed consent was obtained from all parents. The study was approved by the local ethics committee (265–08, 265–08-ff) and is registered in the National Clinical Trials database (NCT02208141).
BMI data were standardized to age- and sex-specific German reference data and are given as BMI SD score (SDS). A cutoff of 1.28 and 1.88 SDS defined overweight and obesity in children. Skinfolds were measured with a Harpenden caliper (Holtain Ltd., Crosswell, Crymych, U.K.). Estimates of the percentage of body fat and total body AT mass were calculated from triceps and subscapular skinfolds according to Slaughter et al..
Fasting blood samples were obtained prior to surgery. Levels of adiponectin, leptin, hs-CRP, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), glucose, and insulin were measured by a certified laboratory. HOMA of insulin resistance (HOMA-IR) was calculated to evaluate insulin resistance. Implausible values were excluded from the analysis (leptin ≤0.2 ng/mL).
Following excision during surgery, subcutaneous AT samples were washed three times in PBS. Approximately 100 mg of AT were immediately frozen in liquid nitrogen for RNA isolation, and 50 mg were fixed in 4% paraformaldehyde for histological analyses. The rest of the sample was weighed and minced, and adipocytes and stromal vascular fraction (SVF) cells were separated by collagenase digestion (1 mg/mL). SVF cells were snap frozen in liquid nitrogen for RNA isolation or subjected to proliferation and differentiation assays. Adipocytes were directly subjected to lipolysis experiments or fixed in osmium tetroxide for analysis of cell size distribution and number using a Coulter counter (Multisizer III; Beckmann Coulter, Krefeld, Germany) with a 560 μm aperture. The effective range of cell sizes analyzed was 50–250 μm. For each participant, the peak diameter of adipocytes (diameter at which frequency of adipocytes reaches maximum) was retrieved from the Multisizer graph according to McLaughlin et al.. We decided to use this approach after methodological comparison with the manual method (Supplementary Data http://diabetes.diabetesjournals.org/content/64/4/1249/suppl/DC1). Total adipocyte number was estimated by dividing adipocyte number per gram sample by total body AT mass.
SVF cells were seeded without preceding passaging at 10,000 cells/cm for proliferation or 33,000 cells/cm for differentiation analyses in 96- or 48-well plates, respectively, and incubated in culture medium (DMEM/F-12, 10% FBS, 100 units penicillin, 0.1 mg/mL streptomycin) at 37°C and 5% CO2. Cell proliferation was assessed by counting Hoechst 33342 (Sigma) stained nuclei at days 2, 4, 6, 8, and 10 after seeding by fluorescence microscopy. Adipocyte differentiation was performed according to the Poietics human adipose-derived stem cell–adipogenesis protocol (Lonza, Cologne, Germany). Differentiation efficiency is given as percent Nile red/Hoechst double-stained cells from the total number of Hoechst-positive cells and as Oil Red O absorbance at 540 nm (FLUOstar OPTIMA; BMG LABTECH, Offenburg, Germany) per well at day 8. Adiponectin in supernatants of differentiated cells was determined by ELISA (Mediagnost, Reutlingen, Germany).
Freshly isolated adipocytes (50 μL) were diluted in 250 μL of serum-free medium (DMEM/F-12, 0.8% BSA) with or without 10 μmol/L isoproterenol for 20 h. The amount of glycerol released into the media was determined using Free Glycerol Reagent (Sigma). Lipolytic activity was normalized to adipocyte number determined by the Coulter counter method and is given as the release of glycerol in ng/mL per 1,000 adipocytes.
Tissue samples were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned (12 μm). Immunohistochemical stainings were performed with a monoclonal CD68 antibody (1:500; M0718, DAKO) using the DAKO REAL APAAP Immunocomplex system according to the manufacturer's protocol.
RNA isolation and quantitative real-time PCR from whole AT samples or isolated SVF cells were performed as previously described. Primer and probe sequences are listed in Supplementary Table 1 http://diabetes.diabetesjournals.org/content/64/4/1249/suppl/DC1.
Data that did not adhere to Gaussian distribution were log-transformed before analyses. Parametric tests (Pearson correlation analysis, Student t test, one-way ANOVA with Dunnett post hoc test) were applied for quantitative traits and χ test for categorical variables. In case of TNF-α and IL-6 mRNA and IL-6 serum levels, log-transformation did not result in Gaussian distribution, and nonparametric tests (Spearman correlation analysis, Mann-Whitney U test) were applied. In the group stratification for obesity, overweight and obese patients were combined. For multiple regression analyses, the stepwise forward model was used. Statistical analysis was performed using Statistica 7.1 (StatSoft, Tulsa, OK).
Research Design and Methods
Subjects and Samples (Leipzig Childhood AT Cohort)
Subcutaneous AT samples were obtained from 171 Caucasian children (0–18 years) undergoing elective orthopedic surgery (n = 98), herniotomy/orchidopexy (n = 54), or other surgeries (n = 19). Obtained tissue samples weighed 0.04 to 16.4 g. Children were free of severe diseases and medication potentially influencing AT biology. The following exclusion criteria were applied: diabetes, generalized inflammation, malignant disease, genetic syndromes, or permanently immobilized children. Written informed consent was obtained from all parents. The study was approved by the local ethics committee (265–08, 265–08-ff) and is registered in the National Clinical Trials database (NCT02208141).
BMI data were standardized to age- and sex-specific German reference data and are given as BMI SD score (SDS). A cutoff of 1.28 and 1.88 SDS defined overweight and obesity in children. Skinfolds were measured with a Harpenden caliper (Holtain Ltd., Crosswell, Crymych, U.K.). Estimates of the percentage of body fat and total body AT mass were calculated from triceps and subscapular skinfolds according to Slaughter et al..
Fasting blood samples were obtained prior to surgery. Levels of adiponectin, leptin, hs-CRP, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), glucose, and insulin were measured by a certified laboratory. HOMA of insulin resistance (HOMA-IR) was calculated to evaluate insulin resistance. Implausible values were excluded from the analysis (leptin ≤0.2 ng/mL).
Isolation of Adipocytes and Cells of the Stromal Vascular Fraction From Human AT Samples
Following excision during surgery, subcutaneous AT samples were washed three times in PBS. Approximately 100 mg of AT were immediately frozen in liquid nitrogen for RNA isolation, and 50 mg were fixed in 4% paraformaldehyde for histological analyses. The rest of the sample was weighed and minced, and adipocytes and stromal vascular fraction (SVF) cells were separated by collagenase digestion (1 mg/mL). SVF cells were snap frozen in liquid nitrogen for RNA isolation or subjected to proliferation and differentiation assays. Adipocytes were directly subjected to lipolysis experiments or fixed in osmium tetroxide for analysis of cell size distribution and number using a Coulter counter (Multisizer III; Beckmann Coulter, Krefeld, Germany) with a 560 μm aperture. The effective range of cell sizes analyzed was 50–250 μm. For each participant, the peak diameter of adipocytes (diameter at which frequency of adipocytes reaches maximum) was retrieved from the Multisizer graph according to McLaughlin et al.. We decided to use this approach after methodological comparison with the manual method (Supplementary Data http://diabetes.diabetesjournals.org/content/64/4/1249/suppl/DC1). Total adipocyte number was estimated by dividing adipocyte number per gram sample by total body AT mass.
Proliferation and Differentiation Capacity
SVF cells were seeded without preceding passaging at 10,000 cells/cm for proliferation or 33,000 cells/cm for differentiation analyses in 96- or 48-well plates, respectively, and incubated in culture medium (DMEM/F-12, 10% FBS, 100 units penicillin, 0.1 mg/mL streptomycin) at 37°C and 5% CO2. Cell proliferation was assessed by counting Hoechst 33342 (Sigma) stained nuclei at days 2, 4, 6, 8, and 10 after seeding by fluorescence microscopy. Adipocyte differentiation was performed according to the Poietics human adipose-derived stem cell–adipogenesis protocol (Lonza, Cologne, Germany). Differentiation efficiency is given as percent Nile red/Hoechst double-stained cells from the total number of Hoechst-positive cells and as Oil Red O absorbance at 540 nm (FLUOstar OPTIMA; BMG LABTECH, Offenburg, Germany) per well at day 8. Adiponectin in supernatants of differentiated cells was determined by ELISA (Mediagnost, Reutlingen, Germany).
Lipolytic Capacity of Isolated Adipocytes
Freshly isolated adipocytes (50 μL) were diluted in 250 μL of serum-free medium (DMEM/F-12, 0.8% BSA) with or without 10 μmol/L isoproterenol for 20 h. The amount of glycerol released into the media was determined using Free Glycerol Reagent (Sigma). Lipolytic activity was normalized to adipocyte number determined by the Coulter counter method and is given as the release of glycerol in ng/mL per 1,000 adipocytes.
Immunohistochemical Analyses
Tissue samples were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned (12 μm). Immunohistochemical stainings were performed with a monoclonal CD68 antibody (1:500; M0718, DAKO) using the DAKO REAL APAAP Immunocomplex system according to the manufacturer's protocol.
RNA Isolation and mRNA Expression Analyses
RNA isolation and quantitative real-time PCR from whole AT samples or isolated SVF cells were performed as previously described. Primer and probe sequences are listed in Supplementary Table 1 http://diabetes.diabetesjournals.org/content/64/4/1249/suppl/DC1.
Statistical Analyses
Data that did not adhere to Gaussian distribution were log-transformed before analyses. Parametric tests (Pearson correlation analysis, Student t test, one-way ANOVA with Dunnett post hoc test) were applied for quantitative traits and χ test for categorical variables. In case of TNF-α and IL-6 mRNA and IL-6 serum levels, log-transformation did not result in Gaussian distribution, and nonparametric tests (Spearman correlation analysis, Mann-Whitney U test) were applied. In the group stratification for obesity, overweight and obese patients were combined. For multiple regression analyses, the stepwise forward model was used. Statistical analysis was performed using Statistica 7.1 (StatSoft, Tulsa, OK).
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