Highly Glycolytic Immortalized Human Dermal Microvascular Endothelial Cells are Able to Grow in Glucose-Starved Conditions

Abstract

Endothelial cells form the inner lining of blood vessels, in a process known as angiogenesis. Excessive angiogenesis is a hallmark of several diseases, including cancer. The number of studies in endothelial cell metabolism has increased in recent years, and new metabolic targets for pharmacological treatment of pathological angiogenesis are being proposed. In this work, we wanted to address experimental evidence of substrate (namely glucose, glutamine and palmitate) dependence in immortalized dermal microvascular endothelial cells in comparison to primary endothelial cells. In addition, due to the lack of information about lactate metabolism in this specific type of endothelial cells, we also checked their capability of utilizing extracellular lactate. For fulfilling these aims, proliferation, migration, Seahorse, substrate uptake/utilization, and mRNA/protein expression experiments were performed. Our results show a high glycolytic capacity of immortalized dermal microvascular endothelial cells, but an early independence of glucose for cell growth, whereas a total dependence of glutamine to proliferate was found. Additionally, in contrast with reported data in other endothelial cell lines, these cells lack monocarboxylate transporter 1 for extracellular lactate incorporation. Therefore, our results point to the change of certain metabolic features depending on the endothelial cell line.

Keywords: MCT1; endothelial cells; glycolysis; lactate; metabolism.

Figure 1

HMEC growth under different nutritional conditions. (a) Cell survival after 6 h incubation in DMEM supplemented with 5 mM glucose (G), 0.5 mM glutamine (Q) and/or 0.5 mM palmitate (P). (b) HMEC growth was monitored in the presence or absence of 5 mM glucose and/or 0.5 mM glutamine and (c) 5 mM glucose and/or 0.5 mM glutamine with and without 1 mM sodium pyruvate. Data are expressed as means ± SD of three independent experiments. *p < 0.05, ***p < 0.001, ****p < 0.0001 versus glucose and glutamine condition (a,b) or condition without pyruvate (c).

Figure 2

HMEC proliferation under different nutritional conditions. 10 μM EdU was added for 2 h to cells already incubated for 22 h in the presence or absence of 5 mM glucose (G) and/or 0.5 mM glutamine (Q). EdU incorporation was detected using a FACS VERSE^TM cytometer. Data are expressed as means ± SD of three independent experiments. **p < 0.01, ***p < 0.001 versus condition with glucose and glutamine.

Figure 3

HMEC migration under different nutritional conditions. Representative images and quantification of wound closure of cells incubated in the presence or absence of 5 mM glucose (G) and/or 0.5 mM glutamine (Q) for 0, 4, and 7 h (scale bar = 200 µm). Data are expressed as means ± SD of three independent experiments. *p < 0.05 versus condition with glucose and glutamine.

Figure 4

Effect of glucose starvation on cell cycle distribution and autophagy. (a) Cell cycle distribution of subpopulations of HMEC grown in the presence or absence of 5 mM glucose for 48 h, stained with propidium iodide and percentage of subG1, G1 and S/G2/M cells were determined using a FACS VERSE^TM cytometer. (b) LC3B-I and LC3B-II protein expression were measured by Western blot in cells grown in the presence or absence of 5 mM glucose. A positive control incubating cells with 5 mM glucose and 50 μM chloroquine (CQ) for 16 h was included. Data from Western blot are normalized against α-tubulin expression. Data are expressed as means ± SD of three independent experiments. Glutamine was added to the media in all experiments.

Figure 5

Flux analysis in HMEC. (a) Oxygen consumption rate (OCR) and (b) extracellular acidification rate (ECAR) in the presence of 5 mM glucose, 0.5 mM glutamine or 0.5 mM palmitate. Data are expressed as means ± SD of three independent experiments with triplicate samples each. *p < 0.05, **p < 0.01, ***p < 0.001 versus control without any metabolic substrate.

Figure 6

Substrate uptake and utilization in HMEC. (a) Glucose uptake, (b) lactate production, (c) glutamine uptake, (d) glutamine oxidation and (e) palmitate uptake were determined in HMEC in the presence or absence of 5 mM glucose (G), 0.5 mM glutamine (Q) and/or 0.5 mM palmitate (P). Data are expressed as means ± SD of three independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001 versus glucose (a,b), glutamine (c,d) or palmitate (e) conditions. ND: non detected.

Figure 7

Substrate uptake and utilization in HUVEC. (a) Glucose uptake, (b) lactate production, (c) glutamine uptake and (d) glutamine oxidation were determined in HUVEC in the presence or absence of 5 mM glucose (G) and/or 0.5 mM glutamine (Q). Data are expressed as means ± SD of three independent experiments. **p < 0.01 versus glutamine condition.

Figure 8

Lactate metabolism in HMEC. (a) Oxygen consumption rate (OCR) and (b) extracellular acidification rate (ECAR) were measured in the presence of 5 mM glucose and/or 10 mM lactate. (c) mRNA expression of LDH-A, LDH-B, MCT4 and MCT1. (d) MCT1 protein expression was determined under normoxia, 200 μM CoCl₂ or 1% hypoxia. (e) MCT1 protein expression was determined in the presence or absence of 10 μM lactate. HL-60 under normoxia were used as positive control. Data are expressed as means ± SD of three independent experiments. *p < 0.05, **p < 0.01 versus no glucose or lactate available (a,b). ND: non detected.