Adipocyte Function. and associated comorbidities Adipose tissue development. Isabel Wagner

Transcription

1 Adipocyte Function Obesity during childhood and adolescents and associated comorbidities Adipose tissue development D. Rockstroh, K. Dittrich, K. Landgraf, R. Tauscher, J. Gesing, M. Neef, M. Wojan, H.Till, W. Kiess, A. Körner

2 Obesity- Accumulation of Adipose Tissue Lean Hypertrophy Hyperplasia increase in fat mass increased lipid storage in (exisiting) adipocytes enlarged fat cells insulin resistance alterations in metabolic profile alterations in adipocytokine profile formation of new adipocytes from preadipocytes adipocyte differentiation

3 Adipogenesis Determination Commitment Terminal Differentiation Phenotype Osteoblasts RunX2, Osx Osteocytes Chondroblasts Chondrocytes Preadipocytes PPARy, C/EBPα White adipocytes? Stem cells Mesenchymal Stem Cells Preadipocytes PRDM16, PPARy Brown adipocytes Myoblasts Myogenin, MyoD Myocytes Fibroblasts, Epithelial cells, Neurons,

4 Determination Commitment Terminal Differentiation Phenotype TG synthesis Glucose uptake Lipolysis Adipocytokines FA uptake Thermogenesis Stem cells Mesenchymal Stem Cells Preadipocytes Immature Adipocytes Mature Adipocytes

5 Transcriptional Regulation of Adipocyte Differentiation Gene Expression Terminal Differentiation KLFs PPARγ KLFs GATAs C/EBPβ C/EBPδ C/EBPα KLFs IRFs Bmal Terminal Differentiation TG synthesis Glucose uptake Lipolysis Adipocytokines

6 Function of Adipocytokines Energy metabolism Food intake Angiogenesis Haematopoesis Sexual development Osteogenesis Immune-System Inflammation Energy metabolism Interleukin-6: Blood pressure Salt-/Water-balance Renin-Angiotensin-System/ Adipotensine Leptin Adiponectin Atherosclerosis Energy metabolism Insulin sensitivity PAI-1 Homeostasis Atherosclerosis Steroidmetabolism Cortisol metabolism Sexual hormones

7 Interactions of Adipocytokines CNS food intake energy expenditure reproduction neuroendocrine function Muscle insulin resistance FFA oxidation Expansion of adipose tissue through hyperplasia and hypertrophy of adipocytes adiponectin leptin cytokines FFA visfatin, resistin? Immune system inflammation proliferation cytokine production oxidative stress Liver insulin resistance FFA oxidation gluconeogenesis NASH? Vascular system atherosclerosis angiogenesis vascular adhesion molecules local inflammation Körner A, Pediatr Res 2007 Körner A, Best Pract Res Clin Endocrinol Metab 2005

8 Obesity prevalence in Germany (CrescNet; n= 68527) 2000 (CrescNet; n= 30029) 1985 (Hesse et al.) th centiles BMI (kg/m 2) th centiles 3 rd centiles Age (yrs) Increasing prevalence of obesity in children and adolescents. Körner A, Pediatr Res 2007

9 The metabolic syndrome in children and adolescents Weiss R, N Engl J Med 2004 de Ferranti SD, Circulation 2004 Lambert M, Int J Obes 2004 Butte A, Pediatr Res 2005 obesity BMI 97 th cent. waist 75 th cent. BMI 85 th cent. waist 90 th cent. triglycerides 95 th cent. 1.1 mmol/l 75 th cent. 90 th cent. HDL 5 th cent. 1.3 mmol/l 25 th cent. 10 th cent. blood pressure 95 th cent. 90 th Perz. 75 th cent. 90 th cent. glucose metab. fasting Glucose IGT 7.8 mmol/l 6.1 mmol/l 6.1 mmol/l 100 mg/dl 38.7% (moderate ob.) 49.7% (morbidly ob.) n=439 (mixed) 31.2% (obese) n=1960 (mixed) 11.5% (normal pop.) n= % (owt.) 30% (obese) n=1030 (Hisp.) High prevalence of metabolic syndrome / cardiovascular risk factors. Körner A, Pediatr Res 2007

10 Impaired metabolism in obese children Data from the Leipzig Obesity cohort Parameter Patients with pathology r to BMI Glucose metabolism insulin resistance Impaired glucose tolerance (2h 6.7 mmol/l) 18.3 % 0.12* ns. r to BMI SDS Hyperinsulinemia Peak Insulin 1000 pmol/l 40.4 % 0.24*** 0.11* Insulin resistance HOMA-IR % 0.46*** 0.18*** Hyperuricemia 25.4 % 0.52*** 0.18*** Hypertriglyceridemia 22.5 % ns. ns. Hypercholesterinemia 12.1 % ns. 0.13** Elevated liver enzymes 16.1 % 0.18*** ns. HOMA-IR n=307 children age: ± 3.7 y ( ) BMI: 1.8 SDS (2.8 ± 0.5) BMI (kg/m 2 ) Körner A, Pediatr Res 2007

11 Impaired glucose metabolism in obese children Blood Glucose (mmol/l) P= ** Obese (n=88) Lean (n=68) Time (min) * Insulin (pmol/l) *** *** P< *** Obese Lean *** *** Time (min) data from Leipzig Atherobesity Childhood Cohort

12 Prevalence P diast. > 95th cent (%) Leipzig Schoolchildren Project: boys girls P<25 P P P P >90 BMI (centiles) Hypertension- not only a problem in adulthood Difference 130 8mmHg! 120 Syst. BP (mmhg) Leipzig Obesity-Cohort: BMI n=2365 children age: y mmhg n=738 children BMI: 2.68±0.02 SDS (Reich A, Int J Obes 2003)

13 Intima Media Thickness (cm) Lean Obese cimt (cm) 0.08 Parameter r P r* P* Intima media thickness BMI SDS cimt (cm) HDL (mmol/l) BMI SDS HDL mean syst. BP HOMA IR ns Matsuda ISI ns *corrected for age, height SDS, BMI SDS

14 occlusion period 5 min ENDOPAT- measure endothelial function pulse wave amplitude (PWA) post-occlusion pulse wave amplitude (PWA) pre- occlusion Reference reactive hyperemia hyperemic reaction: ratio PWA RH / PWA baseline RHI = reactive hyperemia index = PWA RH/ baseline site of measurement PWA RH/ baseline reference pulse wave amplitude (PWA) pre- occlusion pulse wave amplitude (PWA) post-occlusion Normal endothelial function RHI > 1.4 Endothelial dysfunction RHI < 1.4

15 Reactive Hyperemia Index Lean Obese RHI Parameter r P r* P* Endothelial dysfunction in obese children Reactive Hyperemia Index BMI SDS ISI (Matsuda) BMI SDS HDL ns ns mean syst. BP ns ns HOMA IR ns Matsuda ISI *corrected for BMI SDS cimt is increased in relation to BMI and HDL in obese children. FMD is impaired in obese children and associated with insulin sensitivity.

16 Endothelial progenitor cells EPCs homing and proliferation mediated by: Recruitment of EPCs from bone marrow and circulating pool: Morphology, nuclear and AC-LDL LDL- DiL uptake of endothelial progenitor clusters bone marrow derived cells integrate the damaged vessel EPC number and function correlated with - number of cv risk factors - extent coronary disease - future cardiovascular events = independent predictor of morbidity and mortality from cardiovascular disease

17 Changes in EPC number and migration in obese children EPCs / EPCs / Migration CPCs/300mm Lean Obese Lean Obese BMI (kg/m 2 ) The number of circulating EPCs is decreased in obese children. There was no (BMI independent) association with metabolic or cardiovascular parameters.

18 Fat mass and fat cell volume Adipose tissue development Adipocyte numbers in lean and obese Fat cell volume (pl) Body fat mass (kg) Fat mass is determined by both adipocyte number and size Production rate (Re)Generation rate doubled in obese Adipocyte number increases in childhood, Adipocyte number is increased in obese Difference in adipocyte number is established during childhood and remains constant in adulthood Spalding K, Nature (2008)

19 Determination of Adipose Tissue Development in the Progression of Obesity in Children

20 Which changes in expression profile and differentiation potential occur in adipose tissue in relation to age and development during childhood? Infancy early childhood Puberty Adolescence Characterization of the adipose tissue biology Composition Morphology Expressionsanalysis Differentiation Functional analysis amount of cells Multisizer: Osmium labelled cells diameter in µm Log 10 Relative expression ( p p ) Day of differentiation Adiponectin ACACB ACLY PPP2R5A GPX3 GATA6 Latexin CXCL6 AKR1C2 Glycerol in pg/m l pro Zelle 0,0008 0,0007 0,0006 0,0005 0,0004 0,0003 0,0002 0, Lipolyse , human, FG MS 0 1 µm 10 µm 100 µm Isoproterenol Lipolysis Glucoseuptake Triglyceridassay 2 g 5 g

21 Do functional and morphological differences exist during adipogenesis of lean and obese children? Lean Obese Comparison of adipose tissue biology Age groups (yr): - 0-2: infancy - 3-5: obesity rebound - 6-8: early childhood : start puberty : puberty BMI: 2.0 SDS

22 Is there evidence of brown adipose tissue in children? If so, are there any differences of expression and function during childhood development and between lean and obese children? Expression of brown adipose tissue markers : PGC 1-α, UCP-1 PRDM-16, BMP-7 Changes in the oxidative metabolism: Oxygen consumption Amount of Mitochondria Mitochondrial markers: Tfam, NRF-1

23 Sample Distribution Age distribution 34% Number of samples % Girls Boys years 3 5 years 6 8 years 9 11 years years years 20% 10% normal weight overweight obese subcutaneous perirenal omental visceral 70%

24 Microscopic pictures from different depots Subcutaneous, nw 16y., subc., obese, 18y., subc., nw 1y, perirenal, nw 1y., omenatal, nw 0 y. Omental, nw 1.y Lightmicroscopy, satin, haematoxylin and eosin 50 µm ; (by D. Rockstroh)

25 Cell diameter Cell size and diameter and cell size distribution was measured after Osmium labeling of the cells. Adipocytes were analyzed with a Multisizer. Different age groups were compared and showed that size does not vary significantly between the age groups in normal weight children and adolescents.

26 Cell size diameter in lean and obese subjects The maximum of mean diameter was compared between normal weight and overweight children and adolescents aged Significant differences were seen between subjects aged 9-11 and During puberty the fat cell diameter is very similar.

27 Proliferation rate Day 2 Day 4 Day 6 Day 8 Preadipocytes were seede after isolation and stained with Hoechst at day 1,2,4,6 and 8. Cells were counted and a proliferation rate was calculated. The Proliferation rate can be compared between the different age and weight groups. More samples need to be collected to get significant data. relative cell count (in relation to day 1) 8,0 7,0 6,0 5,0 4,0 3,0 2,0 1,0 lean (n=19) obese (n=28) 0,0 d2 d4 d6 d8

28 Differentiation capacity Light microscope of cells stained with either Hoechst and or Nile red to measure the differentiation capacity of preadipocytes Preadipocytes are seeded and differentiation medium is added to evaluate differences in the differentiation capacity between cells from different samples. % of Differentiation lean (n=15) obese (n=16)

29 Triglyceride Assay The amount of Triglycerides were measured in adipocytes after destruction. Differences were noted especially between perirenal, omental and subcutaneous tissues.

30 Hämatoxylin- Eosin staining, 10x by D. Rockstroh; Mixed depot: unilocular and multilocular adipocytes

31 Brown adipose tissue in our samples? Immunhistochemical characterization Heamatoxilin/Eosin (H&E) staining, 10x UCP1 staining, 20x % of UCP1 + positive Staining s 75% of perirenal samples 100% of visceral samples 1,6% of subcutaneous samples data by D. Rockstroh

32 Mean size of cells and UCP-1 Expression 7000 *** *** n= r²= 0,5777 P<0,0001 *** Mean Size (µm²) n=11 n=49 Mean size (µm²] BAT Lean children WAT Lean children WAT overweight/obese children Age in years Data by D. Rockstroh

33 UCP-1 Expression and staining in adipose tissue from children s u b c u t a n e o u s UCP1 Expression n=11 UCP1 + BAT Lean children *** *** n=49 n=33 UCP1 WAT Lean children UCP1 WAT overweight/obese children UCP-1 staining by D. Rockstroh Data by D. Rockstroh

34 PRDM 16 Expression in adipose tissue samples of children Highest expression in UCP-1 positive samples PRDM 16 is another indicator for brown adipose tissue Data by D. Rockstroh

35 Macrophage infiltration CLS/ 100 adipocytes < -1,88 > 1, ,88 > 1,88 BMI/SDS BMI/SDS= < 1,88= underweight BMI/SDS <1,3 = lean BMI/SDS 1,3 1,88 = overweight BMI/SDS >1,88 = obese 5 CD68 staining, 40x, Crown like structures by D. Rockstroh CLS- Existence CLS- Existence in adipocytes. 40% of overweight/ obese showed an increased amount of CLS, but only 5% of lean children Correlation BMI/SDS vs. Macrophage infiltration Positive correlation between BMI/SDS und macrophage infiltration Log(macrophage infiltration/ 100 adipocytes) r²= 0,4501 P>0,0001 log (BMI/SDS +3) Data by D. Rockstroh

36 Summary and Conclusion It is important to understand the adipocyte function to be able to comprehend the pathology of obesity and to be able to treat obesity successfully Obesity during childhood and adolescents has become a major health problem- comorbidities occur already at an early age Brown adipose tissue exists in adipose tissue samples of children- even in subcutaneous depots Adipose tissue development during childhood and adolescents is only partly understood- therefore it is our aim to improve the understanding of the complex pathology of obesity development Successful prevention strategies and treatment options in children and adolescents need to be the goal for the near future

37 Many thanks to all my colleagues Research-Team Prof. KörnerK Denise Rockstroh Dr. Kathrin Landgraf Kathrin Dittrich Daniela Friebe Dr. Julia Gesing Antje Berthold Roy Tauscher Dr. Dennis LöfflerL and Prof. Dr. Antje KörnerK

38 Vielen MANY Dank THANKS für f r FOR Ihre Aufmerksamkeit YOUR ATTENTION Universitätsmedizin Leipzig (2009): Thema, Datum 3

39 Determination of Adipose Tissue Development in the Progression of Obesity in Children

40 Which changes in expression profile and differentiation potential occur in adipose tissue in relation to age and development during childhood? Infancy early childhood Puberty Adolescence Characterisation of the adipose tissue biology Composition Morphology Expressionsanalysis Differentiation Functional analysis ( p p ) Lipolyse , human, FG MS amount of cells Multisizer: Osmium labelled cells diameter in µm Log 10 Relative expression Day of differentiation Adiponectin ACACB ACLY PPP2R5A GPX3 GATA6 Latexin CXCL6 AKR1C2 Glycerol in pg/m l pro Zelle 0,0008 0,0007 0,0006 0,0005 0,0004 0,0003 0,0002 0, µm 10 µm 100 µm Isoproterenol Lipolysis Glucoseuptake Triglyceridassay 2 g 5 g

41 Do functional and morphological differences exist during adipogenesis of lean and obese children? Lean Obese Comparison of adipose tissue biology Age groups (yr): - 0-2: infancy - 3-5: obesity rebound - 6-8: early childhood : start puberty : puberty BMI: 2.0 SDS

42 Is there evidence of brown adipose tissue in children? If so, are there any differences of expression and function during childhood development and between lean and obese children? Expression of brown adipose tissue markers : PGC 1-α, UCP-1 PRDM-16, BMP-7 Changes in the oxidative metabolism: Oxygen consumption Amount of Mitochondria Mitochondrial markers: Tfam, NRF-1

43 Sample Distribution Age distribution Number of samples % 34% Girls Boys years 3 5 years 6 8 years 9 11 years years years 20% 10% normal weight overweight obese subcutaneous perirenal omental visceral 70%

44 Microscopic pictures from different depots Subcutaneous, nw 16y., subc., obese, 18y., subc., nw 1y, perirenal, nw 1y., omenatal, nw 0 y. Omental, nw 1.y Lightmicroscopy, satin, haematoxylin and eosin 50 µm ; (D. Rockstroh)

45 Cell diameter Cell size and diameter and cell size distribution was measured after Osmium labeling of the cells. Adipocytes were analyzed with a Multisizer. Different age groups were compared and showed that size does not vary significantly between the age groups in normal weight children and adolescents.

46 Cell size diameter in lean and obese subjects The maximum of mean diameter was compared between normal weight and overweight children and adolescents aged Significant differences were seen between subjects aged 9-11 and During puberty the fat cell diameter is very similar.

47 Proliferation rate Day 2 Day 4 Day 6 Day 8 Preadipocytes were seede after isolation and stained with Hoechst at day 1,2,4,6 and 8. Cells were Counted and a proliferation rate was calculated. The Proliferation rate can be compared between the Different age and weight groups. More samples need to be collected to get significant data. 600 Proliferationrate: age distribution in obese subjects 1400 Proliferationrate: age distribution in normal weight subjects % of cell number ob ob ob %of cell number nw 9-11nw 12-15nw 16-18nw d2 d4 d6 d8 0 d2 d4 d6 d8

48 Differentiation capacity Light microscope of cells stained with either Hoechst and or Nile red to measure the differentiation capacity of preadipocytes Preadipocytes are seeded and differentiation medium is added to evaluate differences in the differentiation capacity between cells from different samples.

49 Triglyceride Assay The amount of Triglycerides were measured in adipocytes after destruction. Differences were noted especially between perirenal, omental and subcutaneous tissues.

50 Brown adipose tissue Mean Size (µm²) n=11 BAT Lean children *** *** *** *** n=49 n=33 WAT WAT Lean children overweight/obese children UCP1 Expression n=11 UCP1 + BAT Lean children n=49 n=33 UCP1 WAT Lean children UCP1 WAT overweight/obese children

51 Summary and Conclusion It is important to understand the adipocyte function to be able to comprehend the pathology of obesity and to be able to treat obesity successfully Obesity during childhood and adolescence has become a major health problem- comorbidities occur already at an early age Successful prevention strategies and treatment options need to be the goal for the near future Adipose tissue development during childhood and adolescence is only partly understood- therefore it is our aim to unravel the mystery and to understand the complex pathology of obesity development

52 MANY THANKS FOR Vielen Dank für f r Ihre Aufmerksamkeit YOUR ATTENTION Universitätsmedizin Leipzig (2009): Thema, Datum 3