Diabetes Care Publish Ahead of Print, published online October 30, 2009 Hormonal and metabolic factors associated with variations in insulin sensitivity in human pregnancy Harold David McIntyre FRACP1, Allan Mang Zing Chang PhD1, Leonie Kaye Callaway PhD2 , David Michael Cowley FRCPA1, Alan Richard Dyer PhD3, Tatjana Radaelli MD4,5, Kristen Anne Farrell MS 5, Larraine Phyllis Huston-Presley MS5, Saeid Baradaran Amini PhD, JD 5, John Patrick Kirwan PhD 5, Patrick Michael Catalano MD 5, for the HAPO Study Cooperative Research Group* 1 The University of Queensland and Mater Health Services, South Brisbane, Australia; 2 The University of Queensland and Royal Brisbane and Women’s Hospital, Herston, Australia; 3 Department of Preventive Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois; 4 Department of Mother, Child and Neonate “L. Mangiagalli”, IRCCS Ospedale Maggiore Policlinico Mangiagalli and Regina Elena, 20122, Milan, Italy; and 5 Department of Reproductive Biology, Case Western Reserve University at Metro Health Medical Center, Cleveland, Ohio. * Members of the HAPO Study Cooperative Research Group are listed in the Appendix of: HAPO Study Cooperative Research Group. Hyperglycemia and Adverse Pregnancy Outcomes. N Engl J Med. 2008;358:1991-2002. Corresponding Author / Reprint requests: Professor Harold David McIntyre [email protected] Additional information for this article can be found in an online appendix at http://care.diabetesjournals.org Submitted 30 June 2009 and accepted 16 October 2009. This is an uncopyedited electronic version of an article accepted for publication in Diabetes Care. The American Diabetes Association, publisher of Diabetes Care, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher- authenticated version will be available in a future issue of Diabetes Care in print and online at http://care.diabetesjournals.org. Copyright American Diabetes Association, Inc., 2009 Objective: To determine maternal hormonal and metabolic factors associated with insulin sensitivity in human pregnancy Research Design and Methods: Prospective observational cross sectional study of one hundred and eighty normal pregnant women, using samples collected at the time of blinded oral glucose tolerance testing between 24 and 32 weeks gestation as an ancillary to the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study. Setting - two public university teaching hospitals – Cleveland, Ohio, USA and Brisbane, Australia. Methods of assessment - Fasting maternal serum cholesterol, triglycerides, free fatty acids, insulin, leptin, tumour necrosis factor alpha, placental growth hormone (PGH), insulin like growth factors (IGFs) 1 and 2 and insulin like growth factor binding proteins (IGFBPs) 1 and 3 were assayed. Correlation and multiple regression analyses were used to determine factors associated with maternal insulin sensitivity estimated using both OGTT derived (IS OGTT) and fasting (IS HOMA) insulin and glucose concentrations. Results: Insulin sensitivity correlated (r = x, y for IS OGTT , IS HOMA respectively) with fasting maternal serum leptin (-0.44, -0.52 ), IGFBP1 ( 0.42, 0.39) and triglycerides (-0.31, -0.27 ). These factors were significantly associated with insulin sensitivity in multiple regression 2 analyses (adjusted R 0.44 for IS OGTT and IS HOMA). These variables explained more than 40% of the variance in estimates of insulin sensitivity. Conclusions: Maternal hormonal and metabolic factors related to the placenta, adipose tissue and the growth hormone axis are associated with the variation in insulin sensitivity seen during normal human pregnancy. 2 he development of insulin Hyperglycemia and Adverse Pregnancy resistance in pregnancy has Outcome (HAPO) study (8) and subjects T been recognized for many consented in writing both to the main HAPO years, but the causal mechanisms remain study and to this ancillary study. Subjects and unclear. Ryan and colleagues first investigators were blinded to the results of the demonstrated a 40% decrease in insulin oral glucose tolerance test (OGTT), so as not sensitivity (1) in women with gestational to affect the outcome of the primary HAPO diabetes as compared with a control group at project. term. Later, Catalano and colleagues Clinical methods. One hundred confirmed these results describing and eighty (180) women, including 80 from longitudinal changes in insulin sensitivity and Cleveland, enrolled in HAPO were recruited insulin response in women with normal into this ancillary study. Their characteristics glucose tolerance and gestational diabetes are shown in Table 1. A 75g OGTT was before and during pregnancy (2). Despite a performed after 8-10 hours overnight fasting general tendency to attribute whole body in all subjects between 24 and 28 weeks (as insulin resistance in pregnancy to “placental close as possible to the 28th week) of gestation hormones” (3), the precise contribution of according to standardized procedures. The various hormonal factors remains poorly OGTT consisted of fasting (0 min), 60 and defined. Human placental lactogen (hPL) was 120 minute glucose measures and fasting and an early candidate though findings have been 60 minute C-peptide determinations. variable (4). Kirwan et al have suggested an Ancillary study patients had estimations of important role for tumor necrosis factor alpha serum insulin at 0, 60 and 120 mins. As part (TNF∝) (5) whilst placental growth hormone of the HAPO protocol, subjects were (PGH) has been shown to induce insulin unblinded if their fasting plasma glucose level resistance in a mouse model (6) and to (PG) exceeded 105 mg/dL (5.8 mmol/L), if correlate with maternal glycemia in patients the 2-hour OGTT PG exceeded 200 mg/dL with diabetes (7). Our study was designed to (11.1 mmol/L) or if any recorded value was further explore the maternal metabolic and less than 45 mg / dL (2.5 mmol/L). This hormonal correlates of insulin resistance in a study includes only women whose OGTT healthy pregnant population. results were within HAPO limits. Three We hypothesized that factors in women would have been classified as having addition to placental hormones were gestational diabetes by the NDDG criteria and associated with insulin resistance during eight by the Carpenter Coustan criteria. (9) normal pregnancy. However, since all glucose results were blinded, we have not excluded these women MATERIALS AND METHODS from this report. Other hormonal and The protocol was approved by the metabolic factors were measured in the Hospital Institutional Review Board (IRB) fasting state. and the Scientific Review Committee of the Laboratory methods. Glucose General Clinical Research Center (GCRC) at assays in HAPO employed the glucose Metro Health, Cleveland, Ohio, USA and by oxidase method and were carefully the Human Research Ethics Committee of standardized across all HAPO centers under Mater Health Services, South Brisbane, the supervision of the central laboratory in Australia. Both of these centers participated Belfast. The other biochemical and hormonal in the international, multi centre assays for this ancillary study were performed 3 at either the GCRC Cleveland (insulin; leptin; approximate a normal distribution. Natural free fatty acids (FFA); TNF ∝ and insulin logarithms have been used in further analyses. like growth factors one and two [IGF1, We employed linear product moment IGF2]) or Mater Health Services Brisbane (Pearson’s) correlations followed by multiple labs (PGH, insulin like growth factor binding linear regression analysis to explore the proteins one and three [IGFBP1, IGFBP3], relationships between variables. Dependent cholesterol and triglycerides) in one or two variables were ISOGTT and ISHOMA. batches, with one shipment of samples in each Independent variables included all measured direction. Samples with hemolysis were maternal biochemical parameters, maternal excluded prior to testing. All assays were pre pregnancy BMI, BMI at the OGTT, age performed in duplicate. Assay CVs are and centre (Cleveland vs. Brisbane). Results shown in Supplementary Table 1A in the reported include standardized regression online appendix which is available at coefficients (β) with 95 % confidence http://care.diabetesjournals.org. intervals and partial correlation coefficients. Insulin samples were centrifuged at STATA (StataCorp TX, USA) and Statistica 4ºC and stored at -70ºC. Insulin was assayed (StatSoft, OK, USA) were used for statistical using a double-antibody radioimmunoassay analyses. Significance was accepted at the (Linco, St. Charles, MO) as previously 5% level on two tailed testing. described. (2) Leptin, FFA, TNF∝, IGF1, IGF2, PGH, IGFBP1 and IGFBP3 were RESULTS assayed using previously described methods The characteristics (Mean (SD)) of the (5; 10; 11) 180 women who participated in this study are Based on previous work by the outlined in Table 1. The median and Cleveland group (12) the insulin sensitivity interquartile ranges for the biochemical and index calculated from the OGTT according to hormonal variables are also shown in Table 1. the equation first described by Matsuda and Only age at delivery differed between the DeFronzo (ISOGTT) formed our primary Cleveland and Brisbane participants. Non measure of insulin sensitivity. Specifically, Hispanic Whites were the predominant ethnic insulin sensitivity was calculated as follows: group (80%), Hispanics 3%, Asians 9% and ISOGTT