Biochemical Markers of Insulin Sensitivity at Weeks of Gestation in Chinese American Population Vanessa Sy 1, So-Young Kim 1, Takako Araki 1, Diana Huang 2, Anjit Khurana 1, Emilia Liao 1, Doris Tan 2, George Liu 1, Stephen Wan 2, Leonid Poretsky 1, Donna Seto-Young 1 1 Division of Endocrinology and Friedman Diabetes Institute, Department of Medicine; 2 Department of Obstetrics and Gynecology, Beth Israel Medical Center and Albert Einstein College of Medicine, New York, NY 10003

Introduction  Pregnancy is a complex metabolic state that involves remarkable changes in hormone levels (1)  The first trimester is associated with increased insulin sensitivity that promotes adipose tissue accumulation.  During this time, pregnant women are at increased risk for hypoglycemia. Glycogen stores are depleted quickly during the fasting state due to high feto-placental glucose demand. This results in increased lipolysis and ketone production  On the other hand, the second and third trimesters are characterized by insulin resistance.  As levels of cortisol, progesterone, estrogen, prolactin, and human placental lactogen rise, insulin resistance develops (2)

Introduction  Placenta-derived hormones are believed to be a major factor in reprogramming maternal physiology to achieve an insulin-resistant state.  Insulin resistance is the inability of a defined concentration of insulin to achieve a predictable biological response of nutrient metabolism at the level of the target tissue  Insulin resistance results in:  decreased glucose uptake in skeletal muscle, white adipose tissue and liver  decreased suppression of hepatic glucose production  inability of insulin to suppress lipolysis  decreased ability of insulin to suppress amino acid turnover  Pregravid insulin resistance coupled with inadequate insulin response are underlying factors leading to gestational diabetes (3)

Introduction Insulin resistance markers :  Insulin – increased insulin level is involved in developing insulin resistance and GDM (4)  Insulin-like Growth Factor-I (IGF-I) (4)  promotes growth and differentiation  has also been shown to mimic metabolic actions of insulin, including stimulation of glucose and inhibition of gluconeogenesis  circulating free IGF-1 has been shown to be elevated in increasing BMI.  Insulin-like Growth Factor-Binding Protein 1 (IGFBP-1)  hyperinsulinemia suppresses IBFBP-1 production and increases free IGF-I (5) The roles of IGF-I and IGFBP-I in GDM are not yet clearly understood

Introduction Fibroblast Growth Factor-21 (FGF-21)  protein that stimulates glucose uptake in adipocytes  has previously been reported to be associated with markers of insulin resistance (6-7)  A previous study showed that FGF-21 was significantly correlated with decreased adiponectin and dyslipidemia (increased triglycerides, decreased HDL) (6)  In another study, FGF-21 was found to be significantly greater in DM2 patients compared to control patients (7)  The role of FGF-21 in GDM is still unknown

Introduction Inflammatory markers C-Reactive Protein (CRP), and pro- inflammatory cytokines Interleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-  ) are elevated in individuals with DM2 (8)  CRP (9) Produced primarily in the liver under the stimulation of adipocyte- derived IL-6 and TNF-  Studies have shown this to be correlated with parameters relevant to diabetes including obesity, lipogenesis and adiponectin. It has been consistently associated with the incidence of DM2

Introduction  IL-6 (9) Produced by many cell types which include: endothelial cells, monocytes-macrophages, and fibroblasts 15-30% of circulating IL-6 are derived from adipose tissue production, to modulate adipocyte glucose and lipid metabolism Obesity causes IL-6 upregulation in adipocytes Studies have shown IL-6 to be correlated with the extent of insulin resistance  TNF-  (9) Produced by activated mononuclear phagocytes, T-cells, Natural killer cells, and mast cells A previous study has shown a 2.5-fold increase in adipose tissue TNF-  mRNA expression in obese subjects; there was also a strong correlation of TNF-  mRNA expression with hyperinsulinemia

Introduction Monocyte Chemoattractant Protein-1 (MCP-1) (10) –Produced by macrophages and endothelial cells, it is a chemokine with an agonistic effect for monocytes, memory T cells and basophils –It is thought to be a key player in the recruitment of monocytes for atheroma formation –Studies showed that MCP-1 may be released from adipocytes, with an increased secretion and expression in obesity –Obesity being a constituent of the metabolic syndrome (along with DM2), has initiated several studies linking MCP-1 to insulin resistance

Introduction Leptin (11) -An amino acid secreted by adipocytes proportionate to their total tissue mass -A rise in leptin levels signals satiety to the brain through receptors in the hypothalamic and brainstem neurons -Aside from its action on the appetite-regulating center of the brain, previous studies have linked leptin to the CNS regulation of glucose metabolism which may relate to the pathogenesis of insulin resistance and DM2 associated with obesity Retinol Binding Protein 4 (RBP 4 ) (12) -A transport protein for retinol secreted by adipocytes and the liver -Previously published studies suggest that RBP 4 may be one of the key components in explaining the association between obesity and insulin resistance -Elevated RBP 4 levels have been shown to increase hepatic glucose output and cause increased insulin resistance

Introduction Adiponectin –Secreted solely by adipocytes and has been linked to glucose, lipid, and cardiovascular regulation (11) –Reduced adiponectin levels have been associated with obesity, diabetes and, atherosclerosis (13, 11) –Adiponectin circulates in human plasma as a low molecular weight hexamer/monomer and a high molecular weight (HMW) multimer (11) –Some studies suggest that the HMW adiponectin multimer may be an important biomarker for insulin resistance (14)

Introduction Resistin –an adipocyte-derived polypeptide whose role in obesity and DM2 has been the subject of much controversy as different studies have shown contradictory findings (15)

Introduction  Some studies have shown an elevation of the following biomarkers in DM2 and GDM: Insulin IGF-I CRP Leptin Retinol Binding Protein 4 (RBP 4 )  Adiponectin was found to be decreased in GDM & metabolic syndrome  Adiponectin multimer, and not the monomer, is a marker for evaluating DM2  The roles of the following remain unclear in GDM & DM2 as various contradicting findings have been reported: Fibroblast Growth Factor-21 (FGF-21) Resistin Insulin Growth Factor Binding Protein-1 (IGFBP-1)

Introduction  A study found that at a lower BMI, Chinese Americans have: (16) Higher fasting insulin level Increased chronic inflammatory markers Altered circulating adipokines  Our previous studies (unpublished) showed that there were lower levels of adiponectin monomer and multimer in Chinese Americans with GDM  To our knowledge, there has not yet been a report that examined all of the above mentioned biomarkers in Chinese American pregnant women within a single study

Goals of the Study  To analyze the relationships among the following markers in Chinese Americans at weeks of gestation  Insulin  IGF-I  IGFBP-1  Adipocytokines: adiponectin monomer, adiponectin multimer, RBP 4, and leptin  CRP  TNF-   IL-6  MCP-1  Resistin  FGF-21

Methods Beth Israel Medical Center’s Institutional Review Broad approval was obtained for this project and all consent forms were translated into Chinese.  Inclusion Criteria: - 18 to 40 years of age - Pregnant (24-28 weeks of pregnancy) - Chinese ethnicity

Methods  Exclusion Criteria: Cognitive impairment Prior established diagnosis of DM2 Hepatitis B or other infectious diseases Thyroid dysfunction Thalassemia History of miscarriages Infertility with use of In-Vitro Fertilization

Methods  Information collected: Family history of DM2 or previous GDM Age Height Weight Blood pressure 1-hour Glucose Challenge Test (GCT, 50-gram) 3-hour Glucose Tolerance Test (GTT, 100-gram) HgA1C

Methods  For the ELISA procedures, blood samples were drawn 1 hour after the 50-gram oral glucose challenge.  Blood samples were centrifuged at 2000 rpm for 10 mins, and sera were collected and stored at -80 degrees Centigrade until ELISA was performed.  ELISA kits were obtained from the American Laboratory Products Company (ALPCO, Salem, NH), and included the following: Insulin IGF-1 IGFBP-1 Leptin Adiponectin (monomer) Adiponectin (multimer) Resistin RBP4 FGF-21 CRP TNF-  MCP-1 IL-6

Statistical Analysis Data were analyzed using the SPSS system to calculate the Pearson correlation coefficients of glucose, HbA1C,Insulin, BMI and systolic blood pressure (SBP) against the different biomarkers

RESULTS  n = 136  mean age: years  mean glucose levels (1-hr GCT) : mg/dL  mean HbA1c: 5.3%  mean BMI:  Glucose, HbA1c, Insulin, BMI and SBP were each correlated individually with leptin, adiponectin monomer/multimer, resistin, IGF-1, IGFBP-1, FGF-21, RBP4, CRP, TNF- , IL-6 and MCP-1  Statistically significant correlations are presented in the following figures Study Population

RESULTS Figure 1 N=136

Results  Figure 1: Using the entire cohort, Pearson’s correlation analyses were performed. There is positive correlation between glucose level and:  systolic blood pressure (SBP) (R=0.19, p<0.025)  insulin (R=0.489, p<0.001). There is negative correlation between glucose level and:  adiponectin monomer (R= , p<0.001)  adiponectin multimer (R= , p<0.001).

RESULTS Figure 2 N=136

Results  Figure 2: There is positive correlation between HbA1c and  CRP (R=0.2, p<0.025) There is negative correlation between HbA1c and  adiponectin monomer (R= , p<0.003)  resistin (R= , p<0.042).

RESULTS Figure 3 N=136

Results  Figure 3: There is positive correlation between insulin and  IGF-1 (R= 0.346, p<0.001)  FGF-21 (R=0.197, p<0.023) There is negative correlation between insulin and  IGFBP-1 (R=-0.304, p<0.001),  adiponectin monomer (R= -0.23, p<0.007)  adiponectin multimer (R= , p<0.001)

RESULTS Figure 4 N=136

 Figure 4: There is positive correlation between BMI and the following:  SBP (R=0.363, p<0.001)  resistin (R=0.213, p<0.014)  leptin (R= 0.433, p<0.001)  CRP (R=0.216, p<0.016)  RBP 4 (R=0.266, p<0.002)  Insulin (R=0.233, p<0.007)  FGF-21 (R=0.222, p<0.022) There is negative correlation between BMI and the following:  IGFBP-1 (R= , p<0.008),  adiponectin monomer (R= , p<0.037)  adiponectin multimer (R= , p<0.01) Results

Conclusion  For most biomarkers that we examined, the findings in Chinese Americans are similar to those reported in the literature in other populations  The new findings in our study are: Adiponectin monomer is consistently associated with glucose levels, HbA1c, insulin and BMI Resistin is positively correlated with BMI, but negatively correlated with HbA1c IL-6, TNF- , and MCP-1 did not show any correlation with HbA1c and BMI. Only CRP is correlated with both HbA1c and BMI  These findings suggest that the mechanisms underlying the development of diabetes in pregnant Chinese Americans may differ from other populations. Larger future studies are needed to confirm this conclusion

Limitation  Study was limited by: Sample size of 136 patients Lack of a control group

Acknowledgements  The Gerald J. Friedman Diabetes Institute  Chinese American Medical Society & Chinese American Independent Practice Association Research Fund  The Yen Family Foundation

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