13C Metabolic Flux Analysis Indicates Endothelial Cells Attenuate Metabolic Perturbations by Modulating TCA Activity.

Bilal Moiz,Alisa Morss Clyne,Andrew Li,Surya Padmanabhan,Sarah Basehore,Angela Sun,Jonathan Garcia,Cholsoon Jang,Ganesh Sriram,Kaitlyn Albus

doi:10.3390/metabo11040226

Abstract

Disrupted endothelial metabolism is linked to endothelial dysfunction and cardiovascular disease. Targeted metabolic inhibitors are potential therapeutics; however, their systemic impact on endothelial metabolism remains unknown. In this study, we combined stable isotope labeling with 13C metabolic flux analysis (13C MFA) to determine how targeted inhibition of the polyol (fidarestat), pentose phosphate (DHEA), and hexosamine biosynthetic (azaserine) pathways alters endothelial metabolism. Glucose, glutamine, and a four-carbon input to the malate shuttle were important carbon sources in the baseline human umbilical vein endothelial cell (HUVEC) 13C MFA model. We observed two to three times higher glutamine uptake in fidarestat and azaserine-treated cells. Fidarestat and DHEA-treated HUVEC showed decreased 13C enrichment of glycolytic and TCA metabolites and amino acids. Azaserine-treated HUVEC primarily showed 13C enrichment differences in UDP-GlcNAc. 13C MFA estimated decreased pentose phosphate pathway flux and increased TCA activity with reversed malate shuttle direction in fidarestat and DHEA-treated HUVEC. In contrast, 13C MFA estimated increases in both pentose phosphate pathway and TCA activity in azaserine-treated cells. These data show the potential importance of endothelial malate shuttle activity and suggest that inhibiting glycolytic side branch pathways can change the metabolic network, highlighting the need to study systemic metabolic therapeutic effects.

Highlights

Endothelial cells play an important role in pathologies ranging from atherosclerosis to cancer to Alzheimer’s disease
We used metabolic flux analysis to estimate changes in metabolic fluxes with each inhibitor. We show that these metabolic inhibitors can have systemic effects on endothelial cell metabolism, which we speculate may be due to either compensatory mechanisms or signaling pathway activation
This allowed us to estimate metabolic flux values and gain unique insights into human umbilical vein endothelial cell (HUVEC) metabolism that could not have been obtained from analyzing the input and output fluxes and metabolic isotopomer distributions alone

Summary

Introduction

Endothelial cells play an important role in pathologies ranging from atherosclerosis to cancer to Alzheimer’s disease. Endothelial glucose metabolism is disrupted in many of these diseases. Recent efforts demonstrate that manipulating endothelial metabolism can alter cell signaling pathways and thereby cell function [1,2,3]. Many of these studies increased or decreased the activity of a single metabolic enzyme, often focused on glycolysis [4,5,6]. Glycolytic side branch pathways, including polyol, pentose phosphate, and hexosamine biosynthetic pathways, have been implicated in endothelial dysfunction in hyperglycemia; manipulating one of these side branch pathways could have unexpected effects on glycolysis, the tricarboxylic acid (TCA) cycle, or other glycolytic side branch pathways [7,8,9]

Objectives

Methods

Results

Conclusion