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
References (37)
- et al.
Calcium-sensitive probes for the measurement of intracellular calciumeffects of buffer system and magnesium concentration
Biochem Biophys Res Commun
(1995) - et al.
Brain infarction in children with diabetic ketoacidosis
J Diab Complications
(1996) - et al.
The effect of experimentally induced diabetes mellitus on the lipid order and composition of rat cerebral microvessels
Neurosci Lett
(1992) The antioxidative potential of cerebral microvessels in experimental diabetes mellitus
Brain Res
(1995)Central nervous system complications of diabetes mellitus- a perspective from the blood-brain barrier
Brain Res Rev
(1997)- et al.
Cranial computed tomography in children and adolescents with diabetic ketoacidosis
Am J Neuroradiol
(1988) - et al.
Correlates of brain edema in uncontrolled IDDM
Diabetes
(1992) Current Concepts of brain edema
J Neurosurg
(1995)- et al.
Protein kinase C activation and the development of diabetic complications
Diabetes
(1998) - et al.
Physiological roles of ketone bodies as substrates and signals in mammalian tissues
Physiol Rev
(1980)
Acetoacetate and B-hydroxybutyrate kinetics in obese and insulin-dependent diabetic humans
Am J Physiol
Relationship of blood acetoacetate and 3-hydroxybutyrate in diabetes
Diabetes
Vascular permeability factorA unique regulator of blood vessel function
J Cell Biochem
Molecular and biological properties of the vascular endothelial growth factor family of proteins
Endo Rev
Endothelins
N Engl J Med
Vascular endothelial growth factor affects permeability of brain mocrovessel endothelial cells in vitro
Am J Physiol
Influence of ischemia and reperfusion on the course of brain tissue swelling and blood-brain barrier permeability in a rodent model of transient focal cerebral ischemia
Exp Neurol
Diacyglycerol production; Ca 2+ influx, and protein kinase C activation in sustained cellular responses
Endocr Rev
Cited by (71)
Metabolomics comparison of cord and peripheral blood-derived serum eye drops for the treatment of dry eye disease
2021, Transfusion and Apheresis ScienceCitation Excerpt :Interestingly, β-hydroxybutyrate seems to be effective also for the treatment of tear-deficiency in a placebo-controlled study on 65 DED patients [37]. β-hydroxybutyrate stimulates the synthesis of VEGF [38], which is also more concentrated in CBS than in PBS [14]. Another metabolite significantly enriched in CBS is myo-inositol, which has important roles during pregnancy promoting cellular proliferation and organ development [39].
Intracerebral matrix metalloproteinase 9 in fatal diabetic ketoacidosis
2019, Experimental and Molecular PathologyCitation Excerpt :Recent publications by Woo et al. (2016b) and by Garro et al. (2017) reported a positive correlation between DKA severity and the prevalence of MMP8 and MMP9, and hypothesized that these enzymes are playing a role in the disruption of the blood-brain barrier by extracellular matrix cleavage. Activation of MMP9 before DKA treatment likely involves: hyperglycemia, ketones, methylglyoxal and endothelin 1 (ET-1) (Isales et al., 1999; Kim et al., 2012; Koyama et al., 2017); hypercortisolemia and free fatty acids (Boden and Song, 2008), in addition to the bidirectional modulation by inflammatory cytokines and chemokines (Strazielle et al., 2003), whose cleavage by MMP9 can result in a gain of function of some substrates (Van Lint and Libert, 2007). Also, of potential importance is that insulin has been reported to activate MMP9 (Fedorova et al., 2018).
Distinctive metabolomic fingerprint in scleroderma patients with pulmonary arterial hypertension
2017, International Journal of CardiologyThe evolution of diabetic ketoacidosis: An update of its etiology, pathogenesis and management
2016, Metabolism: Clinical and ExperimentalCitation Excerpt :The high H + level allows more Na + into the cell, which attracts water into the cell leading to edema [136]. The ketone bodies acetoacetate and β-hydoxybutyrate may also play a role in the pathogenesis of cerebral edema [137]. Ketone bodies have been shown to affect vascular integrity and permeability and contribute to edema formation.
The Impact of Diabetes on Brain Function in Childhood and Adolescence
2015, Pediatric Clinics of North AmericaCitation Excerpt :In periods of sustained insulinopenia or energy restriction, ketone bodies can serve as the primary cerebral energy source, providing up to 60% of metabolic requirements.53 Ketone bodies can also have direct influences on brain activity by causing increased levels of vascular permeability factor and the vasoconstrictor endothelin-1.54 In vivo animal studies have shown both benefits and injury to the brain from excessive ketosis, which are again related to context.