Original articlesAcetoacetate and β-Hydroxybutyrate Differentially Regulate Endothelin-1 and Vascular Endothelial Growth Factor in Mouse Brain Microvascular Endothelial Cells
Introduction
The metabolic crisis of diabetic ketoacidosis (DKA) can result in the well-recognized, life-threatening, acute complication of brain edema in children and adolescents. There is good evidence that this complication also occurs in a subclinical form in a large percentage of patients, and it is very likely present in some patients even prior to the initiation of DKA treatment.1, 2
The pathogenesis of the brain edema is uncertain, but several theories have been proposed. The most commonly cited theories are (1) a rapid decrease in the plasma osmolality; (2) a disequilibrium between intra- and extracellular pH; (3) an insulin effect on the blood–brain barrier; and/or (4) vasopressin-mediated water retention. These theories suggest that the treatment of the hyperosmolar state and/or the ketoacidosis play a major role in this complication, but the theories do not explain the presence of edema prior to treatment, nor do they address whether the site of the initial insult is vascular (vasogenic) or cellular (cytotoxic).3
We have reported an inverse correlation between the degree of acidosis and the pretreatment presence of brain edema, and a direct correlation between the degree of hyperglycemia and the pretreatment presence of brain edema.2 Therefore, it would seem that the metabolic alterations associated with DKA (ketone bodies, low pH, hyperglycemia and hyperosmolality) would be putative initiators. Koya and King4 also recently reviewed the potential pathways whereby activation of protein kinase C by hyperglycemia may increase vascular permeability. The hyperketonemia and acidosis seen in DKA are the result of increased levels of both acetoacetate (AcAc) and β-hydroxybutyrate (βOHB), with molar concentrations in the blood sometimes being increased over 30-fold.5 Initially, βOHB exceeds AcAc by a ratio of 3:1, possibly due to a selective defect in βOHB utilization;6 but the ratio of βOHB to AcAc gradually decreases during the treatment of DKA.7
Blood–brain barrier integrity is maintained by the endothelial cells (EC) lining the cerebral blood vessels, and thus would be a logical target in any condition leading to brain edema. In order to determine if the brain endothelial cells are a target of ketone bodies, we examined the effects of AcAc and βOHB on the production of vascular permeability factor/vascular endothelial growth factor (VEGF)8 and endothelin-1 (ET-1)9, 10—two peptides important in endothelial cell function and known to be involved in edema formation in the brain.11, 12
Section snippets
Endothelial Cell Culture
Primary mouse brain microvascular endothelial cells (MBMEC) were purchased from In Vitrocyte (Seattle, WA) and used at passages 3–6 for these studies. MBMEC were purified by their ability to metabolize fluorescently labeled, acetylated, low-density lipoprotein (DiI-Ac-LDL), and were examined by their ability to bind to griffonia simplicifolia agglutinin (GSA). Cells were grown to confluence in MBMEC growth medium (Cell Applications, San Diego, CA), supplemented with 10% fetal calf serum (v:v)
Results
Figure 1A–F shows the increase in intracellular calcium in MBMEC to varying concentrations (1–100 nM) of either βOHB and AcAc. In contrast, further increases in the concentrations of either ketone body (up to 100 mM) had no further effect on intracellular calcium. In additional experiments, ketone bodies in medium in which the pH was adjusted between 6.8 and 7.8 were found to have no additional effect on intracellular calcium (data not shown). In separate experiments, MBMEC were grown in a high
Discussion
The relationship between ketone bodies and brain metabolism is well documented in DKA,15 with βOHB and AcAc crossing the blood–brain barrier via a monocarboxylic acid transport system.16 This transport is influenced by increased blood concentrations of ketone bodies and utilized in a region-specific pattern.17 This is supported by more recent studies where proton MR spectroscopy of the brain has demonstrated ketone bodies during treatment of DKA.18 There is clinical data to suggest that the
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