Measuring Brain Endothelial Cell Bioenergetics Via Extracellular Flux Analysis

Abstract

The neurovascular unit (NVU) is an important structural component in the central nervous system (CNS). The NVU consists of multiple cell types that include endothelial cells, pericytes, astrocytes, and others, working collectively as a restrictive interface between blood and neural tissue within the CNS. The NVU functions to transport nutrients, ions, and other substances to and from the blood to maintain homeostasis within the neural cell microenvironment. The NVU is responsible for the regulation of nutrient and ion transport from the blood as neurons require a fastidious supply of nutrients and ions in order to function properly. It is also important to regulate the neural cell microenvironment as many molecules and substrates in blood serum can be detrimental to neural function. This neural dysfunction may, in turn, lead to CNS complications. A dysfunctional NVU is associated with many disease states, including Alzheimer’s, ALS, strokes, multiple sclerosis, epilepsy, and glioblastomas. These disease states are linked to, but not limited to, deregulation of nutrient transport, NVU inflammation and leakage of blood constituents into the neural environment, downregulation of the basal lamina, reduced efficacy and downregulation of ATP-binding cassette (ABC) transporters, and downregulation of tight junction proteins. Therefore, it is important that the NVU possesses mechanisms for which it can restrict passage of detrimental substances into the CNS. The endothelial cell is a principal barrier-forming cell of the NVU because of its direct contact with the blood, its intercellular tight junctions, biotransforming enzymes, and asymmetric distribution of active and carrier-mediated transporters. These properties are important in the separation of blood from neural tissue and regulation of nutrients and ions within the neural environment. Maintaining and regulating these properties requires an abundant supply of energy, in the form of adenosine triphosphate (ATP). Therefore, endothelial cell energy metabolism is a critically important area of study. Cellular energy metabolism is considered the process of exploiting various metabolic substrates to produce ATP. Cells typically utilize glycolysis and oxidative phosphorylation (OXPHOS) as energy producing pathways to maintain cellular ATP demand. OXPHOS is considered the major contributor in ATP production as it produces ~ 33 molecules of ATP per glucose molecule, whereas glycolysis produces only two molecules of ATP per glucose molecule. Glycolysis is often overlooked due to this imbalance of ATP production. However, it is becoming more evident that glycolysis may be a primary energy producing pathway due to its rapid turnover rate and production of molecules that are able to be utilized as building blocks for cellular compartments. Cellular bioenergetics using extracellular flux analysis has been extensively used to study many different cell types such as tumor, immune, and stem cells, but little is known about the energy producing pathways of brain endothelial cells. Here, we characterize the bioenergetics of human brain microvascular endothelial cells by using human brain microvascular endothelial hCMEC/D3 cells as a model. hCMEC/D3 cell bioenergetic properties were characterized by investigating metabolite preference and the effects of various metabolic inhibitors on extracellular acidification and OXPHOS rates. Glycolysis and OXPHOS can be quantitatively measured by using extracellular flux analysis. Using sensitive probes, extracellular flux analysis can measure extracellular acidification and oxygen consumption to quantify glycolytic and OXPHOS rates, respectively. In this study, we show that these cells utilize glycolysis as a primary metabolic pathway and glucose as the preferred metabolite. Although glucose is the primary metabolite hCMEC/D3 cells utilize, they are able to maintain ATP production by utilizing pyruvate and glutamine as well via OXPHOS. Using monocarboxylate transporter 1 (MCT1), mitochondrial pyruvate carrier (MPC), glutaminase (GLS), and glucose transporter 1 (GLUT1) inhibitors, we were able to explore the metabolic flexibility of hCMEC/D3 cells. Nutrient transport inhibition significantly altered glycolytic and oxidative properties of hCMEC/D3 cells. These findings reveal a basic understanding of brain endothelial cell energy production and metabolism. This data may also contribute to our understanding of altered brain endothelial cell function in disease or under conditions of active angiogenesis during development or tumorigenesis. Further understanding of altered brain endothelial cell energy metabolism in a diseased state can allow for the development of therapeutics that target these altered pathways.