(709.4) Differences in Cellular Metabolism and Metabolic Regulation between Non-diabetic and Diabetic Human Coronary Artery Endothelial Cells

Poster Board Number: E28

Cynthia Xu (Cardiovascular Research Center, Rhode Island Hospital, Brown University), Catherine Karbasiafshar (Cardiovascular Research Center, Rhode Island Hospital, Brown University), Karla Braga (Cardiovascular Research Center, Rhode Island Hospital, Brown University), Rayane Teixiera (Cardiovascular Research Center, Rhode Island Hospital, Brown University), Frank Sellke (Cardiovascular Research Center, Rhode Island Hospital, Brown University), M. Abid (Cardiovascular Research Center, Rhode Island Hospital, Brown University)

Objective: To determine the differences in cellular metabolism and metabolic regulation in non-diabetic and diabetic human coronary artery endothelial cells (HCAEC and DM-HCAEC) to further the understanding of the effects of metabolic syndrome on endothelial dysfunction in the setting of cardiovascular disease.

Methods: Both HCAEC (n = 5) and DM-HCAEC (n = 5) were cultured to passage 6. The cells were then starved for 18 hours. After treatment, a Reactive Oxygen Species (ROS) Assay was performed, and cell lysates were made for proteomics analysis and western blotting.

Results: The ROS assay demonstrated that the total ROS was significantly higher in DM-HCAEC (plt;0.0001) than in HCAEC. Proteomics analysis and western blotting showed that DM-HCAECs had significantly altered cellular transport, RNA processing, protein biogenesis, and antioxidant pathways. Proteomics analysis demonstrated that there were about 1,500 proteins significantly upregulated or downregulated in DM-HCAEC compared to HCAEC. Analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed a significant increase in the “vesicle-mediated transport” pathway and significant decrease in the “mRNA metabolic process” pathway in DM-HCAEC with false discovery rates (FDR) of 1.40 x 10-58 and 4.90 x 10-92, respectively. Western blot analysis showed a significant changes in phosphorylated AMPK/total AMPK, phosphorylated mTOR (serine 2481), total mTOR, and several enzymes responsible for the regulation or elimination of ROS (Figure 1). The increased levels of AMPK activation and dysregulation of mTOR may be due to the excessive ROS in DM-HCAEC, which are consistent with the ROS assay findings and significant changes in expression of enzymes that control ROS levels. Further research is needed to determine the mechanisms of downstream effects.

Conclusions: AMPK and mTOR are key regulators of energy homeostasis, cellular growth and metabolism, and are altered in diabetes in the setting of increased oxidative stress. These findings will guide further experiments to assess the effect of metabolic syndrome and diabetes on therapeutics targeted at improving the outcomes of both acute and chronic myocardial ischemia. This also underlines the need for further development of therapies or the use of current therapies to address the metabolic disturbances found in diabetes to improve clinical outcomes in cardiovascular disease.

Funding for this research was provided by the National Heart, Lung, and Blood Institute (NHLBI) 1R01HL133624 (M.R.A.); R01HL46716 and R01HL128831-01A1 (F.W.S.).