Effects of Maternal Obesity on the Placenta and Metabolic Changes in the Fetus. Gernot Desoye

Transcription

1 Gernot Desoye Medical University Graz Effects of Maternal Obesity on the Placenta and Metabolic Changes in the Fetus Clinic of Obstetrics and Gynaecology, Medical University of Graz, Austria

2 Nutrition Oxygen Stress Metabolism Infections Physical Activity Maternal-Fetal Supply Line Mother Fetus Outcome Placenta Genotype Phenotype Metabolism Endocrine Cardiovascular Reproduction Behaviour

3 Neonatal Phenotype in Diabesity

4 Maternal Obesity is Associated with More Subcutaneous Fat in the Newborn Mean skinfold thickness (sum of 8 skinfolds) Normal (10-90th centile) n=179 Obese (> 90th centile) n= ± ± 6.1 p<0.001 Whitelaw AGL, BMJ 1:985, 1976

5 Pedersen Freinkel Concept Expanded maternal Glucose P L A C E N T A fetal Glucose + Insulin White adipose tissue + Growth syncytiotrophoblast endothelium

6 Total Adipocyte Cell Number is Established Early in Life and is Greater in Obese Subjects Adipocyte number (x ) obese lean Age (years) Weight loss does not reduce adipocyte number! Spalding KL et al, Nature; 453: 783, 2008

7 Pedersen Freinkel Concept Expanded maternal Glucose P L A C E N T A fetal Glucose Liver + Lipogenesis TG HDL TG + Insulin Lipogenesis + + LPL White adipose tissue + Growth syncytiotrophoblast endothelium

8 Total SAT thickness (mm) Cord Blood Insulin Association with SAT Thickness Differs by Neonatal Sex Boys Total SAT thickness LN[Insulin (pmol/l)] r=0.26; p<0.001 Total SAT thickness (mm) Girls Total SAT thickness LN[Insulin (pmol/l)] r=0.05; p=0.51 Van Poppel M et al Int J Obes 2016;40:538-42

9 Body Fat at Birth Correlates with Body Fat at 9 Years of Age Catalano P et al Am J Clin Nutr 2009; 90:

10 Elevated Amniotic Fluid Insulin Associates with Obesity and IGT in Childhood Obesity 5 15 years IGT years Weiss et al, Diabetes Care 23: 905 (2000) Silvermann et al, Diabetes Care 18: 611 (1995)

11 Glucose Transfer Across Term Human Transfer characteristics: Rapid Efficient GLUT1 GLUT 1 regulatable (hormones, metabolites, cytokines, oxygen, GFs glyburide) GLUT 1 altered in diabetes & FGR Placenta D-glucose (mmol.min -1.total placental weight -1 ) Ex vivo placental perfusion Maternal glucose: 8 mm 300 Control Diet GDM Insulin Osmond et al, Diabetologia 2001

12 Transplacental Glucose Flux at End of Gestation Not determined at the level of the placenta Depends on the MATERNAL-FETAL glucose concentration gradient Clinical implications Desoye & Nolan, Diabetologia 2016

13 M Placenta F Adiposity HG M Maternal role Placenta Glucose F Contribution to gradient Insulin Glucose M Fetal role Placenta F HI

14 Glucose is Taken up Faster by Neonatal Tissues in Diabetes k-value: Blood glucose disappearance rate constant k - value White class B+C+D+F White class A Normal Birthweight (g) Pedersen J, Postgraduate Medical Journal; 47:66-67, 1971

15 M Placenta F Adiposity M Maternal role Placenta Fetal HG glucose steal phenomenon Glucose F Contribution to gradient Insulin Glucose M Fetal role Placenta F HI Desoye & Nolan, Diabetologia 2016

16 Clinical Implications of Glucose Steal Phenomenon Controlling maternal blood glucose may not be enough to prevent excessive fetal fat accumulation May blunt maternal postload glucose peaks. GDM may become unrecognized Weiss et al, Am J Obstet Gynecol 2001

17 Fetal Hyperinsulinism Blunts Maternal Glucose Levels after ogtt Glucose (mm) AFI 7.0 µu/ml AFI < 7.0 µu/ml 1 hr 2 hrs 0 hr 0 25 wks 31 wks Weiss et al, Am J Obstet Gynecol 2001

18 Fetal Hyperinsulinism Blunts Maternal Glucose Levels after ogtt Glucose (mm) AFI 7.0 µu/ml AFI < 7.0 µu/ml 1 hr 2 hrs 0 hr 0 25 wks 31 wks Weiss et al, Am J Obstet Gynecol 2001

19 At what Time in Gestation Can Fetal Hyperinsulinism Begin?

20 Insulin in Human Fetal Pancreas 9 weeks 14 weeks 24 weeks In t Veld Diabetologia 35, 272 (1992)

21 AF Insulin Present at wks Gestation (n=247) Higher AF insulin (by 1 MOM) associated with 3-fold risk for birth weight > 90th centile Carpenter MW Diabetes Care 24: 1259 (2001)

22 First-Trimester Fasting Glycemia Correlates with Neonatal Weight 6129 women Median GA at fasting glucose: 9.5 weeks Frequency (%) GDM Development LGA and/or Macrosomia Glucose Category Riskin-Mashia S et al Diabetes Care 32: 1639, 2009

23 First-Trimester Fasting Glycemia Correlates with Neonatal Weight 6129 women Median GA at fasting glucose: 9.5 weeks Frequency (%) GDM Development Glucose Category LGA and/or Macrosomia Riskin-Mashia S et al Diabetes Care 32: 1639, 2009

24 Placental Volume (wks 11-14) Associates with Birth Weight Category Effendi M et al Placenta 35:99 102, 2014 Plasencia W et al Fetal Diagn Ther 30:23-28, 2011

25 Insulin Response (IVGTT) and Placental Weight Placental Weight (g) Placental Weight (g) Placental Weight (g) R=0.08, P=n.s R=0.42, P= R=0.26, P=n.s Insulin Response (µu/ml) Pre-pregnancy Early pregnancy (12-14 weeks) Late Pregnancy (34-36 weeks) O Tierney-Ginn et al, JCEM, 2015

26 Insulin Glucose First trimester Maternal Placental Fetal Dialogue Placenta placental development? Desoye & van Poppel, 2014 Insulin Glucose Adipocytes number TG deposition Hypothalamus ß-cell changes 2nd & 3rd trimester Diabesity risk Pregnancy Childhood Adulthood

27 Maternal Placental Fetal Dialogue Insulin resistance Obesity IGT/T2D Stress Environmental pollutants Insulin Glucose First trimester Desoye & van Poppel, 2014 Placenta placental development? Insulin Glucose Adipocytes number TG deposition Hypothalamus ß-cell changes 2nd & 3rd trimester Pregnancy

28 Diabetes in pregnant women shall be carefully managed in EARLY gestation in the hope of preventing excessive body fat deposition in the conceptus Poissonnet CM et al Early Hum Dev 10: 1-11 (1984)

29 Thank you!

30 Extra slides

31 Fetal Fat Lobules Detected at Week 14 Fat Lobule Size (µm) r = Crown Rump Length (mm) Poissonnet et al, Early Human Development, 8:1-11 (1983)

32 Early Changes in Plasma Glucose Concentration by BMI BMI Glucose (mg/100ml/wk) 6-12 wks < >= Mills et al Metabolism 47, ; 1998

33 Neonates born to obese GDM mothers have more intrahepatocellular fat Intrahepatocellular lipid Normal weight Neonatal IHCL (AU) normal weight moms 0.017±0.0045; obese/gdm moms 0.030±0.016 p=0.04 Obese GDM r=0.50, p= Maternal pre-pregnancy BMI (kg/m²) Brumbaugh et al, J Pediatr; 162: , 2013

34 Cord Blood C-Peptide Correlates with 1st Trimester Maternal BMI Cord blood c-peptide (ng/ml) First trimester maternal BMI (kg/m²) p = r = 0.74 Valsamkis G. et al, Hormones 13:87-94, 2014

35 IRI (pg/icc/min) Insulin release by human islet-like cell cultures (ICC) glucose: 2 mm 20 mm 22.5 wk fetuses non-dm IDDM TIME (min) Otonkoski T. et al, Diabetes 37: ,1988

36 insulin secretion (pg/µg dry wt) Basal Insulin Secretion wks wks wks Insulin secretion (pg/µg dry wt) Incremental Insulin Secretion 0 after glucose stimulation (20 mm, 1 hr) controls type-i diabetics controls type-i diabetics poor control tight poor control tight 6 Insulin Secretion from Human Fetal Pancreata Reiher et al, Diabetes Care 6:446, 1983

37 Fetal igtt in subhuman primates Fetal insulin [ng/ml] days; full term: 164 days 250 mg glucose STZ diabetic animals controls Time after injection [min] JCI 48, ; 51, 837, 1972

38 Pedersen Freinkel Concept Expanded maternal Glucose Lipoproteins FFA EL P L A C E N T A fetal Glucose FFA Liver + Lipogenesis TG HDL TG + Insulin Lipogenesis + + LPL White adipose tissue + Growth syncytiotrophoblast endothelium

39 mother placenta fetus Dissociation 1-3% FA Albumin Complex ~~~~~ Free fatty acid ~~~~~ Hydrolysis by Lipases 97-99% FA Lipoproteins EL Lipoprotein Receptor Lipid Droplets Free fatty acid

40 EL is Upregulated by GDM plus Obesity EL mrna expression Endothelial lipase is upregulated in obese GDM only lean obese lean GDM obese GDM * Inflammatory cytokines upregulate placental EL EL mrna expression (fold change compared to control) * TNF-α Leptin * Gauster et al; Diabetes 2011

41 mother placenta fetus Dissociation 1-3% FA Albumin Complex ~~~~~ Free fatty acid ~~~~~ EL 97-99% FA Lipoproteins FABPpm ~~~~~ FATP ~~~~~ FAT/CD36 ~~~~~ Diffusion ~~~~~~ Lipoprotein Receptor Oxidation Mitochondria Peroxisomes Lipid resynthesis ~~~~~ FABP ~~~~~ Storage in Lipid Droplets Lipid hydrolysis Biological activity Signal transduction Gene regulation Eicosanoid formation ~~~~~ Free fatty acid ~~~~~ Diffusion ~~~~~~ FATP ~~~~~ FAT/CD36 ~~~~~ ~~~~~ Free fatty acid ~~~~~ α-fetoprotein Fetal liver: Incorporation into Lipoproteins Desoye & Herrera, 2016

42 Fatty Acid Transfer Across Term Human Transfer characteristics: Slow (3% clearance of H 2 0) Placenta 18: 635, 1997 Inefficient Dependent on chain length Maternal FAs contribute to only ~ 70g (20%) of neonatal fat (normal pregnancy) Pediatr Res 7:192, 1973 Placenta Enrichment in fetal circulation (% of maternal) In vivo stable isotopes Normal GDM 13 C-PA (16:0) 13 C-OA (18:1) 13 C-LA (18:2) * 13 C-DHA (22:6) Pagán A et al, AJP-Endocr Metab 2013

43 Enrichment in fetal effluent (% of maternal) DHA Transfer is More Efficient than Transfer of Non-essential FAs BMI <25kg/m² Non-essential FA Ex vivo placental perfusion Essential FA 0 13 C-PA (16:0) 13 C-OA (18:1) 13 C-LA (18:2) 13 C-DHA (22:6) Hirschmugl et al, unpublished

44 FA Composition of Adipose Tissue in Neonates is Unaltered in Diabetes Fatty Acid (% of Total) Normal (5) Diabetes (12) C 12:0 0.5 ± ± 0.2 C 14:0 4.4 ± ± 1.1 C 16: ± ± 2.7 C 16: ± ± 2.2 C 18:0 5.7 ± ± 1.1 C 18: ± ± 5.2 C 18:2 1.1 ± ± 1.1 King et al, Pediatrics; 47: , 1971

45 Modelling of Perfusion Data Suggest: Two Placental Fatty Acid Pools mother relatively high transfer Slow high capacity route via metabolic pools e.g. phospholipid fast low capacity route?? fetus relatively low transfer Perazzolo S et al J Lipid Res 58: ,

46 Placenta has limited capacity to store excess fatty acids in obesity TG mg/g tissue Triglycerides * < > 40 BMI [kg/m²] Hirschmugl et al, Int J Obes 41: , 2017

47 FATP1 FATP3 Proposed Mechanism lipid droplet TG Obesity lipid droplet ATGL CGI-58 DAG + FA IR ATGL CGI-58 Syncytiotrophoblast Hirschmugl et al, Int J Obes 41: ,

48 Modelling of Perfusion Data Suggest: Two Placental Fatty Acid Pools mother relatively high transfer Slow high capacity route via metabolic pools e.g. phospholipid fast low capacity route?? fetus relatively low transfer Perazzolo S et al J Lipid Res 58: ,

49 Only DHA Transfer is Altered by Maternal Obesity Enrichment in fetal effluent (% of maternal) C-PA (16:0) BMI <25kg/m² BMI >30 kg/m² Non-essential FA 13 C-OA (18:1) Ex vivo placental perfusion 13 C-LA (18:2) Hirschmugl et al, unpublished Essential FA * 13 C-DHA (22:6) Fetus Plasma: PL 6.6% TG 1.6% CE 1.1% NEFA 2.8% Brain: 4.1% Adipose Tissue: 1.6% Haggarty P, Eur J Clin Nutr 2004

50 Fatty Acids in Umbilical Cord Plasma POLYUNSATURATED (%) a b V a A PUFA *** a b V b A SATURATED (%) a Saturated Fatty Acids N=24 N=17 b b b b a V A V A 0 AGA GDM 0 AGA GDM Ortega-Senovilla H et al, Diabetes Care 2009

51 Low Cord Plasma DHA Levels May Indicate Fetal Insulin Resistance Zhao JP et al, PLoS One; 9(1):e85054, 2014.

52 Summary Fatty Acids Obesity does not Affect Transplacental Transfer of Nonessential Fatty Acids Obesity enhances DHA Transfer Does obesity enhance DHA extraction into fetal tissues Is this an insulin driven phenomeon? Consequence: fetal DHA steal? (Desoye & Nolan Diabetologia, 2016) 52

53 Umbilical cord C-peptide associated with arterial stiffness at 15 years N= 129/42 GDM; 87 controls; no difference p=0,03 p = 0,01 C-peptide: < or 75 percentile p = 0,03-8 central aortic pressure wave reflection pulse wave velocity Tam et al, Diabetes Res Clin Pract 95: 169 (2012)

54 Fetal hyperinsulinism leads to multiple changes maternal compartments stroma fetal glucose glucose insulin Vascularization oxygen Growth Orexigenic NPY/ AGRP ARC neurons Brain/Hypothalamus Liver / Skeletal muscle Glucose uptake Differentiation Anorexigenic Ventromedial nucleus Adipocyte Proliferation TG storage syncytiotrophoblast endothelium Metabolism

55 Early changes in plasma glucose concentration by BMI BMI Glucose (mg/100ml/wk) 6-12 wks < >= Mills et al Metabolism 47, ; 1998

56 Summary - Glucose The placenta does not appear to actively contribute to fetal oversupply with glucose (at the end of gestation)

57 Late in pregnancy: Summary Glucose transfer driven by maternal-fetal concentration gradient Mechanisms of lipid/fatty acid transfer unclear No evidence for direct contribution of placenta to fetal overnutrition (maternal diabetes, obesity)

58 Insulin Glucose First trimester Maternal Placental Fetal Dialogue Placenta placental development? Desoye & van Poppel, 2014 Insulin Glucose Adipocyte number TG deposition Hypothalamus ß-cell changes 2nd & 3rd trimester Diabesity risk Pregnancy Childhood Adulthood

59 Special Role of DHA in Materno-Fetal Dialogue Transfer mechanism Fetal/neonatal insulin sensitivity

60 Special Role of DHA in Materno-Fetal Dialogue Transfer mechanism Fetal/neonatal insulin sensitivity Fetal/neonatal brain development

61 Cord DHA Associates with Psychomotor Development Index (PDI) at 6 months Cord blood DHA (%) PDI 6 months r=0.27 P=0.049 Zornoza-Moreno M et al, Eur J Clin Nutr 2014

62 mother placenta fetus Dissociation 1-3% FA Albumin Complex ~~~~~ Free fatty acid ~~~~~ EL 97-99% FA Lipoproteins FABPpm ~~~~~ FATP ~~~~~ FAT/CD36 ~~~~~ Diffusion ~~~~~~ Lipoprotein Receptor Oxidation Mitochondria Peroxisomes Lipid resynthesis ~~~~~ FABP ~~~~~ Storage in Lipid Droplets Lipid hydrolysis Biological activity Signal transduction Gene regulation Eicosanoid formation ~~~~~ Free fatty acid ~~~~~ Diffusion ~~~~~~ FATP ~~~~~ FAT/CD36 ~~~~~ ~~~~~ Free fatty acid ~~~~~ α-fetoprotein Fetal liver: Incorporation into Lipoproteins Desoye & Herrera, 2016

63 Lipids Free fatty acids (1-3%) Lipoproteins Apoproteins Cholesterol/esters Triglycerides Phospholipids Vitamins

64 Tree-like Structure of Human Placenta

65 The human placenta is compartimentalized 1-4 μm ia. g tolo be a i i D a.it d na ditali a i l a si à CT t a cie ditali o S si a l a l a Ca ta ivere u r d c e c di s Stroma ST e E a OY preg S MVM DE le si T BM O igina N ER e or G da rsion a t e Sinusoid CT ara e la v p e r r p eve a v i sit er ric o Ca P ap i D Stroma Courtesy Dr C. Jones, Univ Manchester, UK ST Lewis et al Ann Nutr Metab 63:208,

66 Compartimentalization of Fatty Acid Utilization 66

67 FATP1 FATP3 Obesity Effect on 90 Placental Target Genes Related to Lipids lipid droplet ATGL TG CGI-58 Syncytiotrophoblast Hirschmugl B et al, Int J Obes 41: ,

68 Maternal Glucose is Essential for Fetal Development The fetus does not produce glucose The fetus requires ~ 40 g glucose per day Placental GLUT1 is major glucose transporter for transfer

69 GLUT 1 in Term Placentas Hahn et al, Cell Tiss Res 1995

70 Placental Glucose Transporters in vitro Regulation Hyperglycemia in vitro downregulates glucose uptake and GLUT1 in human term trophoblasts Hahn et al., FASEB J 12: 1221, 1998 Hyperglycemia in vitro induces GLUT1 translocation in term human trophoblasts Hahn et al., Diabetologia 43: 173, 2000

71 Transplacental glucose transport depends on maternal and fetal blood flow Maternal blood flow Fetal blood flow Illsley et al. Trophoblast Res. 2, 535, 1987 Illsley et al, Trophoblast Res 1987

72 Determinants of Materno-Fetal Transfer Mother Uteroplacental Blood flow Concentration gradient Structure and morphology Metabolism Transport activity Umbilical Blood flow Fetus

73 TG and acetyl coa carboxylase activity in human fetal subcutaneous tissue (1) Triglyceride (mg/g) 13 (4) Acetyl CoA carboxylase (nmol/min/g) 16 (5) 18 (4) Gestational age (weeks) 20 (5) >20 (1) Dunlop M. et al, Early Human Dev; 2: , 1978

74 Body Fat (%) Body Fat (%) in Offspring of Women with GDM and Obesity P=0.002 P=0.002 NGT GDM Fat Free Mass (g) NGT Col 2 Col 3 P= P=0.006 P= LGA AGA SGA LGA BMI<25 AGA BMI 25 Petersen 1988; Catalano AJOG 2003; Durnwald AJOG 2004; Sewell AJOG 2006

75 Triglycerides in Placental Tissue TG mg/g tissue * TG < > 40 BMI [kg/m²] < > 40 BMI [kg/m²] lean (20-25 kg/m 2 n=18) vs. obese (30-64 kg/m 2 n=55), placenta specimen collected in Cleveland (USA) * P <0.05, *** P <0.001 HOMA-IR HOMA-IR *** *** ***

76 CGI-58 relative expression Maternal BMI and Insulin Associate with Placental CGI BMI [kg/m²] BMI vs CGI58 protein R = P < BMI vs CGI58 RNA R = P = CGI-58 relative expression 6 Insulin vs CGI58 protein R = P < Insulin vs CGI58 RNA 4 R = P = Insulin [mu/l] Hirschmugl B et al, Int J Obes 41: ,

77 Villus tree is made up by various types of villi with different functions ia. g tolo be a i i D a.it d na ditali a i l à t a cie ditali o S si a l a l a ta ivere u ed i scr c E e ga d Y SO si pre E T D inale O RN orig E a G sione d r a rat la ve a rep vere p tiva r rice i s po Pe a i D Terminal villi Kaufmann P. & Burton G, The Physiology of Reproduction, 1994

78 Human Newborns have Highest % Body Fat among Mammals % Body fat at birth Human Guinea pig Harp seal Fur seal Sea lions Reindeer Baboon Lamb Calf Foal Black bear Mouse Elephant seal Rabbit Cat Caribou Pig Rat Hamster Kuzawa CW, Yearbook Physical Anthropol 1998

79 Maternal and Fetal Fat During Gestation Fetal lipid accretion maximum at term of gestation: 7 g/day EM Widdowson, 1968; P Haggarty, Ann Rev Nutr 2010

80 The Human Term Placenta Rohan Lewis & MuVis, University of Southampton

81 The human placenta is compartimentalized 1-4 μm ia. g tolo be a i i D a.it d na ditali a i l a si à CT et talia i c o idi S s a all re a a Ca t u crive d e c di s Stroma ST e E a OY preg S MVM DE le si T BM O igina N ER e or G da rsion a t e Sinusoid CT ara e la v p e r r p eve a v i sit er ric o Ca P ap i D Stroma ST Courtesy Dr C. Jones, Univ Manchester, UK Lewis et al Ann Nutr Metab 63:208,

82 Pedersen Freinkel Concept Expanded maternal Glucose Lipoproteins FFA EL P L A C E N T A fetal Glucose FFA Liver + Lipogenesis TG HDL TG + Insulin Lipogenesis + + LPL White adipose tissue syncytiotrophoblast endothelium

83 FA Transfer is not Altered by Maternal Obesity Enrichment in fetal effluent (% of maternal) BMI <25kg/m² BMI >30 kg/m² Non-essential FA Ex vivo placental perfusion * 0 13 C-PA (16:0) 13 C-OA (18:1) 13 C-LA (18:2) 13 C-DHA (22:6) Hirschmugl et al, unpublished

84 maternal fatty acids Fetal Hyperinsulinism May Stimulate Extraction of Polyunsaturated Fatty Acids compartments stroma fetal fatty acids fatty acids vein glucose glucose insulin arteries PUFA Brain Retina Liver syncytiotrophoblast endothelium

85 The Placenta has Two Surfaces which Interact with the Environment of Mother and Fetus Endothelium Fetal Circulation Syncytiotrophoblast Maternal Circulation

86 Computer Model Predicts Transfer Lewis R et al, Southampton, UK 86

87 Metabolism Compartimentalization of Fatty Acid Utilization FATP FATP thioesterase FA FA Acyl-CoA Storage, Conversion, FA Oxidation FA Transfer FA FA FA:FABP FA FA Diffusion Diffusion??? 87

88 Gestational Changes in Maternal and Fetal FC TG PL Lipids TG - NEFA Desoye G et al. JCEM 1987 P. Haggarty Ann Rev Nutr 30:237, 2010

89 D-glucose (mmol.min -1.total placental weight -1 ) 300 Control Total Transplacental Net Transfer of Glucose 50 Maternal glucose: 8 mm GDM 0 Diet Insulin Osmond et al, Diabetologia 2001

90 Term Placenta GLUT3 GLUT4 be a i i D a.it d na ditali a i l à t a cie ditali o S si a l a l a ta ivere u ed i scr c E e ga d Y SO si pre E T D inale O RN orig E a G sione d r a rat la ve a rep vere p tiva r rice i s po Pe a i D Mol Hum Reprod :1173 JCEM : :4097 JCEM 1998 ia. g tolo

91 Glucose 1/3 Triglyceride- FA Glycerol Gly cogen Placental Glucose Utilisation Desoye & Shafrir 1994 Mother Lactate 3/4 1/4 CO 2 2% 3% 10% 80% 5% 2/3 Fetus

92 Placental glucose consumption Pediatric Res 20: 269 (1986)

93 Glucose uptake and transfer depend on maternal-fetal concentration gradient Glucose (µmoles/min/g) Hauguel S et al. Pediatric Res 20: 269, 1986 uptake transfer Maternal - fetal glucose gradient (mm)

94 Maternal-fetal glucose transfer is unaltered in FGR Glucose transfer (µmol min -1 g -1 ) B1 B2 H1 H2 B3 B4 B4 G Experimental Period Preterm FGR Preterm normal Term normal Daniel EC et al, Ped Res; 47: , 2000

95 Transfer routes for maternal nutrients and oxygen SEM of terminal villus Charnock-Jones DS & Burton G Bailliére s Clin Obst Gyn 14:953, 2000

96 Nutrient transfer across the placenta: Oxygen

97 Human Newborns have Distinct Features among Mammals Cerebral O 2 uptake (% of total body metabolism) % total O 2 to brain Human (birth) Human (adult) Sheep (birth) Spider monkey Chimpanzee Rabbit Dog Elephant Rat Horse Sheep (birth) Mouse Guinea Pig Pig Kuzawa CW. Yearbook Physical Anthropol 41: , 1998

98 The fetus can be Hypoxic in GDM Log Ep [mu/ml] Umbilical erythropoietin levels 0 P<0.01 Control (16) GDM (20) White A & A/B RBC x 10 9 /L Nucleated red blood cells Control AGA LGA diabetes Widness et al JCI 67:637 (1981) Yeruchimovich et al Obstet Gynecol 95:85 (2000)

99 Fetal Hyperinsulinism Contributes to Hypoxia compartments maternal fetal stroma Adipocyte glucose glucose insulin Glucose uptake oxygen Metabolism syncytiotrophoblast endothelium

100 The Placenta in Diabetes is Hypervascularized So-called monster villus, diabetes mellitus, wk 40 (Desoye, Kaufmann 2005)

101 maternal Fetal Hypoxia and Insulin Stimulate Vascularization compartments stroma fetal Adipocyte glucose glucose insulin Vascularization Glucose uptake VEGF oxygen Metabolism syncytiotrophoblast endothelium

102 Diffusion-dependent transport Oxygen in diabetes diffusive conductance (ml/min.kpa) Control 29 ± 1.4 White A-C 36 ± 1.8* White D,F,R 31 ± 1.6 * Mayhew et al. Diabetologia 36, 955, 1993

103 Fetal Phenotype in Diabesity Body Fat (%) Normal (10-90th centile) GDM NGT GDM LGA AGA SGA Obesity Mean skinfold thickness (sum of 8 skinfolds) Obese (> 90th centile) 28.6 ± ± 6.1 p<0.001 Catalano AJOG 2003; Durnwald AJOG 2004; Whitelaw AGL, 1976

104 Human Newborns Have Highest % Body Fat Among Mammals % Body fat at birth Human Guinea pig Harp seal Fur seal Sea lions Reindeer Baboon Lamb Calf Kuzawa CW. Yearbook Physical Anthropol 41: , 1998 Foal Black bear Mouse Elephant seal Rabbit Cat Caribou Pig Rat Hamster

105 Pedersen Freinkel Concept Expanded maternal Glucose Lipoproteins FFA EL P L A C E N T A fetal Glucose FFA Insulin White adipose tissue + syncytiotrophoblast endothelium

106 Pedersen Freinkel Concept Expanded maternal Glucose Lipoproteins FFA EL P L A C E N T A fetal Glucose FFA Insulin White adipose tissue + syncytiotrophoblast endothelium

107 Maternal and Fetal Fat During Gestation Fetal lipid accretion maximum at term of gestation: 7 g/day EM Widdowson, 1968; P Haggarty Ann Rev Nutr 30:237, 2010

108 Gestational Changes in Maternal and Foetal Lipids FC TG PL TG - NEFA Desoye G et al. JCEM 1987 Haggarty P Ann Rev Nutr 30:237, 2010

109 Lipids Free fatty acids (1-3%) Lipoproteins Apoproteins Cholesterol/esters Triglycerides Phospholipids Vitamins

110 mother placenta foetus Dissociation 1-3% FA Albumin Complex ~~~~~ Free fatty acid ~~~~~ Hydrolysis by Lipases 97-99% FA Lipoproteins EL Lipoprotein Receptor Lipid Droplets Free fatty acid

111 EL is Upregulated by GDM plus Obesity EL mrna expression Endothelial lipase is upregulated in obese GDM only lean obese lean GDM obese GDM * Inflammatory cytokines upregulate placental EL EL mrna expression (fold change compared to control) * TNF-α Leptin * Gauster et al. Diabetes, 2011

112 mother placenta foetus Dissociation 1-3% FA Albumin Complex ~~~~~ Free fatty acid ~~~~~ EL 97-99% FA Lipoproteins FABPpm ~~~~~ FATP ~~~~~ FAT/CD36 ~~~~~ Diffusion ~~~~~~ Lipoprotein Receptor ~~~~~ FABP ~~~~~ Lipid Droplets ~~~~~ Free fatty acid ~~~~~ Free fatty acid Diffusion ~~~~~~??? ~~~~~ FAT/CD36 ~~~~~ ~~~~~ Free fatty acid ~~~~~ α-fetoprotein Incorporation into Lipoproteins

113 Non è possibile visualizzare l'immagine. mother placenta foetus Dissociation 1-3% FA Albumin Complex ~~~~~ Free fatty acid ~~~~~ EL 97-99% FA Lipoproteins FABPpm ~~~~~ FATP ~~~~~ FAT/CD36 ~~~~~ Diffusion ~~~~~~ Lipoprotein Receptor Oxidation Mitochondria Peroxisomes Lipid resynthesis ~~~~~ FABP ~~~~~ Storage in Lipid Droplets Droplets Biological activity Signal transduction Gene regulation Eicosanoid formation ~~~~~ Free fatty acid ~~~~~ Free fatty acid Diffusion ~~~~~~??? ~~~~~ FAT/CD36 ~~~~~ ~~~~~ Free fatty acid ~~~~~ α-fetoprotein Incorporation into Lipoproteins

114 Fatty Acid Transfer Across Term Human Placenta Transfer characteristics: Slow (3% clearance of H 2 0) Placenta 18: 635, 1997 Inefficient Dependent on chain length Maternal FAs contribute to only ~ 70g (20%) of neonatal fat (normal pregnancy) Pediatr Res 7:192, 1973 Enrichment in fetal circulation (% of maternal) In vivo stable isotopes Normal GDM 13 C-PA (16:0) 13 C-OA (18:1) 13 C-LA (18:2) * 13 C-DHA (22:6) Pagán A et al, AJP-Endocr Metab 2013

115 FA Transfer is not Altered by Maternal Obesity Enrichment in fetal effluent (% of maternal) BMI <25kg/m² BMI >30 kg/m² Non-essential FA Ex vivo placental perfusion * 0 13 C-PA (16:0) 13 C-OA (18:1) 13 C-LA (18:2) 13 C-DHA (22:6) Hirschmugl et al, unpublished

116 FA Composition of Adipose Tissue in Neonates is Unaltered in Diabetes Fatty Acid (% of Total) Normal (5) Diabetes (12) C 12:0 0.5 ± ± 0.2 C 14:0 4.4 ± ± 1.1 C 16: ± ± 2.7 C 16: ± ± 2.2 C 18:0 5.7 ± ± 1.1 C 18: ± ± 5.2 C 18:2 1.1 ± ± 1.1 King et al, Pediatrics; 47: , 1971

117 Pedersen Freinkel Concept Expanded maternal Glucose Lipoproteins FFA EL P L A C E N T A fetal Glucose FFA Liver + Lipogenesis TG HDL TG + Insulin Lipogenesis + + LPL White adipose tissue syncytiotrophoblast endothelium

118 Only DHA Transfer is Altered by Maternal Obesity Enrichment in fetal effluent (% of maternal) C-PA (16:0) BMI <25kg/m² BMI >30 kg/m² Non-essential FA 13 C-OA (18:1) Ex vivo placental perfusion 13 C-LA (18:2) Hirschmugl et al, unpublished Essential FA * 13 C-DHA (22:6) Fetus Plasma: PL 6.6% TG 1.6% CE 1.1% NEFA 2.8% Brain: 4.1% Adipose Tissue: 1.6% Haggarty P Eur J Clin Nutr 58: , 2004

119 Fatty Acids in Umbilical Cord Plasma POLYUNSATURATED (%) a b V a A PUFA *** a b V b A SATURATED (%) a Saturated Fatty Acids N=24 N=17 b b b b a V A V A 0 AGA GDM 0 AGA GDM Ortega-Senovilla H et al Diabetes Care 32: 120, 2009

120 Fetal Hyperinsulinism May Stimulate Extraction of Polyunsaturated Fatty Acids maternal fatty acids compartments stroma fetal fatty acids fatty acids vein glucose glucose insulin arteries PUFA Brain Retina Liver syncytiotrophoblast endothelium

121 Computer Model Predicts Transfer Lewis R et al, Southampton, UK 121

122 Villus tree is made up by various types of villi with different functions ia. g tolo be a i i D a.it d na ditali a i l à t a cie ditali o S si a l a l a ta ivere u ed i scr c E e ga d Y SO si pre E T D inale O RN orig E a G sione d r a rat la ve a rep vere p tiva r rice i s po Pe a i D Terminal villi Kaufmann P. & Burton G, The Physiology of Reproduction, 1994

123 Compartimentalization of Fatty Acid Utilization 123

124 Compartimentalization of Fatty Acid Utilization Metabolism FATP FATP thioesterase FA FA Acyl-CoA Storage, Conversion, FA Oxidation FA Transfer FA FA FA:FABP FA FA Diffusion Diffusion??? 124

125 Nature of the metabolic pool? How does the placenta handle the fatty acid excess of GDM & obesity?

126 Human placenta contains lipid bodies in the syncytiotrophoblast * * normal pregnancy GDM Type-1 lipids lipids * TG (nmole) PL (g) Cholesterol (g) TG (nmole) PL (g) * Cholesterol (g) Jones & Fox, Electron Microsc Res 4: 129, 1991 Shafrir et al, AJOG 144: 5, 1982

127 Cleveland Cohort with Various Degrees of Obesity BMI Number of samples Lean: Obese: RNA extracted Quality control on bioanalyzer, RIN>7.5 Gene expression measured by Nanostring technology

128 Pathways and Genes examined House keeping genes 34 Lipid and FA uptake/binding 16 Lipid storage 5 Lipases/Hydrolases 11 FA elongation/desaturation/oxidation 16 Hormones and hormone receptors 5 Lipoprotein associated proteins 11 Transcription factors/regulators 8 Cholesterol and Steroid Hormone Transport/Biosynthesis

129 Correlation of Maternal BMI with Lipid Related mrnas in Placenta N=80; 90 targets ACAT ACAT CGI PLIN ApoE ELOVL PPARG Spearman Correlation

130 Location of LD-associated Proteins in Human Term Placenta ATGL PLIN2 PLIN3 CK7 Neg control (rabbit) Neg control (mouse) Hirschmugl B et al, Int J Obes, in press

131 Regression of placental lipid related mrnas with % neonatal fat A-FABP/FABP FATP Adjusted for maternal smoking and fetal sex 131

132 mother placenta foetus maternal obesity Lipids/ Fatty acids Metabolism transfer Lipid Droplets Free fatty acid Fatty acids fetal adiposity

133 Summary and Conclusion Maternal-fetal transfer of non-essential fatty acids is unaltered in GDM and obesity Fatty acids are stored as TG in the placenta (trophoblast) in lipid droplets; more TG stored in GDM and obesity Activity of TG synthesis and lipolysis determine the net amount of TG stored It is unclear what happens in extreme conditions of maternal overnutrition

134 University of Southampton, UK Rohan Lewis Simone Perazzolo Bram Senger Keith Godfrey Sarah Crozier University of Cleveland, OH, USA Pat Catalano Sylvie Hauguel De Mouzon Collaborators University of Murcia, ES Antonio Gázquez García Elvira Larque University of Granada, ES Cristina Campos Maite Segura King's College London, UK Lucilla Poston

135 The Team Christian Wadsack Eva Kitzinger Birgit Hirschmugl

136 Funding: European Commission

137 Thank you for your attention!

138

139 Pedersen Freinkel Concept Expanded maternal Glucose Lipoproteins FFA EL P L A C E N T A fetal Glucose FFA Liver + Lipogenesis TG HDL TG + Insulin Lipogenesis + + LPL White adipose tissue syncytiotrophoblast endothelium

140 DHA is enriched in placenta and cord blood 12 hr after maternal 13 C-FA administration Cord blood/ Maternal Placenta/ Maternal (%) (%) C-PA 13 C-OA 13 C-LA 13 C-DHA * * 0 13 C-PA 13 C-OA 13 C-LA 13 C-DHA Gil-Sánchez A et al., Am J Clin Nutr 92:115, 2010

141 Pedersen Freinkel Concept Expanded maternal Glucose Lipoproteins FFA EL P L A C E N T A fetal Glucose FFA Liver + Lipogenesis TG HDL TG + Insulin Lipogenesis + + LPL White adipose tissue syncytiotrophoblast endothelium

142 Villus tree is made up by various types of villi with different functions 142

143 Regression of Placental Lipid Related mrnas with % Neonatal Fat A-FABP/FABP FATP Adjusted for maternal smoking and fetal sex Hirschmugl B et al, unpublished 143

144 The role of maternal lipids in driving fetal fat accretion is still unknown 144

145 Regression of Placental Lipid Related mrnas with % Neonatal Fat A-FABP/FABP FATP Adjusted for maternal smoking and fetal sex Hirschmugl B et al, unpublished 145

146 In Maternal Obesity the Human Placenta Stores More Triglycerides Jones & Fox, Electron Microsc Res 4: 129, 1991 Hirschmugl B et al, Int J Obes 41: , 2017

147 Late Pregnancy

148 Nutrient transfer across the placenta: Glucose

149 Pathways of Materno-Fetal Transport Glucose: * [gluc] >[gluc] m f * saturable * stereospecific * Na -indep. * GLUT1 * mvm:bm ~ 3:1 intervillous space fetal blood

150 Nutrient transfer across the placenta: Lipids Fatty Acids

151 The Placenta Stores Glucose glycogen g/total placenta normal pregnancy * GDM * T1DM Desoye et al., Diabetologia 1994 Br J Ob Gyn 83: 43 (1976)

152 Term Placenta GLUT3 Glycogeninetologia. iab D i d lia.it a an idita i l a s à t a i e l oci idita S lla e a s a uta criver d ce di s e YE rega O ES si p D e OT iginal N ER e or G da rsion a t e ara e la v p pre ever a v siti er ric o p P Dia

153 Fetal glucose can be stored in the placenta

154 Only the placenta has the capacity to store excess fetal glucose Placenta + 40% muscle Fetus liver

155 The human placenta contains more DNA and stores glycogen and lipids in diabetes amount in total placenta * * * * lipids lipids * * * * normal pregnancy GDM Type-1 0 DNA (g) Glycogen (g) TG (nmole) PL (g) Cholesterol (g)

156 Lipids - Fatty Acids

157 Placental distribution of AA taken up % of total AA taken up * controls Type-I PL DOHFA DG FFA TG * * Kuhn et al. Diabetes 39, 914 (1990)

158 Type-I increases AA uptake and conversion into eicosanoids resulting in a lower PGI /TxA Control * AA uptake AA conversion PGI2/TxA2 * * 2 2 Type-I Kuhn et al, Prostaglandins 40: , 1990

159 Placenta is Robust to Maternal Obesity N=90 Lipid related genes Cleveland cohort: 6 modified (BMI) SWS cohort: 4 modified (BMI) N=17 Candidate genes UPBEAT cohort: 17 genes, 1 modified NIGO-Health cohort: 17 genes,???? modified Hirschmugl et al, Int J Obes 41: , 2017 Lewis R et al, in prep Gill C et al, in prep

160 mother placenta foetus maternal obesity lipids NO transfer NO YES metabolism NO Lipid Droplets Free fatty acid lipids neonatal adiposity

161 Effects of a diabesogenic environment on early placental development Lower levels of human placental lactogen in T1DM in the first trimester (Pedersen et al, Acta Obstet Gynecol Scand 1998) High glucose levels reduce proliferation of first trimester trophoblasts in vitro (Fröhlich et al, Am J Pathol 2012) Insulin and TNFα upregulate MMP14 in first trimester trophoblasts in vitro (Hiden et al, Diabetes 2008)