Characterization of differential gene expression profiles in diabetic embryopathy using DNA microarray analysis

Objective The molecular mechanisms by which maternal diabetes impairs embryogenesis are not established. This study aimed to determine the developmental genes and molecular pathways that are involved in diabetic embryopathy, by comparing gene expression profiles in the yolk sacs between the embryos of diabetic and control rats by using DNA microarray analysis. Study design Diabetes was induced in female rats by injecting streptozotocin (65 mg/kg) intravenously. Glucose levels were controlled by subcutaneously implanting insulin pellets. The female rats were mated with normal male rats. At gestation day 4, the insulin pellets were removed from a group of animals, making them hyperglycemic. The animals with insulin pellets served as controls. At gestational day 12, embryos were explanted, and yolk sacs were collected. Total RNA, free of DNA contamination, was extracted from the yolk sacs. Complementary DNA probes were synthesized, labeled with Cy3 and Cy5 fluorescent dyes, and used to hybridize rat oligo-array containing 10,000 genes. Data were analyzed by using 1-sample t test on log2 ratios, with P< .05 representing a significant difference. The changes in expression levels of important genes were verified with the use of a real-time polymerase chain reaction (PCR). Results Five microarray experiments produced consistent results. A total of 101 genes were found to be differentially expressed between the embryos of diabetic and control rats. Analyses that used PathwayAssist (Ariadne Genomics, Rockville, MD) revealed a number of potential signaling pathways and genes involved in insulin signaling and stress response (insulin 2, insulin-binding protein 1, GST π1), cell growth (GAP43, CSF1R, HGF), calcium signaling (calbindin 3, CBP A6), and PKC signaling (PKCBP β15, FABP5), in concert with prior biochemical and molecular findings. Conclusion These observations show significant alterations in expression of developmental and stress response genes in diabetic embryopathy, and demonstrate, for the first time, that the yolk sac cells express insulin during early development. In addition, these data also demonstrate that hyperglycemia induces altered gene expression, resulting in aberrant cell signaling, morphogenesis, and embryopathy.