95 ALTERED PROTEIN AND AMINO-ACID METABOLISM IN PREIMPLANTATION EMBRYOS FROM DIABETIC RABBITS

Abstract

During pregnancy the preimplantation period is a critical ontogenetic stage in embryo development. As the embryo is highly sensitive to its surrounding milieu and vulnerable to dysregulations by external stimuli, we investigated the influence of a maternal diabetes mellitus type 1 on blastocyst metabolism and protein modifications. Here we report on protein and amino acid metabolism in preimplantation embryos. A diabetes mellitus type 1 rabbit model was used to measure the protein and amino acid concentrations in blastocyst cavity fluid at Day 6 postcoitum. The protein concentration was enhanced in embryos from diabetic rabbits. The level of the various proteinogenic amino acids was unchanged except for the branched-chain amino acids (BCAAs; l-leucine, l-isoleucine, l-valine). The concentration of l-leucine [153.9 µM ± 60.14] and l-valine [231.95 µM ± 75.67] increased 2-fold and of l-isoleucine [99.85 µM ± 42.92] increased 3-fold. Due to the altered BCAA levels, we assumed a disturbed biodegradation. The expression of the BCAA oxidising enzymes (Bcat2, Bckdha, Dbt, and Dld) and BCAA transporters was determined by real-time PCR. Embryos grown in diabetic rabbits revealed a decreased expression of BCAA oxidizing enzymes and of the BCAA transporter LAT-2. The BCAA transporter LAT-2 was mainly localized in the embryoblast. The quality of proteins in blastocysts from diabetic rabbits was evaluated by analyzing protein glycation (advanced glycation end products, AGEs). Two specific AGE modifications, namely arg-pyrimidine and pentosidine, were detected in Day-6 blastocysts by Western Blot analysis. In blastocyst cavity fluid the AGE-specific fluorescence at 440ex/535em nm was significantly enhanced. Our findings show that not just the quantity of proteins but also the quality is affected in rabbit embryos grown in diabetic mothers. There is evidence to suggest that vital protein interactions and signalling pathways are misprogrammed with likely negative consequences for further life. Supported by EU FP 7 EpiHealth (N°278418).