Neurochem Res (2017) 42:35–49
DOI 10.1007/s11064-016-2099-2
ORIGINAL PAPER
β-Hydroxybutyrate in the Brain: One Molecule, Multiple
Mechanisms
Lavanya B. Achanta1,2 · Caroline D. Rae1,2  
Received: 29 September 2016 / Revised: 31 October 2016 / Accepted: 2 November 2016 / Published online: 8 November 2016
© Springer Science+Business Media New York 2016
Abstract β-Hydroxybutyrate (βOHB), a ketone body,
is oxidised as a brain fuel. Although its contribution to
energy metabolism in the healthy brain is minimal, it is
an interesting metabolite which is not only oxidised but
also has other direct and collateral effects which make it
a molecule of interest for therapeutic purposes. In brain
βOHB can be produced in astrocytes from oxidation of
fatty acids or catabolism of amino acids and is metabo-
lised in the mitochondria of all brain cell types although
uptake across the blood brain barrier is a metabolic con-
trol point. βOHB possesses an intrinsic high heat of com-
bustion, making it an efficient mitochondrial fuel, where it
can alter the NAD+/NADH and Q/QH2 couples and reduce
production of mitochondrial reactive oxygen species. It
can directly interact as a signalling molecule influencing
opening of K+ channels and regulation of Ca2+ channels.
βOHB is an inhibitor of histone deacetylases resulting in
upregulation of genes involved in protection against oxida-
tive stress and regulation of metabolism. It interacts with
an inflammasome in immune cells to reduce production of
inflammatory cytokines and reduce inflammation. Use of
βOHB as an efficient neurotherapeutic relies on increasing
blood βOHB levels so as to encourage entry of βOHB to
the brain. While use of βOHB as a sole therapeutic is cur-
rently limited, with employment of a ketogenic diet a more
widely used approach, recent development and testing of
esterified forms of βOHB have shown great promise, with
* Caroline D. Rae
c.rae@unsw.edu.au
1
Neuroscience Research Australia, Barker St, Randwick,
NSW 2031, Australia
2
School of Medical Sciences, The University of New South
Wales, Kensington, NSW 2052, Australia
the approach elevating plasma βOHB while allowing con-
sumption of normal diet. An improved understanding of the
mechanisms by which βOHB acts will allow better design
of both diet and supplemental interventions.
Keywords Ketone body metabolism · Histone
deacetylase inhibition: K+ channels · Brain energy
metabolism
Introduction
βHydroxybutyrate, (3-hydroxybutanoic acid; C4H8O3;
βOHB) is a water soluble principal ketone body formed
reversibly from acetoacetate [1]. It constitutes up to 70%
of the ketone bodies (βOHB, acetoacetate and their break-
down product acetone) synthesized in liver mitochondria
from the oxidation of fatty acids derived from adipose
tissues [2] or human milk [3] and from the conversion of
ketogenic amino acids [4]. Unlike acetone, βOHB is a non-
volatile and stable compound that, once synthesized, may
be released into the blood stream.
It is well-accepted that the preferred substrate to be oxi-
dised for brain energy supply is glucose. However, even
under normal circumstances, brain will metabolise alter-
native substrates such as monocarboxylates like pyruvate
[5], lactate [6], acetate [7] and βOHB [8]. Most of βOHB
oxidised by the brain is supplied by the liver via the blood-
stream. βOHB is also synthesised endogenously in astro-
cytes which are the only cell type in the brain capable of
oxidising fatty acids [9] and this βOHB can be released and
supplied to other brain cell types as fuel.
There are several intriguing aspects of βOHB metabo-
lism and chemistry that potentially make it a more useful
substrate than other alternative substrates; indeed some
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have argued that there is likely a benefit to the brain in the
body being mildly ketotic [10]. Here, we review what is
currently known about the biochemistry of βOHB and how
it could potentially benefit brain function.
Brain Transport of βOHB
The concentration of βOHB in plasma under healthy,
fasted conditions is relatively low with reference values
reported ~0.04  mmol/L ranging as high as 0.08  mmol/L
[11] although this may increase in circumstances such as
fasting or starvation (5–6  mM; [12]), dietary interven-
tion or diabetic ketosis (~25  mM; [13]). In diabetic keto-
sis, the amount of plasma βOHB may increase more than
one hundred fold with values in excess of 10  mmol/L
reported in the literature [14]. In non-diabetic subjects, a
three-day fast has been reported to increase plasma levels
from 0.03 ± 0.04 to 3.15 ± 0.67. The brain concentration of
βOHB in these same subjects increased from 0.05 ± 0.05
to 0.98 ± 0.16  mmol/L across the same time span [15]. In
adolescent mice a ketogenic diet has been found to increase
plasma βOHB levels to an average of 1.1 ± 0.2  mM [16].
The same mice showed a concentration of βOHB in hip-
pocampal extracellular fluid of only 50.7 ± 5.5 μM indicat-
ing a βOHB plasma/hippocampal extracellular fluid ratio
of about 1:25, with the authors calculating an intracellu-
lar βOHB concentration around threefold higher than the
extracellular concentration [16].
Penetration of βOHB into the brain is carrier dependent.
As a monocarboxylate, βOHB is a substrate for monocar-
boxylate transporters, members of the SLC16 family which
are proton symporters found widely throughout the brain
[17]. MCT1 (SLC16A1) is a relatively low affinity trans-
porter located mainly in the blood brain barrier with lower
levels in astrocytes [18]. MCT2 (SLC16A7) is a higher
affinity transporter mostly located in neurons in the post
synaptic density [19]. MCT4 (SLC16A3) is a low affinity
transporter localised exclusively to astrocytes [20]. Each
of these transporters display different affinities for βOHB
(Table  1) with the neuronally located MCT2 having the
highest affinity for βOHB [21] and displaying little if any
stereospecificity for D-βOHB over L-βOHB [18]. MCT1
has much less affinity for βOHB [22] while MCT4 has
Table 1 Monocarboxylate Transporter
transporter (MCT) affinities for
βOHB and acetoacetate MCT1 [22]
MCT2 [21]
MCT4 [23]
SMCT1(SLC5A8) [24]
a
DL-βOHB used in this work
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Neurochem Res (2017) 42:35–49
very little affinity at all [23]. βOHB is also transported by a
sodium-dependent monocarboxylate transporter (SMCT1,
SLC5A8) which is located in neurons [24]. Another
sodium-dependent transporter (SMCT2, SLC5A12) has
been identified in astrocytes but its affinity for βOHB has
not yet been established [25]. Taken together, it would seem
that neuronal cells are well equipped to take up βOHB, but
that glial cells also have capacity to take up and release
βOHB. Uptake of βOHB has been measured in cultured rat
astrocytes with a reported KM of 6.03 mM [26].
Uptake of βOHB is generally a metabolic control point
in the metabolism of βOHB with limited capacity and
expression of MCT1 under normal dietary circumstances
and low plasma βOHB concentrations. Expression of
MCT1 and hence uptake of βOHB by the brain is increased
significantly by ingestion of a high-fat (ketogenic) diet [27,
28] or with fasting. In rats, a ketogenic diet has been shown
to increase βOHB influx forty-fold due to increases in cap-
illary density and threefold increase in MCT1 staining with
a doubling of the number of capillaries staining positive for
MCT1 and no measurable increase in blood flow [29]. In
humans, increasing the plasma concentration of βOHB up
to 12 times normal levels has been shown to have a linear
relationship with brain oxidation of D-βOHB up to concen-
trations of ~2  mmol/L [30]. Similarly, in adult humans, a
linear relationship has been demonstrated between plasma
acetoacetate concentration and the rate of brain acetoace-
tate consumption using positron emission tomography [31].
These authors also showed an inverse relationship between
the rate of glucose consumption and the rate of ketone body
(acetoacetate) consumption after induction of mild ketosis
via 4 days of ketogenic diet. Interestingly, there seems to be
no obvious relationship in rat brain between the amount of
energy able to be extracted from ketones following starva-
tion and pre-starvation glucose utilisation rates [32].
MCT1 blood brain barrier expression is also age-
dependent, being high in neonates and decreasing with
age [33, 34]. A “developmental switch” has been identi-
fied, where the brain shifts from metabolising fatty acids,
ketones and monocarboxylates, to metabolising glucose
[35], In neonatal rats this is accompanied by a decrease in
the vascular expression of MCT1 in the brain, an increase
in the blood brain barrier expression of the glucose trans-
porter GLUT1 and an increase in the non-vascular brain
D-βOHB (mM) L-βOHB (mM) Acetoacetate (mM)
10.1 ± 0.53 11.4 ± 0.82 5.5 [140]
1.2 ± 0.2 1.2 ± 0.2a 0.8 ± 0.1
130 ± 96 824 ± 64 216 ± 27
1.442 ± 0.124 2.33 ± 0.17 0.213 ± 0.039
Neurochem Res (2017) 42:35–49
expression of GLUT3. Expression of non-vascular brain
monocarboxylate transporters MCT1 and MCT2 is not
altered by development [35].
Expression of MCT1 in the blood brain barrier shows
significant species difference, being much lower in adult
monkey (0.834 ± 0.368 fmol/μg protein) than in adult
mouse (23.7 ± 0.87 fmol/μg protein) [33] and similarly
lower in human than in mouse (2.27 ± 0.85 vs 23.7 ± 1.6
fmol/μg protein, respectively); [36].
The question of how βOHB enters the mitochondrion,
the site at which it is most likely metabolised, is less set-
tled. βOHB is poor substrate for the mitochondrial pyru-
vate carrier (KM = 5.6  mM; [37]) although its metabolite
acetoacetate is carried with reasonable affinity (0.56  mM;
[37]). Under concentrations found in  vivo it is unlikely
that this carrier contributes significantly to βOHB uptake.
βOHB is taken up into mitochondria, by a mechanism dis-
tinct from that of pyruvate, with reasonably high affinity.
Uptake is reported to be 50% saturated at 0.9 mM and capa-
ble of accumulating high concentrations of βOHB inside
the mitochondrion, suggestive of active uptake [38, 39].
The Chemistry of β-Hydroxybutyrate
βOHB has an inherently high heat of combustion compared
with the end product of glycolysis, pyruvate, for exam-
ple, producing 243.6  kcal/mol of C2 units compared with
185.7  kcal/mol in the case of pyruvate; this derives from
the fact that βOHB is more reduced than pyruvate [40]. In
rat heart, addition of a physiological ratio of ketone bod-
ies resulted in an improvement in the mitochondrial redox
potential of the half reaction (Eh) NAD+/NADH couple
from −280 to −300 mV and an increase in the Coenzyme
Q couple EhQ/QH2 from −4 to +12 mV indicating that the
chemistry of ketone bodies themselves is capable of driving
mitochondrial efficiency ([41]).
Oxidation of βOHB to Acetyl-CoA
β-Hydroybutyrate Dehydrogenase Catalyses the First
Step in βOHB Oxidation
Once βOHB has entered the brain, it is first oxidised
by the mitochondrial enzyme β-hydroxybutyrate dehy-
drogenase (BDH, E.C. 1.1.1.30) in a reaction requir-
ing NAD+, producing acetoacetate and NADH (Fig.  1).
D-β-Hydroxybutyrate is the naturally occurring isoform
and the substrate for BDH in the brain; the inactive L-β-
hydroxybutyrate isomer does not support mitochondrial
respiration [42]. This means that the two ketone bodies,
βOHB and acetoacetate are in near equilibrium with the
37
mitochondrial NAD+/NADH ratio such that introduction
of βOHB has the potential to shift this ratio. The intercon-
version of βOHB and acetoacetate is rapid [43] making it
difficult to study the effects of one without considering the
other.
BDH is subject to lysine acetylation, a process whereby
proteins can be modified post-translation and their activity
may possibly be altered as a result. It is known that BDH
acetylation can be altered by SIRT3 (silent information reg-
ulator 3) as SIRT3 knockout mice have altered BHD acety-
lation [44, 45] but it is not known what the effect of acety-
lation or de-acetylation may have on the enzyme as this has
not yet been examined.
A cytosolic form of BDH has been reported in humans;
DHRS6, a short form of dehydrogenase with structural
homologies to bacterial hydroxybutyrate dehydrogenases.
DHRS6 substrate screening studies showed sole NAD+-
dependent conversion of (R)-β-hydroxybutyrate to ace-
toacetate with a low affinity KM around 10 mM [46]. This
enzyme is sometimes designated BHB2 and has been
reported as elevated in malignant gliomas [47] but little
is known yet about its expression or role in healthy brain
tissue.
Pathways for Conversion of Acetoacetate to Acetyl-CoA
There are several enzymes in mammalian systems catalys-
ing the interconversion of acetoacetate and acetyl-CoA. In
liver (Fig.  1), acetoacetate can be converted from acetyl-
CoA through the combined activity of hydroxymethylglu-
taryl-CoA synthase (E.C. 2.3.3.10) and 3-hydroxy-3-meth-
ylglutaryl-CoA lyase (HMG-CoA lyase; E.C. 4.3.1.4).
These enzymes are key in the production of ketone bodies
from fatty acids. HMG-CoA is also an intermediate in the
breakdown pathway of branched chain amino acids such as
leucine. Ketone bodies can be produced from leucine by
glial cells, showing an overlap between ketone body metab-
olism and metabolism of branched chain amino acids [48].
In tissues that produce no or little fatty acids, these
enzymes may be silenced by alternative slicing mechanisms
that produce inactive variants of both enzymes [49]. In
brain and other tissue which do not produce ketone bodies
in any appreciable amounts under normal circumstances,
the conversion of acetoacetate to acetyl-CoA is catalysed
by the mitochondrially located succinyl-CoA: 3-oxoacid-
CoA transferase (SCOT; E.C. 2.3.8.5) and acetylacetylCoA
thiolase (E.C. 2.3.1.9) which has both mitochondrial (T2)
and cytosolic (CT) forms [50] (Fig. 1).
A cytosolic pathway for the direct activation of ace-
toacetate exists in brain [51], in the reaction catalysed by
acetoacetylCo-A synthetase (AACS; E.C. 6.2.1.16). This
reaction requires ATP and produces AMP and PPi [52].
The enzyme is highly expressed in the brain of humans
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Fig. 1 Pathways for synthesis and catabolism of ketone bodies in ketogenic and ketone body metabolising tissues
and rats and enriched in neuronal-like cells in the cortical
and hippocampal regions [53] where its expression patterns
are different to that of SCOT [51], suggesting that the two
pathways play different but complementary roles.
βOHB Metabolising Enzyme Deficiencies have Similar
Outcomes and Therapies
There have been case reports of patients with hereditary
succinyl-CoA: 3-oxoacid-CoA transferase (SCOT) defi-
ciency, first described in 1972 [54], which is characterised
by intermittent ketoacidotic attacks and permanent hyperke-
tonaemia [55]. The gene encoding SCOT occupies a large
100 kb region on chromosome 5p13 [56] and deficiencies
are relatively rare: fewer than 30 affected individuals were
reported in 2011 [55]. Despite the ketotic episodes, the out-
come is generally good. Hyperketonaemia is eliminated by
restricting leucine intake and providing intravenous glu-
cose. Less than optimal outcomes can be eliminated by fast
diagnosis and prevention of hypoglycaemia [57].
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Neurochem Res (2017) 42:35–49
Similar issues are present in 3-hydroxy-3-methylglutaryl
coenzyme A (HMG CoA) lyase deficiency where leucine
metabolism and ketone body production are deranged.
Life-threatening episodes include hypoglycaemia, acidosis
and hyperammonemia but not ketosis [58]. The occurrence
of this disorder is similarly rare to that of SCOT deficiency
with around 18 patients identified in 1988 [58]. Brain
imaging studies of these patients show diffuse white mat-
ter abnormalities with more focal lesions reported related
to the degree of metabolic derangement experienced by the
patient [59, 60].
Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency
has also been reported in a similar number of patients and
again, the outcomes are good if the metabolic derange-
ment is detected early and managed appropriately [61].
T2 is generally co-expressed in tissues along with SCOT
although the SCOT/T2 ratio is higher in brain than any
other tissue [50]. A case of deficiency in the cytosolic form
of acetoacetyl-CoA thiolase (CT) has also been reported
associated with mental retardation, which was subsequently
stabilised with diet [62].
Neurochem Res (2017) 42:35–49
Metabolism and Metabolic Effects of βOHB
Compared to other alternative substrates for brain, such
as lactate, pyruvate and acetate, less is known about
metabolism of βOHB. It was established in the 1960s that
brain switches to metabolism of ketone bodies, includ-
ing βOHB, during fasting, when glucose is in short sup-
ply and the body resorts to metabolism of fat and pro-
tein [12]. It was also noted that ketone body oxidation
was greater in suckling than in adult rats [63] and Louis
Sokoloff noted in 1973 that the previous idea of the brain
as being exclusively restricted to metabolism of glucose
must be reconsidered, in view of the fact that “under
certain conditions and during certain periods of life the
brain can and does rely, at least in part, on other sub-
strates, particularly ketone bodies” [64]. Indeed ketones
can comprise as much as 60–70% of brain energy needs
during prolonged fasting and starvation [65].
Ketones are preferred over glucose in the developing
brain. Newborn rats incorporated significantly more radi-
oactivity from [3-14C]βOHB into glutamate and aspartate
than from [2-14C]glucose [66]. It has been suggested [67]
that developing brain preferentially uses ketones to spare
glucose for metabolism in the pentose phosphate path-
way, for example. The pentose phosphate pathway leads
to the biosynthesis of ribose for DNA and to the produc-
tion of NADPH for lipid biosynthesis, both of which are
crucial in brain development. The activities of BDH and
glucose-6-phosphate dehydrogenase, the enzyme which
generates NADPH [68], peak in rats around postnatal
day 18 and 21, respectively [69]. The changes in ketone
body metabolism during development in are detailed in
the excellent review from the laboratory of Astrid Nehlig
[70].
The switch to metabolism of ketones over glucose
under conditions of starvation, or in switching to a
ketogenic diet, requires modification of transporters and
enzyme activities. Ketogenic diets and their effects on
brain metabolism and brain disorders have been reviewed
extensively by others (e.g. [71, 72]) and are not the sub-
ject of this review.
Examination of metabolism of βOHB under “normal”
conditions in brain can be a very informative and useful
tool for researchers interested in metabolic compartmen-
tation and metabolite interaction within the brain. βOHB,
along with competing substrates, or analysis of metabolism
of labelled βOHB (either radio-labelled or stable isotope)
has been employed successfully to give us unique insights
into mitochondrial and cellular processes. From the first
observation that respiration rates were altered in brain
when metabolising βOHB it was apparent that metabolism
of this ketone body was sufficiently different to that of glu-
cose to warrant further attention.
39
Neurons, Astrocytes and Oligodendrocytes all
Metabolise Ketone Bodies
Edmond et al. studied the capacity of brain cells in culture
to use glucose or ketone bodies for respiration [9]. They
found that all cell types use βOHB in respiration but that
neurons and oligodendrocytes use βOHB about threefold
more efficiently than astrocytes, as do synaptic terminals
[73]. Perhaps surprisingly, investigation of the activities of
the three major ketone body metabolising enzymes, BDH,
SCOT and T2 (Fig.  1) have been found to be highest in
astrocytes although each of the cell types is quite capable
of using ketone bodies as substrate [74].
In contrast to acetate, where metabolism gives rise to a
higher specific activity in glutamine compared to glutamate
due to metabolic compartmentation of its metabolism [75,
76], metabolism of βOHB has been shown to give higher
specific activity in glutamate compared to glutamine [77].
Modelling of the relative distribution of label from gluta-
mate and glutamine in human brain has given rise to the
suggestion that metabolism of βOHB is predominantly neu-
ronal [78]. This contrasts with the determination by others
in rat brain that βOHB is more preferentially used in glial
cells than neurons [8].
It is not immediately clear why these differences in cel-
lular preference may have been found. The infusion time
(2  h) was identical between the work of Pan and that of
Künnecke, with Pan et  al. infusing a higher concentration
for the first 20  min then a steady, lower level for the rest
of the time, while Künnecke et al. used a single, constantly
infused concentration. The two papers also used different
metabolic models and made different assumptions about
pool sizes.
Enzyme Activities Vary with Age and Across Different
Species
In the rat, there are quite large developmental changes in
enzyme activity, with this effect being largest in astrocytes,
which lose SCOT and T2 activity appreciably across the
15  days since birth. BDH activity remains relatively con-
stant from birth to adulthood [74] but has been reported
to decrease in adulthood with aging [79]. The activities
of these enzymes also varies appreciably between spe-
cies, with quite high levels of activity reported in rodents
such as mice and rats, with appreciably (ten-fold) lower
SCOT activity in guinea pig and sheep. These authors
also reported that the activities of these enzymes were not
altered in rats by 48  h of starvation or alloxan diabetes
[80]. Levels of the three enzymes are lower in human brain
than in rat and there is little reported change with devel-
opment between 32 week gestation foetus and 70 year old
adults [81], although regional differences in the ability to
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metabolise acetoacetate have been demonstrated to vary
with age in humans [82]. Page and Williamson [81] con-
clude that there is sufficient enzyme activity in human
brain to account for consumption of ketone bodies, what-
ever their concentration in the blood. This is also sup-
ported in humans by tracer studies showing that transport
of βOHB across the blood brain barrier is the limiting fac-
tor, with βOHB utilisation found to increase almost linearly
with concentration, with the rate of use higher in gray than
in white matter [83].
βOHB Metabolism Varies with Brain Region
A study injecting 3-14C βOHB intravenously into male
Sprague–Dawley rats, followed by freezing and sectioning
of the brain after 10 min, showed regional variation in the
uptake of βOHB with the lower layers of the cortex show-
ing greater uptake than the upper layers. The striatum and
globus pallidus showed low uptake while the area of the
hypothalamic arcuate nucleus-median eminence took up
large amounts of label, with the pituitary and pineal gland
taking up the most [84].
BDH expression has been shown to vary across the
brain. Its expression in cerebellum has been reported to
be patchy with intense staining in Purkinje cells and den-
drites postnatally, with less prominent staining here and in
the surrounding molecular layer later in life, while the extra
germinal layer of the cerebellum did not show appreciable
staining [85].
The cerebral metabolic rate of acetoacetate (CMRa) has
recently been measured using positron emission tomogra-
phy (PET) in humans and found to be reasonably constant
across brain regions, with the lowest rate in hippocam-
pus (CMRa = 0.4 ± 0.0  μmol/100  g/min) and the high-
est rate in regions of the parietal and occipital cortices
(CMRa = 0.9 ± 0.1  μmol/100  g/min). These rates increase
appreciably after four days on a ketogenic diet, rising to
3.3 ± 0.3  μmol/100  g/min in hippocampus and as high as
6.5 ± 0.7 in frontal pole and to 6.2–6.4 μmol/100 g/min in
parietal and occipital regions [31].
βOHB can Supply Baseline Energy Needs but may not
Support Synaptic Activity
Early experiments suggested that, in Guinea pig hippocam-
pal slices, βOHB was able to support maintenance of high
energy phosphate levels, but unable to support synaptic
potentials [86]. This was also shown in the rat, although
there was an age-dependent effect, with βOHB able to sup-
port ~90% maintenance of synaptic potential compared
to initial values in the p4 rat and ~20% in the p7 rat [87].
The interpretation of this work is complicated by the use of
DL-βOHB rather than the stereoscopically preferred isomer
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Neurochem Res (2017) 42:35–49
D-βOHB. L-βOHB competes with D-βOHB for uptake via
the various MCTs (Table 1).
As detailed above, βOHB is directly metabolised in
mitochondria, being used to produce acetyl-CoA for oxi-
dation in the Krebs cycle for the ultimate production of
ATP. Glucose, on the other hand, generates cytosolic ATP
via substrate level phosphorylation, before also providing
carbon units for mitochondrial oxidative phosphorylation.
It has been suggested that there is compartmentalisation of
ATP in cells, with the cytosolic component being used to
power ion channels, such as the Na+/K+-ATPase or KATP
channel activity [88–90]. Neither glycolysis nor oxidative
phosphorylation alone are adequate to power ion gradients
in the brain [90].
In keeping with this, the contribution of βOHB to basal
(housekeeping) needs in the brain vs fuelling neuronal
activity has been estimated after 36  h of fasting in rats.
Provided the blood concentration of βOHB is high enough,
βOHB is capable of supplying all basal requirements and
around half of the energy needs of neuronal activity [91].
βOHB Metabolism Alters Glycolytic Activity,
Mitochondrial NAD+/NADH and Malate Aspartate
Shuttle Activity
βOHB has been shown to alter the activity of pyruvate
dehydrogenase through conversion of the enzyme to the
inactive form [92] and to inhibit glycolysis through an
indirect mechanism at the level of phosphofructokinase
[93]. When βOHB was added to synaptosomes metabo-
lising [6-14C]-glucose the rate of 14CO2 production was
reduced by ~35% but when βOHB was added to mitochon-
dria this rate increased slightly by 12% [94]. In the reverse
case, when [3-14C]-3-hydroxybutyrate was incubated with
mitochondria and synaptosomes and glucose was added
(5 mM), production of 14CO2 was reduced by 65 and 70%,
respectively. One possible explanation for this may lie in
the alteration of mitochondrial NAD+/NADH ratio due
to the 2  h of pre-incubation with βOHB. Recently, it has
been demonstrated that the NAD+/NADH ratio is shifted
in cortical neurons preincubated for two hours with 8 mM
βOHB [95]. The increased mitochondrial respiration drives
changes in BDNF expression through increased genera-
tion of reactive oxygen species, activation of NF-ĸB tran-
scription factor and activity of the histone acetyltransferase
p300/EP300 [95].
Effects of βOHB on metabolism are seen through the
increase in acetyl-CoA levels which rise by 15- to 18-fold
as a result of addition of ketone bodies such as βOHB or
acetoacetate to cells [41]. The direct consequence of this
is a reduction in levels of “free” Coenzyme A and an effect
on the steady-state equilibrium of 2-oxoglutarate dehy-
drogenase, which requires unesterified CoA as a cofactor
Neurochem Res (2017) 42:35–49
(Fig.  2); levels of 2-oxoglutarate rise while those of suc-
cinate and oxaloacetate fall. The changes in 2-oxoglutarate
and oxaloacetate then result in concomitant increased glu-
tamate and reduced aspartate levels. In compartments with
glutamate decarboxylase this also results in a rise in GABA
levels due to stimulation of GAD by increased glutamate
[96] (Fig. 2).
In astrocytes, the presence of βOHB (5  mM) in the
absence of glucose with 15N-glutamate as substrate has
been shown to result in decreased transamination of gluta-
mate to aspartate, as well as a reduction in aspartate levels
[97]. By contrast, this reduction in aspartate levels is not
seen when 15N-GABA is used as substrate. βOHB has no
measureable direct effect on aspartate aminotransferase so
the authors suggested that the effect was due to diversion
of oxaloacetate away from transamination and towards syn-
thesis of citrate by combination with acetyl-CoA (which
would be elevated in the presence of βOHB). The authors
noted elevation in astrocytic citrate levels when βOHB was
supplied. Secondarily, they noted that glutamine synthetase
is inhibited by citrate [97] as are both the cytosolic and
mitochondrial forms of malic enzyme [98]. GABA could
reasonably be expected to supply the section of the Krebs
Fig. 2 βOHB metabolism in brain. Metabolism of βOHB by BDH
(βOHB dehydrogenase) produces NADH inside the mitochondria
while the second step of its metabolism catalysed by SCOT (succinyl-
CoA: 3-oxoacid-CoA transferase) produces succinate. Together, these
have the potential to alter mitochondrial redox potentials through
shifting the NAD+/NADH and Q/QH2 couples. βOHB metabolism
also reduces glycolysis, thereby reducing the flux through cytosolic
malate dehydrogenase as the drive to regenerate NAD+ is less when
41
cycle via succinic semialdehyde dehydrogenase, thus main-
taining levels of oxaloacetate and hence aspartate [97].
Ketone bodies such as βOHB have been reported to not
be able to be used to synthesise pyruvate [99] but authors
have reported pyruvate recycling from βOHB [8] with
pyruvate recycling capability increasing after hypoglycae-
mia [100]. This has important implications for anaplerosis
in the Krebs cycle and for the ability to synthesise Krebs
cycle by-products, such as the neurotransmitters glutamate
and GABA, as well as aspartate.
A reduction in the synthesis of neurotransmitter gluta-
mate has been reported in cultured mouse glutamatergic
neurons, with significantly lower levels of glutamate and
correspondingly higher levels of aspartate present when
superfused in the presence of 1  mM βOHB compared to
1 mM glucose [101]. Adding 1 mM βOHB to the neurons
along with 1  mM glucose somewhat counterintuitively
resulted in further increases in glutamate and further reduc-
tions in aspartate. Depolarising the neurons with 300  μM
NMDA, 10  μM glycine and 15  mM K+ had no effect on
glutamate or aspartate levels in the presence of 1  mM
βOHB while significant reductions in glutamate and total
glutamate + aspartate were seen under depolarisation in the
glycolysis is decreased. This, in turn, reduces the exchange of malate
and α-ketoglutarate across the mitochondrial membrane, result-
ing potentially in lower cytosolic α-ketoglutarate levels and reduced
ability to synthesise glutamate and aspartate via cytosolic aspartate
aminotransferase. AcAc acetoacetate, CoASH Coenzyme-A, OAA
oxaloacetate, AcAcCoA acetylacetoCo-A, MPC mitochondrial pyru-
vate carrier, MCT1 monocarboxylate transporter 1
13
42
presence of 1  mM glucose. The failure to increase βOHB
metabolism under depolarising conditions was attributed
to increased mitochondrial Ca2+ under depolarising con-
ditions with concomitant effects on NAD+/NADH and
therefore on BDH activity [101]. Failure to increase βOHB
metabolism under acoustic activation has also been demon-
strated using βOHB tracer [102].
There has been some speculation as to the role of the
malate aspartate shuttle when glycolysis is reduced during
ketone metabolism. The role of this shuttle is the transfer
of reducing equivalents between the cytosol and mitochon-
drion and it is required because the inner mitochondrial
membrane is impermeable to NADH and NAD+. In the
presence of βOHB, glycolysis is reduced resulting in less
requirement to transfer NADH to the mitochondrion, and
mitochondria are able to regenerate NADH via the mito-
chondrial BDH-catalysed reaction. This may be expected to
reduce the activity of both the cytosolic and mitochondrial
malate dehydrogenases (Fig. 2), with associated effects on
the movement of 2-oxoglutarate (α-ketoglutarate), a key
intermediate in the Krebs cycle but also a key metabolite
in cytosolic transaminase reactions. Further complicat-
ing matters, the metabolism of βOHB via SCOT generates
succinate from succinyl-CoA, which are also Krebs cycle
intermediates, but bypassing the succinylCo-A synthetase
step (Fig. 2).
βOHB Reduces Production of Reactive Oxygen Species
via Action at Complex II
The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
treated mouse is a model of Parkinson’s disease. MPTP
exerts its effects through blocking complex I (NADH
dehydrogenase) in mitochondria [103]. Mice, treated with
MPTP, were infused with βOHB which conferred partial
protection against the dopaminergic neurodegeneration and
motor deficits induced by MPTP. The authors suggested
that this effect was due to effects of βOHB on complex II
(succinate dehydrogenase), thus increasing mitochondrial
respiration and bypassing the complex I deficits [104].
This is because increased SCOT activity in metabolising
the βOHB drives increased production of succinate; the
authors showed that succinate was protective against MPTP
toxicity, although to a higher degree than βOHB, and that
this effect was not driven by increased NADH, since added
NADH had little effect. Investigation of this hypothesis
in C. elegans using worms with complex I or II defective
mutants however, suggested that βOHB required com-
plex I to exert its effects and that these were independent
of complex II [105] although they were also able to show
succinate and other TCA cycle intermediate dependent
effects. The finding of βOHB protection via Complex I is
13
Neurochem Res (2017) 42:35–49
also supported in purified mitochondria from G93A-SOD1
transgenic mice, a model of amyotrophic lateral sclerosis,
where βOHB was protective in maintaining ATP produc-
tion against a rotenone (Complex I inhibitor) challenge but
not against a complex II inhibitor, malonate [106].
Neurons treated with MPP+ (1-methyl-4-phenylpyridin-
ium) which produces a Parkinson’s disease like syndrome,
or with Aβ1-42, a fragment of amyloid protein which is
lethal to hippocampal cells and produces an Alzheimer’s
like condition, have been shown to be protected from
these toxic effects by pre-treatment with βOHB [107]. The
authors suggest that this effect is due to alteration in the
Q/QH2 couple, with resultant reduction in the capacity of
Complex II to produce reactive oxygen species while at the
same time relieving the product inhibition of Complex I.
The capability of βOHB to reduce production of reactive
oxygen species and prevent neuronal death was also dem-
onstrated in rats in a model of hypoglycaemia induced by
glucose deprivation [108].
βOHB Metabolism Alters Activity of K+-ATP Channels
Supply of βOHB along with glucose has been shown to
reduce the use of glucose in brain cellular preparations
[101, 109] and this is likely due to a reduction in glycolytic
activity, with addition of 1  mM βOHB to neurons in the
presence of glucose reducing glycolysis by around a third
[110]. The effect of 1 mM βOHB was further examined and
found to alter the response of NMDA receptors which dis-
played both a higher EC50 to NMDA and a lower maximal
calcium response in the presence vs the absence of βOHB,
but no effect on the release of neurotransmitter. By study-
ing responses induced by the KATP channel blocker gliben-
clamide and the KATP channel opener diazoxide the authors
concluded that these effects of βOHB were via indirect
effects on KATP channels. The presence of βOHB and the
resultant reduction in glycolytic activity and reduced avail-
ability of ATP would result in either a higher opening prob-
ability for KATP channels under these circumstances, or a
longer opening time [110]. Application of ketone bodies
at supra-physiological concentrations (0.4 mM βOHB) has
been shown to reduce the spontaneous firing rates of sub-
stantia nigra pars reticulate neurons, and to reduce the vul-
nerability of these neurons to a challenge that caused them
to fire faster. This effect was mediated by D-βOHB but not
by the inactive L-βOHB. Further, this protective effect on
the neurons was abolished when the KATP channels were
ablated, either through pharmacological approaches or
gene knockout [111]. The effect of ketone bodies on KATP
channels may be mediated via Bcl-2 associated agonist
of cell death (BAD) protein, which acts as a regulator of
glucose metabolism, including in brain [112]. Ablation of
BAD through knockout results in significantly increased
Neurochem Res (2017) 42:35–49
mitochondrial use of βOHB and reduction in use of glu-
cose. Knockout mice were also significantly more resistant
to kainite-induced seizures. This effect is likely mediated
through phosphorylation of the BH3 domain [112].
βOHB Metabolism Alters Activity of Ca2+ Channels
There is considerable evidence that βOHB causes elevation
in Ca2+. A study adding βOHB to glial cells showed dra-
matic and immediate elevations in cytosolic Ca2+ through a
mechanism involving L-type Ca2+ channels [113]. In sym-
pathetic neurons, βOHB has been shown to directly inter-
act with a G-protein coupled fatty acid receptor (GPR41;
FFA3) and to modulate sympathetic activity, although
some have reported it to be an antagonist at this receptor
[114] and others an agonist [115] with EC50 2.0 ± 1.2 mM.
This receptor (GPR41) is coupled to N-type (Cav2.2) Ca2+
channels and βOHB is capable of modulating these cur-
rents at concentrations which are achievable in vivo [115].
Acetoacetate, by contrast, has no effect.
βOHB is an Inhibitor of Histone Deacetylases HDAC 1,
3 and 4 at Concentrations Achievable In Vivo
βOHB has been shown to improve defence against oxida-
tive stress through its direct inhibition of histone deacety-
lase (HDAC) activity. βOHB inhibits HDAC1, HDAC3 and
HDAC4 with IC50s of 5.3, 2.4 and 5.4  mM, respectively.
This inhibition results in direct upregulation of genes in
the FOXO3A network, including catalase, mitochondrial
superoxide dismutase (Mn-SOD) and metallothionein 2.
The authors also treated mice with βOHB infusion for 24 h
with and challenged them with paraquat. They found sig-
nificantly reduced evidence of oxidative stress, measured
by lipid peroxidation and protein carbonylation, in kidney
from βOHB-treated compared to control mice [116]. βOHB
has also been shown to reduce lipoperoxidation caused by
three days of intrastriatal injections of glutamate in mice
[117].
βOHB Increases Lifespan in Worms and in Cultured
Cells
Supplementation with βOHB has been shown to extend the
lifespan of C. elegans by 20%. This was suggested to be
via two possible mechanisms. Firstly, via HDAC inhibition
through an AMPK and Sir2-dependent effect (Sir2 is the C.
elegans equivalent of the mammalian SIRT1), or through
increasing the concentration of acetyl-CoA, again resulting
in increased histone acetylation [105]. Secondly the effects
of βOHB may be mediated through inhibition of the insulin
signalling pathway. In mice, administration of βOHB has
been shown to yield a 50% reduced phosphorylation and
43
activity of Akt/protein kinase B downstream of the insulin
receptor, decreasing insulin signalling [118].
Extension of life span in glial cells has also been demon-
strated following addition of 5 or 10 mM βOHB with sig-
nificant reductions in apoptosis in the presence of βOHB.
This effect was greater using methyl-βOHB, which has
improved membrane permeability compared to βOHB
[113].
βOHB Reduces Neuroinflammation Generated
by Immune Cells
βOHB has been reported to reduce inflammation gener-
ated by macrophages via a mechanism independent of
any of those reported above [119]. The NLR family, pyrin
domain-containing 3 (NLRP3) inflammasome is a multi-
protein complex that controls the activation of caspase-1
and the release of the pro-inflammatory cytokines IL-1β
and IL-18 in macrophages [120]. This inflammasome,
which is expressed mainly in immune cells [121], is acti-
vated by a shift to low cytoplasmic K+ levels. Youm et al.
noted that βOHB prevented the decline in K+ levels and
prevented activation of NLRP3. They worked thoroughly
through a list of known βOHB mechanisms to see if these
were responsible for preventing inflammasome activation.
They were able to rule them all out but did not identify
what mechanism actually was responsible [119]. Ca2+ sig-
nalling was not tested as a possible cause although the Ca2+
sensing receptor in mice has been shown to be involved in
NLRP3 activation [122].
Summary of Metabolic Effects of βOHB
• βOHB is taken up in to the brain via the monocarboxy-
late transporter. Brain uptake can be a metabolic con-
trol point. βOHB can also be produced endogenously in
brain by astrocytes which have the capacity to metabo-
lise free fatty acids.
• βOHB is a high energy substrate and its addition to
mitochondria will alter the NAD+/NADH and Q/QH2
couples. Glycolysis is reduced in the presence of βOHB
and mitochondria have less capacity to generate reactive
oxygen species from Complex II.
• βOHB can bind to BAD protein and influence the open-
ing of K+ channels. This effect may also be exacerbated
under metabolically active conditions by reduced glyco-
lysis, leading to reduced cytosolic ATP to power the K+
channels.
• βOHB is an agonist at free fatty acid receptor GPR41,
directly modulating the activity of N-type Ca2+ chan-
nels.
13
44
• βOHB is an inhibitor of histone deacetylases HDAC
1, 3 and 4 at concentrations achievable in  vivo. This
inhibition results in direct upregulation of genes in the
FOXO3A network, including catalase, mitochondrial
superoxide dismutase (Mn-SOD) and metallothionein 2.
• βOHB has specific anti-inflammatory effects on NLRP3
inflammasome via a direct but as yet unidentified mech-
anism.
Measurement of βOHB in Brain Using Magnetic
Resonance Spectroscopy
βOHB is detectable in human and rat brain in vivo. Under
normal circumstances, the concentrations of βOHB in
brain are very low and difficult to detect reliably using
standard, or even edited, magnetic resonance spectros-
copy. The main reported confound is in confusing the
methyl resonance from βOHB with that from lactate.
Lactate is chemically similar with a methyl resonance
linked to an hydroxymethyl group, but the two can be
resolved at field strengths as low as 1.5 T (Fig. 3). βOHB
has been measured in human brain using a range of dif-
ferent sequences. Early attempts to measure brain βOHB
in diabetes mellitus used short-echo STEAM (STimu-
lated Echo, Acquisition Mode; [123]) spectroscopy
(TE = 30  ms) at 1.5  T and detected peaks arising from
acetoacetate and acetone but not βOHB [124]. At 1.5  T
the methyl peaks from βOHB and lactate overlap con-
siderably and the presence of significant lipid or macro-
molecule resonances at short echo times can also make
it difficult to resolve the βOHB doublet from the back-
ground resonance; this is a similar issue to that found
when trying to measure lactate levels using short-echo
spectroscopy. Others have detected βOHB at 3  T using
Fig. 3 High field section of
1
H magnetic resonance spectra
of βOHB and lactate at 3 T.
Spectra were generated using
NMRScope in jMRUI (v 4,
build 162) using published
coupling constants and chemi-
cal shifts and a PRESS (Point
RESolved Spectroscopy)
sequence (TE = 144 ms)
13
Neurochem Res (2017) 42:35–49
short-echo (TE = 20 ms) STEAM in infants supplemented
with DL-βOHB [125].
In 1998, an editing method was published to allow
resolution of the methyl doublets from lactate and βOHB
from background lipid signal, and used to measure brain
levels of these compounds in epilepsy both before and
after treatment with a ketogenic diet. The authors suc-
cessfully detected both lactate and βOHB at 2.1  T; no
βOHB was detected in the patient with epilepsy at base-
line but a significant elevation in this compound was
noted after implementation of a ketogenic diet [126].
This method, which used ISIS for localisation [127],
was further modified by this group and used to detect
βOHB in healthy humans and following fasting-induced
ketosis [15]. The authors were able to quantify the βOHB
with a reasonable reliability.
In the absence of access to editing methods, others
have detected βOHB in  vivo in diabetic ketosis using
single voxel PRESS (Point RESolved Spectroscopy)
with a long (144  ms) echo-time; this allows full inver-
sion of the βOHB doublet and removes any contaminat-
ing lipid signal due to relaxation [128]. A short echo
(TE = 35  ms, plus a further TE = 144  ms acquisition)
PRESS method has also been used to detect βOHB fol-
lowing a ketogenic diet, recovering the βOHB signal by
fitting the spectrum with a basis set of known priors,
including βOHB [129].
The editing sequence MEGA-PRESS [130] has been
adapted for measurement of lactate in  vivo [131] and
could also be used successfully to measure βOHB with
improved signal to noise over ISIS localisation methods.
In summary, βOHB is difficult to detect reliably in
healthy human brain and is best measured using an edit-
ing sequence to resolve the βOHB resonances from back-
ground signals.
Neurochem Res (2017) 42:35–49
Therapeutic Effects of βOHB
Much of the literature about the effects of ketone bodies
on disease outcomes is focused on the ketogenic diet, mak-
ing it difficult to divorce the effects of βOHB alone from
the more complicated systems effects induced by this diet.
Here, we examine the literature where βOHB alone has
been used as a potential therapeutic.
The main problem with using βOHB as a therapeutic lies
in being able to deliver sufficient βOHB to the brain, due to
the limited uptake of βOHB across the blood brain barrier
and the difficulty in sustaining high enough levels of βOHB
in the blood. As detailed above, uptake can be increased by
instigation of dietary changes, such as a high fat diet or reg-
ular ingestion of medium chain fatty acids. Ketogenic diets,
which can raise plasma βOHB to higher levels, are difficult
to adhere to, both for those consuming them and for their
care-givers. Medium chain triacylglycerols, on the other
hand, can be consumed along with the usual diet, making
them more attractive. Consumption of medium chain tria-
cylglycerols produces a mild ketosis which has been esti-
mated to contribute up to 8–9% to brain energy metabolism
and it is well tolerated [132].
The effect was tested of a one-off drink of medium chain
triglycerides on the cognition of 20 adults with impaired
cognition (diagnosed with Alzheimer’s disease or mild
cognitive impairment) compared to consumption of a pla-
cebo on a separate day. βOHB levels were measured in
blood subsequent to consumption. The authors reported
a different response in cognitive performance and blood
βOHB levels depending on the subjects ApoE status; those
with the APOE-ε4 allele responded to MCT consump-
tion with continually increasing plasma βOHB levels, no
improvement on the Alzheimer disease assessment scale
cognitive subscale (ADAS-cog) compared to plateauing of
plasma βOHB levels and improvement on the ADAS-cog
in those without APOE-ε4 allele. All subjects performance
on paragraph recall improved with MCT consumption but
performance on the Stroop colour word test, which meas-
ures selective attention, was unchanged [133]. This study
had only 20 participants, meaning that statistical power was
limited, especially after subdividing the group by APOE
status.
Recently, attention has been given to esterified versions
of βOHB, such as (R)-3-hydroxybutyl (R)-3-hydroxybu-
tyrate. This compound can be consumed with the normal
diet and is hydrolysed completely in the small intestine
producing βOHB and 1,3-butanediol which is converted
to βOHB and acetoacetate in the liver, producing a “thera-
peutic ketosis”. This method results in blood βOHB levels
higher than those achieved after consuming medium chain
fatty acids and is well tolerated [134]. Supplementation
with (R)-3-hydroxybutyl (R)-3-hydroxybutyrate has shown
45
promise in a case of Alzheimer disease [135] and it is plain
that the use of ketone bodies such as (R)-3-hydroxybutyl
(R)-3-hydroxybutyrate deserves further investigation [136].
βOHB has been used experimentally in rats to try and
prevent blood brain barrier disruption due to traumatic
brain injury. βOHB, administered by infusion prior to
injury, not only failed to provide protection against blood
brain barrier disruption in brain injured rats but it caused
blood brain barrier disruption in healthy animals [137]
resulting in decreased GSH and malondialdehyde levels
and reduced glucose transporter (GLUT1) expression. GSH
levels are maintained by reduction with NADPH, which is
mostly produced via the pentose phosphate pathway [68].
Although it might be expected that NADPH levels might be
altered if glycolytic activity is reduced, with concomitant
reduction in GSH, the ketogenic diet has been shown to
increase GSH levels through increased GSH synthesis and
reduction in production of reactive oxygen species [138].
In this regard, it should be borne in mind that acute and
chronic exposure to βOHB have different outcomes. Indeed
exposure to a ketogenic diet after contusion injury in rats
has been shown to reduce contusion volume and improve
neuronal survival, with better responses in younger rats
[139].
Conclusion
βOHB is a high energy metabolite that has significant met-
abolic impact and, if well understood, could be used as a
well-tolerated therapeutic agent in a range of neurodegen-
erative, inflammatory and traumatic brain disorders.
Acknowledgements The authors would like to thank Ben Rowlands
of Neuroscience Research Australia and Don Thomas of the UNSW
Mark Wainwright Analytical Centre for their assistance in the prepa-
ration of this manuscript. The authors would like to acknowledge the
lifetime commitment of Professor Mary McKenna to the pursuit of
scientific excellence and education in neurochemistry and to dedicate
this article to her contributions to this field. We have greatly enjoyed
and also benefitted from our interactions over the years.
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