1. Vitamin D Adipocytokines ….and GDM pregnancies

2. Objectives:  To review the role for Vit D as an insulin-sensitizing hormone, with particular reference to GDM pregnancy  To review some of the roles for adipocytokines in insulin resistance and GDM pregnancy  To review our local experience with Vit D/adipocytokines in GDM women and their neonates

3. But first….back to med school for a moment

4. Vitamin D: a few reminders  Ergocalciferol (D2): provitamin form  Cholecalciferol (D3): inactivated, unhydroxylated form  25(OH) Vit D3: this from is the least variable, and considered best measure of sufficiency. Therefore, most common form measured.  1, 25(OH) Vit D3: this form is variable. Measured in odd rare conditions such as Vit D resistant rickets

5. Vitamin D: a few reminders  Ergocalciferol (D2): provitamin form  Cholecalciferol (D3): inactivated, unhydroxylated form  25(OH) Vit D3: this form is the least variable, and considered best measure of sufficiency. Therefore, most common form measured. This is the form that will appear in this talk as VitD  1, 25(OH) Vit D3: this form is variable. Measured in odd rare conditions such as Vit D resistant rickets

7. Vitamin D: recent oddities  We usually think of Vit D as promoting bone health  The last few decades have illustrated that Vit D has hormone properties   structural similarities with testosterone, steroids, cholesterol

8. ….cholecalciferol

9. …….cholesterol

10. Vit D and GDM: what do we know?  Vit D deficiency suspected to be a risk factor for glucose intolerance  For instance:  54 GDM, 39 IGT; 11 controls, matched for age, BMI, pregnancy week 24-28  [NB: Iranian study] Soheilykhah Nutr Clin Pract 2012. 25. 524

11. CONCLUSIONS:  83% GDM and IGT women had VitD <20ng/ml  vs  71.2% Controls (p=0.03)  Lowest VitD levels were in GDM women compared to Controls 

12. Does Vit D status predict GDM risk?  953 pregnant women in USA  Nested case/control study  VitD level taken at 16 weeks gestation  57 women developed GDM  Zhang Plos One 2008.3.e3753

13. CONCLUSIONS  VitD at 16 wks in women developing GDM: 24.2 ng/ml  vs  Controls: 30.1 ng/ml (p<0.001)  [Difference remained significant after adjusting for weight, age, race, family history of DM, prepreg BMI]

14. Does VitD predict adverse preg outcomes?  Meta-analysis up to Oct 2012  24 studies fit criteria (Vitamin D/status/deficiency/insufficiency/pregnancy)  Outcome: women with VitD <50 nm/l:   OR 2.09 risk pre-eclampsia (CI: 1.5-2.9)   OR 1.38 risk GDM (1.12-1.7)   OR 1.57 risk preterm birth (1.08-2.31)   OR 1.52 risk SGA (1.08-2.15)  Wei.Mat-Fetal Medicine 2013.26.889

15. What about interventional studies?  ………..wait……………………

16. What links VitD and insulin resistance?  1. No one really knows  BUT  2. Perhaps: through inflammation:  VitD can be shown to be associated with anti-inflammatory properties;  and insulin resistant states are also pro-inflammatory states  inflammation  endothelial dysfunction  pre-eclampsia  inflammation  immune modulation (IL-1 and IL-6 in particular)

17. Links between VitD and insulin resistance  3. Perhaps:  VitD receptors are present in the placenta and fetal tissues  VitD regulates genes involved in trophoblast invasion/angiogenesis

18. Links between VitD and insulin resistance  4. Perhaps:  pancreatic beta cells have VitD receptors   may regulate insulin secretion  Vit D stimulates insulin receptor expression  promotes insulin sensitivity

19. VitD and inflammatory markers  So: holding onto VitD considerations but switching over to thinking about adipocytokines

20. What are Adipocytokines  Proteins produced by adipose cells  [note: adipose is not just unwanted insulation, but rather a large endocrine organ]  Many, many, many known and still unknown associations and effects

21. Adipocytokines You know some of these already:  leptin  adiponectin  TNF- α  interleukins  resistin …etc….

22. Just a few metabolic associations of adipokines  Low adiponectin levels have been associated with an increased incidence of Type 2 DM  Adiponectin increased insulin sensitivity, fatty acid oxidation and reduces liver glucose production  Leptin reflects total body adipose mass  Resistin levels increase with fat mass and correlate with insulin resistance  TNF α and IL-6 increased in obesity and are linked to insulin resistance and type 2 DM

23. Functions of Adipokines? Endocrine; paracrine; autocrine roles in:  hemostasis  lipids metabolism  atherosclerosis  BP regulation  insulin sensitivity  angiogenesis  immunity  inflammation  Miehle. Clin Endocrinology 2012.76.2

25. Adipocytokines and GDM  TNF- α correlates with insulin resistance in pregnancy  TNF- α is released from maternal side of placenta  Leptin rises during pregnancy and falls after delivery  Leptin correlates with insulin resistance in pregnancy  Adiponectin levels are lower in GDM than control preg women  Lacroix. Curr Diab Rep 2013. 13. 238

26. For instance: Normal PregGDMPre-eclampsia LEPTIN Rises, peak at 28 wksIncreased ADIPONECTIN Declines thru out pregnancy DecreasedIncreased RESISTIN Higher than nonprgtIncreased VISFATIN Peaks 19-26 weeks then drops Increased

28. ..So, cooking up a study in London ON INTERESTING UNKNOWNS:  What are VitD levels in offspring of GDM pregnancies?  What are the profiles of adipocytokines in GDM women AND their offspring?  Are maternal and neonatal VitD levels correlated?  Are maternal and neonatal adipokine levels correlated?  Do corralations exist between VitD and inflammatory adiokines (maternal and neonatal)?

29. …..we had the following building blocks… LUCK Kelly Summers’ adipokine assay GDM women

31. PSI grant  Maternal, umbilical arterial and umbilical venous 25 hydroxyvitamin D and adipocytokine concentrations in pregnancies with and without gestational diabetes  R McManus, K Summers, B DeVrijer, N Cohen, A Thompson, I Giroux Clinical Endocrinology 2014; 80:635-641.

32. Methods  Case control  GDM diagnosed before clinic referral  no recruitment during Nov-Mar months  GDM and Controls recruited at 31 weeks  Did 48 hour dietary and supplement recall [before GDM saw RD]

33. Methods  Maternal blood taken/spun/frozen at 31 weeks for  Ca  Phosphate  BG  CRP  PTH  Adipocytokines (adiponectin, resistin, PAI-1, IL-6; Il-8, leptin, TNF α, MCP-1)

34. Methods  On day of delivery:  Neonatal umbilical artery and umbilical vein bloods were taken for DR staff for:  Ca  Phosphate  BG  CRP  PTH  Adipocytokines (adiponectin, resistin, PAI-1, IL-6; Il-8, leptin, TNF α, MCP-1)

35. ….do you want to guess? Umbilical artery flows: Umbilical vein flows:

36. So  umbilical arterial blood would reflect fetal chemistry  umbilical vein blood would reflect maternal AND placental chemistry

38. Demographics Maternal  age  pre-pregnancy weight  maternal weight at time of blood taking Infant  birth weight  infant gestational age  apgar scores  duration of hospital stay/complications

39. A few stats Sample size of 24 X2=48 would allow for detecting a 30 nm/l difference between [VitD] in GDM women vs C

40. Results  73 women  36 GDM; 37 C  Matched for week of gestation; present weight; pre-preg weight; maternal VitD intake

41. MATERNAL CHARACTERISTICS AND BIOCHEMISTRY CONTROLGDMp N3736 Age (yrs)30.2±4.131.6±5.00.19 Weeks Gestation31.4±3.631.6±2.90.84 Current Weight (kg)85.8±21.389.1±16.00.47 Pre-pregnancy Weight (kg) 74.4±18.977.7±17.40.44 Pre-pregnancy BMI (kg/m 2 ) 27.2±7.228.7±5.50.34 Maternal Vitamin D Intake (ug/day) 14.4±6.415.8±2.90.44 ‡

42. MATERNAL CHARACTERIST ICS AND BIOCHEMISTRY CONTROLGDMp 25(OH)D (nm/L) [range]93.2±19.2 [55-135]77.3±24.3 [33-128]0.009 PTH (pm/L)4.33±16.571.78±1.100.732 † Calcium (mm/L)2.20±0.132.20±0.090.898 Phosphate (mm/L)1.06±0.181.04±0.170.519 GLUCOSE (MM/L)4.68±0.895.46±1.290.008 Alkaline Phosphatase (u/L)82.2±27.189.5±18.80.234 CRP (mg/L)6.03±4.996.00±5.360.983 ADIPONECTIN ( Μ G/ML) 34.1±20.317.5±11.8<0.001 † RESISTIN (NG/ML)31.9±12.125.4±9.10.045 PAI-1 (NG/ML)21.0±12.613.9±10.00.038 IL-6 (pg/ml)1.93±1.321.76±1.000.627 IL-8 (pg/ml)2.39±0.982.25±1.920.185 † Leptin (ng/ml)41.2±33.740.1±26.40.899 TNF- α (pg/ml) 4.99±2.085.83±2.460.196 MCP-1 (pg/ml)115.6±52.8115.9±81.10.688 †

43. DELIVERY OUTCOMES CONTROLGDMp Infant Weight (g) GESTATIONAL AGE (weeks) 3457.8 ± 455.2 39.5±0.9 3384.6 ± 504.2 38.2±1.2 0.547 <0.001 Apgar 18.2 ± 1.78.0 ± 2.10.749 Apgar 28.8 ± 0.58.9 ± 0.60.689 Labour Duration (hours) 8.1 ± 5.87.6 ± 4.00.891 † Placental Weight (g)677.2 ± 169.7746.0 ± 197.60.159 Post partum Stay (hours) 46.8 ± 15.545.5 ± 25.40.353 ‡ Sex – Male (%)19 (59)19 (61)0.877 Induced Labour (%)17 (53)20 (64)0.359 Caesarian Section (%)8 (22)6 (16)0.51

44. INFANT ARTERIAL UMBILICAL CHEMISTRY CONTROLGDMp 25(OH)D (nm/L)65.6±17.658.0±20.80.195 Calcium (mm/L)2.54±0.212.46±0.350.420 Glucose (mm/L)3.67±0.813.44±1.500.227 ‡ ADIPONECTIN (ΜG/ML) 100.0±52.257.0±31.70.006 RESISTIN (NG/ML)222.4±456.557.1±34.50.030 ‡ PAI-1 (NG/ML)21.5±22.711.2±6.60.049 † IL-6 (pg/ml)37.8±105.716.9±22.30.779 † IL-8 (pg/ml)20.7±23.011.8±5.80.784 ‡ Leptin (ng/ml)44.7±46.446.1±37.90.910 TNF-α (pg/ml)10.7±2.111.7±3.00.209 MCP-1 (pg/ml)690.6±552.6574.8±275.40.608 †

45. INFANT VENOUS UMBILICAL CHEMISTRY CONTROLGDMp 25(OH)D (nm/L)64.8±11.566.3±19.50.952 ‡ Calcium (mm/L)2.62±0.252.50±0.180.086 Glucose (mm/L)3.70±1.243.96±0.840.422 ADIPONECTIN ( Μ G /ML) 109.9±49.564.0±33.70.004 RESISTIN (NG/ML)237.4±529.247.5±17.9<0.001 ‡ PAI-1 (NG/ML)15.5±13.98.4±8.20.009 † IL-6 (pg/ml)38.4±109.411.2±12.40.871 † IL-8 (pg/ml)15.7±18.68.7±5.00.464 ‡ Leptin (ng/ml)49.9±40.155.4±48.60.675 TNF- α (pg/ml) 10.9±2.511.8±2.80.253 MCP-1 (pg/ml)457.4±289.7425.0±247.90.690

46. Searching for correlations [we limited correlations to r>0.4 or r<-0.4; p<0.05]  Maternal Control VitD levels: + correlated with resistin only  Maternal GDM VitD: + correlated with PAI-1; IL-8; TNF- α

47. Searching for Correlations..cont  Neonatal VitD levels were not correlated with any of:  infant weight  placental weight  Apgar scores  labour duration/hospital stay  adipocytokines

49. Admitting our limitations  Big picture: no one knows what level of VitD is “ideal” for the non-osteomalacial actions of VitD  Our women were not as VitD deficient as in some studies so differing conclusions might occur if there was a wider range of serum levels  Our GDM women were generally only mildly hyperglycemic (ie) no one was on insulin when maternal bloods were taken: again a wider range of insulin impairement may have uncovered differing results

51. So…what…? #1: As expected:  GDM women had lower adiponectin than Controls  however, this finding was present despite being matched for weight and pregnancy week  lower adiponectin levels would be consistent with increased GDM maternal inflammation…but….

52. So what #2  GDM had lower resistin and PAI-1 levels [argues against inflammatory biochemical profile]  GDM leptin was not different from C  GDM CRP, ILs, TNF α, MCP-1 not different as well   overall, no conclusive evidence for inflammatory chemistry in GDM women

53. So what #3:  GDM maternal VitD lower than Controls  But:  Umbilical arterial and venous VitD showed no difference between GDM and C offspring

54. So what #4  Maternal GDM VitD levels were positively (not negatively as expected) correlated with some adipokines thought to be associated with inflammation (PAI-1; IL-8, TNF α)  Neonatal VitD levels did not correlate with inflammatory markers

55. So what #5  Neonates born to mothers with GDM also manifested lower adiponectin and resistin levels  even in umbilical arterial bloods   suggesting that there are adverse adipokine profiles present at birth

57. So what #6  We learned a very important life lesson:  Never ever again do a study where blood samples from babies have to be centrifuged and frozen at any time of day, night, holidays……

59. Just an aside  Enthusiasm for VitD as THE miracle metabolic hormone has waned  Much of what our study was built upon (remember, grant applied for <2008) tantalizing hints and correlations around VitD effects  However, interventional studies have suggested perhaps some effect attributable to VitD, although final word not yet in…..

60. For instance  54 women with GDM (Iran)  randomized to placebo or cholecalciferol 50,000 u at study entry and day 21  fasting samples for BG and insulin taken at study onset as well as after 6 weeks

61.  Results:  VitD supplementation:  was correlated with lower FG (-17.1±14.8 mg/dl vs -0.9±16.6 mg/dl, p<0.001)  was correlated with lower serum insulin  was correlated with improved QUICKI index Asemi. Am J Clin Nutr 2013.98.1425

62. one more…  120 Iranian women <12 weeks of pregnancy  randomized to:  200 u VitD OD;  50,000 u VitD monthly  50,000 u Vit D Q2 weeks  until delivery

63. measured:  FBG,  insulin,  Ca,  VitD  before and after intervention

64. Results  Group C receiving 50,000 u every 2 weeks had biggest rise in VitD level  FBG dropped 2.02 mg/dl in Group C (NS)  Insulin level in group C went up less than Group A (NS)  comparing all 3 groups: insulin and HOMA IR were improved with higher doses of VitD supplementation  Soheilyhkhah Gynec Endocrinol 2013. 29. 396

65. ..one last London connection  DALI study  Vitamin D and lifestyle intervention for gestational diabetes mellitus (GDM) prevention: an European multicentre, randomized trial-study protocol  9 countries  <20 weeks gestation  8 intervention arms (placebo, healthy diet, healthy activity, Vit D combos)

66. DALI  Vit D dose is 1600 u OD until delivery  primary outcome: gestational weight gain, fasting glucose and insulin sensitivity, OB outcomes  [biorepository blood is being stored at Lawson/David Hill]  Jelsma BMC Pregnancy Childbirth 2013. 13.124

67. that’s all ….