The relationship between glucose metabolism byproduct, D-lactate, and vascular endothelial cell dysfunction and possible role in diabetes

Abstract

Diabetes mellitus is a chronic disease associated with vascular complications. Vascular endothelial dysfunction caused by increased endothelial cell apoptosis contributes to diabetic cardiovascular complications. The glucose metabolic by-product, D-lactate, is elevated in diabetics and it is unknown whether it contributes to endothelial cell apoptosis. We hypothesized that diabetic D-lactate levels induce apoptosis in human vascular endothelial cells (HUV-EC-C). HUV-EC-C were incubated with 0.2 mM D-lactate (DLA) and mRNA expression of PI3K/AKT pathway members (AKT1, Bcl-2, BAD, eNOS, PI3K) were measured using Quantitative RT-PCR. DLA downregulated all genes at 6 and 24 hours, followed by increase in expression after 48 hours except PI3K, which remained below control. To further investigate apoptosis, the Human Apoptosis PCR Array was used and expression of all proapoptotic genes (TNF family members) and antiapoptotic genes (IAP family members) were decreased and increased, respectively, at 24 hours followed by an increase and decrease, respectively, at 48 hours. Caspase activity, measured using the Caspase-Glo® 3/7 Assay after HUV-EC-C exposure to 0.2 mM DLA alone or in combination with 20 mM glucose (GLU) or 5 µM methylglyoxal (MG), was increased after 1, 72, and 96 hours. Furthermore, to know whether DLA (0.2 mM) and DLA (0.2 mM), GLU (20 mM) and MG (5 µM) combined cause changes in cellular energy metabolism, creatine (Cr) and high-energy phosphate substrates (CrP, ATP, ADP, AMP) were quantified using HPLC and no changes were observed. We further measured ROS production in HUV-EC-C treated with 0.06-2 mM DLA alone or 0.2 mM DLA with 5-30 mM GLU or 5-160 μM MG. All DLA concentrations increased ROS production by 160% to 216%. DLA with GLU or MG significantly increased ROS production compared to GLU or MG alone. Lastly, D-lactate dehydrogenase (D-LDH) expression was determined using Quantitative RT-PCR and D-LDH was not detected in HUV-EC-C. In conclusion, DLA altered expression of different pro- and anti-apoptotic genes in HUV-EC-C. Furthermore, exposure of HUV-EC-C to DLA levels typically present in diabetics resulted in time-dependent changes in caspase activity, possibly due to excessive ROS production. Whether these changes eventually lead to endothelial dysfunction in diabetes needs further investigation.