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Effects of exogenous ketone supplementation on blood ketone, glucose, triglyceride, and lipoprotein levels in Sprague–Dawley rats


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Nutritional ketosis induced by the ketogenic diet (KD) has therapeutic applications for many disease states. We hypothesized that oral administration of exogenous ketone supplements could produce sustained nutritional ketosis (>0.5 mM) without carbohydrate restriction. We tested the effects of 28-day administration of five ketone supplements on blood glucose, ketones, and lipids in male Sprague–Dawley rats. The supplements included: 1,3-butanediol (BD), a sodium/potassium β-hydroxybutyrate (βHB) mineral salt (BMS), medium chain triglyceride oil (MCT), BMS + MCT 1:1 mixture, and 1,3 butanediol acetoacetate diester (KE). Rats received a daily 5–10 g/kg dose of their respective ketone supplement via intragastric gavage during treatment. Weekly whole blood samples were taken for analysis of glucose and βHB at baseline and, 0.5, 1, 4, 8, and 12 h post-gavage, or until βHB returned to baseline. At 28 days, triglycerides, total cholesterol and high-density lipoprotein (HDL) were measured. Exogenous ketone supplementation caused a rapid and sustained elevation of βHB, reduction of glucose, and little change to lipid biomarkers compared to control animals. This study demonstrates the efficacy and tolerability of oral exogenous ketone supplementation in inducing nutritional ketosis independent of dietary restriction.
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R E S E A R C H Open Access
Effects of exogenous ketone
supplementation on blood ketone, glucose,
triglyceride, and lipoprotein levels in
SpragueDawley rats
Shannon L. Kesl
, Angela M. Poff
, Nathan P. Ward
, Tina N. Fiorelli
, Csilla Ari
, Ashley J. Van Putten
Jacob W. Sherwood
, Patrick Arnold
and Dominic P. DAgostino
Background: Nutritional ketosis induced by the ketogenic diet (KD) has therapeutic applications for many disease
states. We hypothesized that oral administration of exogenous ketone supplements could produce sustained
nutritional ketosis (>0.5 mM) without carbohydrate restriction.
Methods: We tested the effects of 28-day administration of five ketone supplements on blood glucose, ketones,
and lipids in male SpragueDawley rats. The supplements included: 1,3-butanediol (BD), a sodium/potassium β-
hydroxybutyrate (βHB) mineral salt (BMS), medium chain triglyceride oil (MCT), BMS + MCT 1:1 mixture, and 1,3
butanediol acetoacetate diester (KE). Rats received a daily 510 g/kg dose of their respective ketone supplement via
intragastric gavage during treatment. Weekly whole blood samples were taken for analysis of glucose and βHB at
baseline and, 0.5, 1, 4, 8, and 12 h post-gavage, or until βHB returned to baseline. At 28 days, triglycerides, total
cholesterol and high-density lipoprotein (HDL) were measured.
Results: Exogenous ketone supplementation caused a rapid and sustained elevation of βHB, reduction of glucose,
and little change to lipid biomarkers compared to control animals.
Conclusions: This study demonstrates the efficacy and tolerability of oral exogenous ketone supplementation in
inducing nutritional ketosis independent of dietary restriction.
Keywords: Ketogenic diet, Ketone ester, Ketone supplement, Appetite, β-hydroxybutyrate, Hyperketonemia,
Emerging evidence supports the therapeutic potential of
the ketogenic diet (KD) for a variety of disease states,
leading investigators to research methods of harnessing
the benefits of nutritional ketosis without the dietary
restrictions. The KD has been used as an effective non-
pharmacological therapy for pediatric intractable sei-
zures since the 1920s [13]. In addition to epilepsy, the
ketogenic diet has elicited significant therapeutic effects
for weight loss and type-2 diabetes (T2D) [4]. Several
studies have shown significant weight loss on a high fat,
low carbohydrate diet without significant elevations of
serum cholesterol [512]. Another study demonstrated
the safety and benefits of long-term application of the
KD in T2D patients. Patients exhibited significant weight
loss, reduction of blood glucose, and improvement of
lipid markers after eating a well-formulated KD for
56 weeks [13]. Recently, researchers have begun to
investigate the use of the KD as a treatment for acne,
polycystic ovary syndrome (PCOS), cancer, amyotrophic
lateral sclerosis (ALS), traumatic brain injury (TBI) and
Alzheimers disease (AD) with promising preliminary
results [1426].
* Correspondence:
Department of Molecular Pharmacology and Physiology, Morsani College of
Medicine, University of South Florida, 12901 Bruce B. Downs Blvd. MDC8,
Tampa, FL 33612, USA
Full list of author information is available at the end of the article
© 2016 Kesl et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (, which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
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Kesl et al. Nutrition & Metabolism (2016) 13:9
DOI 10.1186/s12986-016-0069-y
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The classical KD consists of a 4:1 ratio of fat to pro-
tein and carbohydrate, with 8090 % of total calories de-
rived from fat [27]. The macronutrient ratio of the KD
induces a metabolic shift towards fatty acid oxidation
and hepatic ketogenesis, elevating the ketone bodies
acetoacetate (AcAc) and β-hydroxybutyrate (βHB) in the
blood. Acetone, generated by decarboxylation of AcAc, has
been shown to have anticonvulsant properties [2832].
Ketone bodies are naturally elevated to serve as alternative
metabolic substrates for extra-hepatic tissues during the
prolonged reduction of glucose availability, suppression of
insulin, and depletion of liver glycogen, such as occurs
during starvation, fasting, vigorous exercise, calorie
restriction, or the KD. Although the KD has clear
therapeutic potential, several factors limit the efficacy
and utility of this metabolic therapy for widespread
clinical use. Patient compliance to the KD can be low
due to the severe dietary restriction - the diet being
generally perceived as unpalatable - and intolerance to
high-fat ingestion. Maintaining ketosis can be difficult
as consumption of even a small quantity of carbohydrates
or excess protein can rapidly inhibit ketogenesis [33, 34].
Furthermore, enhanced ketone body production and tissue
utilization by the tissues can take several weeks (keto-adap-
tation), and patients may experience mild hypoglycemic
symptoms during this transitional period [35].
Recent studies suggest that many of the benefits of the
KD are due to the effects of ketone body metabolism.
Interestingly, in studies on T2D patients, improved gly-
cemic control, improved lipid markers, and retraction of
insulin and other medications occurred before weight
loss became significant. Both βHB and AcAc have been
shown to decrease mitochondrial reactive oxygen species
(ROS) production [3639]. Veech et al. have summarized
the potential therapeutic uses for ketone bodies [28, 40].
They have demonstrated that exogenous ketones favorably
alter mitochondrial bioenergetics to reduce the mitochon-
drial NAD couple, oxidize the co-enzyme Q, and increase
the ΔG(free enthalpy) of ATP hydrolysis [41]. Ketone
bodies have been shown to increase the hydraulic effi-
ciency of the heart by 28 %, simultaneously decreasing
oxygen consumption while increasing ATP production
[42]. Thus, elevated ketone bodies increase metabolic effi-
ciency and as a consequence, reduce superoxide production
and increase reduced glutathione [28]. Sullivan et al. demon-
strated that mice fed a KD for 1012 days showed increased
hippocampal uncoupling proteins, indicative of decreased
mitochondrial-produced ROS [43]. Bough et al. showed an
increase of mitochondrial biogenesis in rats maintained on a
KD for 46 weeks [44, 45]. Recently, Shimazu et al. reported
that βHB is an exogenous and specific inhibitor of class I
histone deacetylases (HDACs), which confers protection
against oxidative stress [38]. Ketone bodies have also been
shown to suppress inflammation by decreasing the
inflammatory markers TNF-a, IL-6, IL-8, MCP-1, E-selectin,
I-CAM, and PAI-1 [8, 46, 47]. Therefore, it is thought that
ketone bodies themselves confer many of the benefits asso-
ciated with the KD.
Considering both the broad therapeutic potential and
limitations of the KD, an oral exogenous ketone supple-
ment capable of inducing sustained therapeutic ketosis
without the need for dietary restriction would serve as a
practical alternative. Several natural and synthetic ketone
supplements capable of inducing nutritional ketosis have
been identified. Desrochers et al. elevated ketone bodies
in the blood of pigs (>0.5 mM) using exogenous ketone
supplements: (R, S)-1,3 butanediol and (R, S)-1,3
butanediol-acetoacetate monoesters and diester [48]. In
2012, Clarke et al. demonstrated the safety and efficacy
of chronic oral administration of a ketone monoester of
R-βHB in rats and humans [49, 50]. Subjects maintained
elevated blood ketones without dietary restriction and
experienced little to no adverse side effects, demonstrating
the potential to circumvent the restrictive diet typically
needed to achieve therapeutic ketosis. We hypothesized that
exogenous ketone supplements could produce sustained
hyperketonemia (>0.5 mM) without dietary restriction and
without negatively influencing metabolic biomarkers, such
as blood glucose, total cholesterol, HDL, LDL, and triglycer-
ides. Thus, we measured these biomarkers during a 28-day
administration of the following ketone supplements in rats:
naturally-derived ketogenic supplements included medium
chain triglyceride oil (MCT), sodium/potassium -βHB
mineral salt (BMS), and sodium/potassium -βHB mineral
salt + medium chain triglyceride oil 1:1 mixture (BMS +
MCT) and synthetically produced ketogenic supplements
included 1, 3-butanediol (BD), 1, 3-butanediol acetoace-
tate diester/ ketone ester (KE).
Synthesis and formulation of ketone supplements
KE was synthesized as previously described [29]. BMS is
a novel agent (sodium/potassium- βHB mineral salt)
supplied as a 50 % solution containing approximately
375 mg/g of pure βHB and 125 mg/g of sodium/potas-
sium. Both KE and BMS were developed and synthesized
in collaboration with Savind Inc. Pharmaceutical grade
MCT oil (~65 % caprylic triglyceride; 45 % capric trigly-
ceride) was purchased from Now Foods (Bloomingdale,
IL). BMS was formulated in a 1:1 ratio with MCT at the
University of South Florida (USF), yielding a final mix-
ture of 25 % water, 25 % pure βHB mineral salt and
50 % MCT. BD was purchased from Sigma-Aldrich
(Prod # B84785, Milwaukee, WI).
Daily gavage to induce dietary ketosis
Animal procedures were performed in accordance with
the University of South Florida Institutional Animal
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 2 of 15
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Care and Use Committee (IACUC) guidelines (Protocol
#0006R). Juvenile male SpragueDawley rats (275325 g,
Harlan Laboratories) were randomly assigned to one of
six study groups: control (water, n=11), BD (n=11), KE
(n= 11), MCT (n= 10), BMS (n= 11), or BMS + MCT
(n= 12). Caloric density of standard rodent chow and
dose of ketone supplements are listed in Table 1. On
days 114, rats received a 5 g/kg body weight dose of
their respective treatments via intragastric gavage.
Dosage was increased to 10 g/kg body weight for the
second half of the study (days 1528) for all groups
except BD and KE to prevent excessive hyperketone-
mia (ketoacidosis). Each daily dose of BMS would equal
~10001500 mg of βHB, depending on the weight of the
animal. Intragastric gavage was performed at the same time
daily, and animals had ad libitum access to standard
rodent chow 2018 (Harlan Teklad) for the duration of
the study. The macronutrient ratio the standard rodent
chow was 62.2, 23.8 and 14 % of carbohydrates, protein
and fat respectively.
Measurement and analysis of blood glucose, ketones,
and lipids
Every 7 days, animals were briefly fasted (4 h, water
available) prior to intragastric gavage to standardize
levels of blood metabolites prior to glucose and βHB
measurements at baseline. Baseline (time 0) was imme-
diately prior to gavage. Whole blood samples (10 μL)
were taken from the saphenous vein for analysis of
glucose and βHB levels with the commercially avail-
able glucose and ketone monitoring system Precision
Xtra(Abbott Laboratories, Abbott Park, IL). Blood
glucose and βHB were measured at 0, 0.5, 1, 4, 8,
and 12 h after test substance administration, or until
βHB returned to baseline levels. Food was returned to
animals after blood analysis at time 0 and gavage. At
baseline and week 4, whole blood samples (10 μL)
were taken from the saphenous vein immediately
prior to gavage (time 0) for analysis of total choles-
terol, high-density lipoprotein (HDL), and triglycerides
with the commercially available CardioChekblood
lipid analyzer (Polymer Technology Systems, Inc., In-
dianapolis, IN). Low-density lipoprotein (LDL)
cholesterol was calculated from the three measured
lipid levels using the Friedewald equation: (LDL Choles-
terol = Total Cholesterol - HDL - (Triglycerides/5)) [51,
52]. Animals were weighed once per week to track
changes in body weight associated with hyperketonemia.
Organ weight and collection
On day 29, rats were sacrificed via deep isoflurane
anesthesia, exsanguination by cardiac puncture, and de-
capitation 48 h after intragastric gavage, which correlated
to the time range where the most significantly elevated
blood βHB levels were observed. Brain, lungs, liver,
kidneys, spleen and heart were harvested, weighed (AWS-
1000 1 kg portable digital scale (AWS, Charleston, SC)),
and flash-frozen in liquid nitrogen or preserved in 4 %
paraformaldehyde for future analysis.
All data are presented as the mean ± standard deviation
(SD). Data analysis was performed using GraphPad
PRISMversion 6.0a and IBM SPSS Statistics 22.0. Re-
sults were considered significant when p < 0.05. Trigly-
ceride and lipoprotein profile data were analyzed using
One-Way ANOVA. Blood ketone and blood glucose
were compared to control at the applicable time points
using a Two-Way ANOVA. Correlation between blood
βHB and glucose levels in ketone supplemented rats was
compared to controls using ANCOVA analysis. Organ
and body weights were analyzed using One-Way
ANOVA. Basal blood ketone and blood glucose levels
were analyzed using Two-Way ANOVA. All mean com-
parisons were carried out using Tukeys multiple com-
parisons post-hoc test.
Effect of ketone supplementation on triglycerides and
Baseline measurements showed no significant changes
in triglycerides or the lipoproteins (data not shown).
Data represent triglyceride and lipoprotein concentra-
tions measured after 4 weeks of daily exogenous ketone
supplementation. No significant change in total choles-
terol was observed at 4 weeks for any of the ketone
treatment groups compared to control. (Fig. 1a). No
Table 1 Caloric density and dose of ketone supplements
Macronutrient Information Standard Diet Water BMS + MCT BMS MCT KE BD
% Cal from Fat 18.0 0.0 50.0 N/A 100.0 N/A N/A
% Cal from Protein 24.0 0.0 N/A N/A 0.0 N/A N/A
% Cal from Carbohydrates 58.0 0.0 N/A N/A 0.0 N/A N/A
Total Caloric Density (Kcal/g) 3.1 0.0 5.1 1.9 8.3 5.6 6.0
Dose 014 Days (g/kg) ad libitum N/A 5.0 5.0 5.0 5.0 5.0
Dose 1528 Days (g/kg) ad libitum N/A 10.0 10.0 10.0 5.0 5.0
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 3 of 15
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significant difference was detected in triglycerides for
any ketone supplement compared to control (Fig. 1b).
MCT supplemented animals had a significant reduction
in HDL blood levels compared to control (p < 0.001)
(Fig. 1c). LDL levels in ketone-supplemented animals did
not significantly differ from controls (Fig. 1d).
Ketone supplementation causes rapid and sustained
elevation of βHB
Over the 28-day experiment, ketone supplements ad-
ministered daily significantly elevated blood ketone
levels without dietary restriction (Fig. 2a, b). Naturally
derived ketogenic supplements including MCT (5 g/kg)
elicited a significant rapid elevation in blood βHB within
3060 min that was sustained for 8 h. BMS + MCT (5 g/
kg) elicited a significant elevation in blood βHB at 4 h,
which was no longer significant at 8 h. BMS (5 g/kg) did
not elicit a significant elevation in blood βHB at any
time point. For days 1428, BMS + MCT (10 g/kg) and
MCT (10 g/kg) elevated blood βHB levels within 30 min
and remained significantly elevated for up to 12 h. We
observed a delay in the peak elevation of blood βHB:
BMS + MCT peaked at 8 h instead of at 4 h and MCT at
4 h instead of at 1 h. Blood βHB levels in the BMS group
did not show significant elevation at any time point,
even after dose escalation (Fig. 2a). Synthetically derived
ketogenic supplements including KE and BD supple-
mentation rapidly elevated blood βHB within 30 min
and was sustained for 8 h. For the rats receiving ketone
supplementation in the form of BD or the KE, dosage
was kept at 5 g/kg to prevent adverse effects associated
with hyperketonemia. The Precision Xtraketone moni-
toring system measures βHB only; therefore, total blood
ketone levels (βHB + AcAc) would be higher than mea-
sured. For each of these groups, the blood βHB profile
remained consistent following daily ketone supplementa-
tion administration over the 4-week duration. (Fig. 2b).
Ketone supplementation causes a significant decrease of
blood glucose
Administration of ketone supplementation significantly
reduced blood glucose over the course of the study
(Fig. 3a, b). MCT (5 g/kg) decreased blood glucose com-
pared to control within 30 min which was sustained for
Fig. 1 Effects of ketone supplementation on triglycerides and lipoproteins: Ketone supplementation causes little change in triglycerides and
lipoproteins over a 4-week study. Graphs show concentrations at 4-weeks of total cholesterol (a), Triglycerides (b), LDL (c), and HDL (d).
MCT supplemented rats had signfiicantly reduced concentration of HDL blood levels compared to control (p< 0.001) (b). One-Way ANOVA
with Tukeys post hoc test, results considered significant if p< 0.05. Error bars represent mean (SD)
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 4 of 15
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Fig. 2 Effects of ketone supplementation on blood βHB. a,bBlood βHB levels at times 0, 0.5, 1, 4, 8, and 12 h post intragastric gavage for ketone
supplements tested. aBMS + MCT and MCT supplementation rapidly elevated and sustained significant βHB elevation compared to controls for
the duration of the 4-week dose escalation study. BMS did not significantly elevate βHB at any time point tested compared to controls. bBD and
KE supplements, maintained at 5 g/kg, significantly elevated βHB levels for the duration of the 4-week study. Two-Way ANOVA with Tukeys post
hoc test, results considered significant if p< 0.05. Error bars represent mean (SD)
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 5 of 15
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Fig. 3 Effects of ketone supplementation on blood glucose. a,bBlood glucose levels at times 0, 0.5, 1, 4, 8, and 12 h (for 10 dose) post intragastric
gavage for ketone supplements tested. aKetone supplements BMS + MCT and MCT significantly reduced blood glucose levels compared to controls
for the duration of the 4-week study. BMS significantly lowered blood glucose only at 8 h/week 1 and 12 h/week 3 (b) KE, maintained at
5 g/kg, significantly reduced blood glucose compared to controls from week 14. BD did not significantly affect blood glucose levels at
any time point during the 4-week study. Two-Way ANOVA with Tukeys post hoc test, results considered significant if p< 0.05. Error bars
represent mean (SD)
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8 h at baseline and at week 1. MCT (10 g/kg) likewise
decreased blood glucose within 30 min and lasted
through the 12 h time point during weeks 2, 3, and 4.
BMS + MCT (5 g/kg) lowered blood glucose compared
to control from hours 18 only at week 1. BMS + MCT
(10 g/kg) lowered blood glucose compared to control
within 30 min and remained low through the 12 h time
point at weeks 2, 3, and 4. Rats supplemented with BMS
had lower blood glucose compared to control at 12 h in
week 4 (10) (Fig. 3a). Administration of BD did not sig-
nificantly change blood glucose levels at any time point
during the 4-week study. KE (5 g/kg) significantly low-
ered blood glucose levels at 30 min for week 1, 2, 3, and
4 and was sustained through 1 h at weeks 24 and sus-
tained to 4 h at week 3. (Fig. 3b).
Hyperketonemia suppresses blood glucose levels
At baseline, 4 h after intragastric gavage, the elevation of
blood ketones was inversely related to the reduction of blood
glucose compared to controls following the administration
of MCT (5 g/kg) (p = 0.008) and BMS + MCT (5 g/kg) (p =
0.039) . There was no significant correlation between blood
ketone levels and blood glucose levels compared to controls
for any other ketone supplemented group at baseline
significant correlation between blood ketone levels and
Fig. 4 Relationship between blood ketone and glucose levels: aBMS + MCT (5 g/kg) supplemented rats demonstrated a significant inverse relationship
between elevated blood ketone levels and decreased blood ketone levels (r
= 0.4314, p = 0.0203). bAt week 4, BMS + MCT (10 g/kg) and MCT (10 g/kg)
showed a significant correlation between blood ketone levels and blood glucose levels (r2 = 0.8619, p< 0.0001; r
= 0.6365, p= 0.0057). Linear regression
analysis, results considered significant if p<0.05
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 7 of 15
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blood glucose levels compared to controls in MCT (10 g/kg)
and BMS + MCT (10 g/kg) (p< 0.0001, p< 0.0001) (Fig. 4b).
Ketone supplementation changes organ weight and
decreases body weight
At day 29 of the study, animals were euthanized and
brain, lungs, liver, kidneys, spleen and heart were har-
vested and weighed. Organ weights were normalized to
body weight. Ketone supplementation did not signifi-
cantly change brain, lung, kidney, or heart weights
compared to controls (Fig. 5a, b, d, f). MCT supple-
mented animals had significantly larger livers compared
to their body weight (p< 0.05) (Fig. 5c). Ketone supple-
ments BMS + MCT, MCT and BD caused a significant
reduction in spleen size (BMS + MCT p< 0.05, MCT p
< 0.001, BD p< 0.05) (Fig. 5e). Rats administered KE
gained significantly less weight over the entire study
compared to controls. BMS + MCT, BMS, and BD sup-
plemented rats gained significantly less weight than con-
trols during weeks 2 4, and MCT animals gained less
Fig. 5 Effects of ketone supplementation on organ weight: Data is represented as a percentage of organ weight to body weight. a,b,d,f
Ketone supplements did not significantly affect the weight of the brain, lungs, kidneys or heart. cLiver weight was significantly increased as
compared to body weight in response to administered MCT ketone supplement compared to control at the end of the study (day 29) (p< 0.001).
eRats supplemented with BMS+ MCT, MCT, and BD had significantly smaller spleen percentage as compared to controls (p<0.05,p< 0.001, p< 0.05).
Two-Way ANOVA with Tukeys post-hoc test; results considered significant if p< 0.05. Error bars represent mean (SD)
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 8 of 15
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weight than controls at weeks 3 4 (Fig. 6). Increased
gastric motility (increased bowel evacuation and changes
to fecal consistency) was visually observed in rats sup-
plemented with 10 g/kg MCT, most notably at the 8 and
12-h time points. All animals remained in healthy weight
range for their age even though the rate of weight gain
changed with ketone supplementation [5354]. Food in-
take was not measured in this study. However, there was
not a significant change in basal blood glucose or basal
blood ketone levels over the 4 week study in any of the
rats supplemented with ketones (Fig. 7).
Nutritional ketosis induced with the KD has proven
effective for the metabolic management of seizures and
potentially other disorders [126]. Here we present
evidence that chronic administration of ketone supple-
ments can induce a state of nutritional ketosis without
the need for dietary carbohydrate restriction and with
little or no effect on lipid biomarkers. The notion that
we can produce the therapeutic effects of the KD with
exogenous ketone supplementation is supported by our
previous study which demonstrated that acutely admin-
istered KE supplementation delays central nervous
system (CNS) oxygen toxicity seizures without the
need for dietary restriction [29]. We propose that
exogenous ketone supplementation could provide an
alternative method of attaining the therapeutic
benefits of nutritional ketosis, and as a means to
further augment the therapeutic potential of the KD.
Ketone supplementation causes little to no change in
triglycerides and lipoproteins
One common concern regarding the KD is its purported
potential to increase the risk of atherosclerosis by elevat-
ing blood cholesterol and triglyceride levels [55, 56]. This
topic remains controversial as some, but not all, studies
have demonstrated that the KD elevates blood levels of
cholesterol and triglycerides [5762]. Kwitervich and col-
leagues demonstrated an increase in low-density lipopro-
tein (LDL) and a decrease in high-density lipoprotein
(HDL) in epileptic children fed the classical KD for
two years [27]. In this study, total cholesterol in-
creased by ~130 %, and stabilized at the elevated level
over the 2-year period. A similar study demonstrated that
the lipid profile returned to baseline in children who
remained on the KD for six years [63]. Children typically
remain on the diet for approximately two years then re-
turn to a diet of common fat and carbohydrate ingestion
[64]. The implications of these findings are unclear, since
the influence of cholesterol on cardiovascular health is
controversial and macronutrient sources of the diet vary
per study. In contrast to these studies, the majority of re-
cent studies have suggested that the KD can actually lead
to significant benefits in biomarkers of metabolic health,
including blood lipid profiles [6572]. In these studies, the
Fig. 6 Effects of ketone supplementation on body weight: Rats administered ketone supplements gained less weight over the 4-week period;
however, did not lose weight and maintained healthy range for age. KE supplemented rats gained significantly less weight during the entire
4-week study compared to controls. BMS +MCT, BMS, and BD supplemented rats gained significantly less weight than controls over weeks 24.MCT
supplemented rats gained significantly less weight than controls over weeks 34, Two-Way ANOVA with Tukeys post hoc test, results considered
significant if p< 0.05. Error bars represent mean (SD)
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KD positively altered blood lipids, decreasing total triglyc-
erides and cholesterol while increasing the ratio of HDL
to LDL [6877]. Although, the KD is well-established in
children, it has only recently been utilized as a strategy to
control seizures in adults. In 2014, Schoeler and col-
leagues reported on the feasibility of the KD for adults,
concluding that 39 % of individuals achieved > 50 % reduc-
tion in seizure frequency, similar to the results reported in
pediatric studies. Patients experienced similar gastrointes-
tinal adverse advents that have been previously described
in pediatric patients, but they did not lead to discontinu-
ation of the diet in any patient [78].
With oral ketone supplementation, we observed a sig-
nificant elevation in blood βHB without dietary restric-
tion and with little change in lipid biomarkers (Fig. 1).
Over the 4 week study, MCT-supplemented rats demon-
strated decreased HDL compared to controls. No signifi-
cant changes were observed in any of the triglycerides or
lipoproteins (HDL, LDL) with any of the remaining ex-
ogenously applied ketone supplements. It should be
noted that the rats used for this study had not yet
reached full adult body size [79]. Their normal growth
rate and maturation was likely responsible for the
changes in triglyceride and lipoprotein levels observed in
the control animals over the 4 week study (baseline data
not shown, no significant differences) [80, 81]. Future
studies are needed to investigate the effect of ketone
supplementation on fully mature and aged animals.
Overall, our study suggests that oral ketone supplemen-
tation has little effect on the triglyceride or lipoprotein
profile after 4 weeks. However, it is currently unknown if
ketone supplementation would affect lipid biomarkers
after a longer duration of consumption. Further studies
are needed to determine the effects of ketone supplements
on blood triglyceride and lipoproteins after chronic
administration and as a means to further enhance the
hyperketonemia and improve the lipid profile of the clinic-
ally implemented (4:1) KD.
LDL is the lipoprotein particle that is most often associ-
ated with atherosclerosis. LDL particles exist in different
sizes: large molecules (Pattern A) or small molecules
(Pattern B). Recent studies have investigated the im-
portance of LDL-particle type and size rather than
total concentration as being the source for cardiovascular
risk [56]. Patients whose LDL particles are predominantly
small and dense (Pattern B) have a greater risk of
Fig. 7 Effects of ketone supplementation on basal blood ketone and basal blood glucose levels: Rats administered ketone supplements did not
have a significant change in basal blood ketone levels (a) or basal blood glucose levels (b) for the four week study. Two-Way ANOVA with Tukeys
post-hoc test, results considered significant if p< 0.05. Error bars represent mean (SD)
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 10 of 15
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cardiovascular disease (CVD). It is thought that small,
dense LDL particles are more able to penetrate the endo-
thelium and cause in damage and inflammation [8285].
Volek et al. reported that the KD increased the pattern
and volume of LDL particles, which is considered to re-
duce cardiovascular risk [73]. Though we did not show a
significant effect on LDL levels for ketone supplements,
future chronic feeding studies will investigate the effects
of ketone supplementation on lipidomic profile and LDL
particle type and size.
Therapeutic levels of hyperketonemia suppress blood
glucose levels
We demonstrated that therapeutic ketosis could be
induced without dietary (calorie or carbohydrate) restric-
tion and that this acute elevation in blood ketones was
significantly correlated with a reduction in blood glucose
(Figs. 2, 3 and 4). The BMS ketone supplement did not
significantly induce blood hyperketonemia or reduced
glucose in the rats. The KE supplemented rats trended
towards reduced glucose levels; however, the lower dose
of this agent did not lower glucose significantly, as re-
ported previously in acute response of mice [59]. MCTs
have previously been shown to elicit a slight hypoglycemic
effect by enhancing glucose utilization in both diabetic
and non-diabetic patients [8688]. Kashiwaya et al. dem-
onstrated that both blood glucose and blood insulin
decreased by approximately 50 % in rats fed a diet where
30 % of calories from starch were replaced with ketone es-
ters for 14 days, suggesting that ketone supplementation
increases insulin sensitivity or reduced hepatic glucose
output [89]. This ketone-induced hypoglycemic effect has
been previously reported in humans with IV infusions of
ketone bodies [90, 91]. Recently, Mikkelsen et al. showed
that a small increase in βHB concentration decreases glu-
cose production by 14 % in post-absorptive health males
[92]. However, this has not been previously reported with
any of the oral exogenous ketone supplements we studied.
Ketones are an efficient and sufficient energy substrate for
the brain, and will therefore prevent side effects of
hypoglycemia when blood levels are elevated and the pa-
tient is keto-adapted. This was most famously demon-
strated by Owen et al. in 1967 wherein keto-adapted
patients (starvation induced therapeutic ketosis) were
given 20 IU of insulin. The blood glucose of fasted pa-
tients dropped to 12 mM, but they exhibited no
hypoglycemic symptoms due to brain utilization of ke-
tones for energy [93]. Therefore, ketones maintain brain
metabolism and are neuroprotective during severe
hypoglycemia. The rats in the MCT group had a correl-
ation of blood ketone and glucose levels at week 4,
whereas the combination of BMS + MCT produced a sig-
nificant hypoglycemic correlation both at baseline and at
week 4. No hypoglycemic symptoms were observed in the
rats during this study. Insulin levels were not measured in
this study; however, future ketone supplementation stud-
ies should measure the effects of exogenous ketones on
insulin sensitivity with a glucose tolerance test. An
increase in insulin sensitivity in combination with our
observed hypoglycemic effect has potential therapy impli-
cations for glycemic control in T2D [40]. Furthermore, it
should be noted that the KE metabolizes to both AcAc
and βHB in 1:1 ratio [29]. The ketone monitor used in this
study only measures βHB as levels of AcAc are more diffi-
cult to measure due to spontaneous decarboxylation to
acetone; therefore, the total ketone levels (βHB + AcAc)
measured were likely higher, specifically for the KE [14].
Interestingly, the 10 g/kg dose produced a delayed blood
βHB peak for ketone supplements MCT and BMS + MCT.
The higher dose of the ketogenic supplements elevated
blood levels more substantially, and thus reached their
maximum blood concentration later due to prolonged
metabolic clearance. It must be noted that the dosage used
in this study does not translate to human patients, since
the metabolic physiology of rats is considerably higher.
Future studies will be needed to determine optimal dosing
for human patients.
Effects of ketone supplementation on organ weight and
body weight percentage
Ketone supplementation did not affect the size of the
brain, lungs, kidneys or heart of rats. As previously men-
tioned, the rats were still growing during the experimen-
tal time frame; therefore, organ weights were normalized
to body weight to determine if organ weight changed in-
dependently to growth. There could be several reasons
why ketones influenced liver and spleen weight. The ra-
tio of liver to body weight was significantly higher in the
MCT supplemented animals (Fig. 5). MCTs are readily
absorbed in the intestinal lumen and transported directly
to the liver via hepatic portal circulation. When given a
large bolus, such as in this study, the amount of MCTs
in the liver will likely exceed the β-oxidation rate, caus-
ing the MCTs to be deposited in the liver as fat droplets
[94]. The accumulated MCT droplets in the liver could
explain the higher liver weight to body weight percentage
observed with MCT supplemented rats. Future toxicology
and histological studies will be needed to determine the
cause of the observed hepatomegaly. It should be empha-
sized that the dose in this study is not optimized in
humans. We speculate that an optimized human dose
would be lower and may not cause hepatomegaly or po-
tential fat accumulation. Nutritional ketosis achieved with
the KD has been shown to decrease inflammatory markers
such as TNF-α, IL-6, IL-8, MCP-1, E-selectin, I-CAM, and
PAI-1 [8, 46], which may account for the observed
decrease in spleen weight. As previously mentioned,
Veech and colleagues demonstrated that exogenous
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 11 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
supplementation of 5 mM βHB resulted in a 28 % increase
in hydraulic work in the working perfused rat heart and a
significant decrease in oxygen consumption [28, 41, 42].
Ketone bodies have been shown to increase cerebral blood
flow and perfusion [95]. Also, ketone bodies have been
shown to increase ATP synthesis and enhance the effi-
ciency of ATP production [14, 28, 40]. It is possible that
sustained ketosis results in enhanced cardiac efficiency
and O
consumption. Even though the size of the
heart did not change for any of the ketone supple-
ments, further analysis of tissues harvested from the
ketone-supplemented rats will be needed to determine
any morphological changes and to understand changes in
organ size. It should be noted that the Harlan standard
rodent chow 2018 is nutritionally complete and formu-
lated with high-quality ingredients to optimize gestation,
lactation, growth, and overall health of the animals. The
same cannot be said for the standard American diet
(SAD). Therefore, we plan to investigate the effects of
ketone supplements administered with the SAD to deter-
mine if similar effects will be seen when the micronutrient
deficiencies and macronutrient profile mimics what most
Americans consume.
MCT oil has recently been used to induce nutritional
ketosis although it produces dose-dependent gastrointes-
tinal (GI) side effects in humans that limit the potential
for its use to significantly elevate ketones (>0.5 mM).
Despite these limitations, Azzam and colleagues pub-
lished a case report in which a 43-year-old-man had a
significant decrease in seizure frequency after supple-
menting his diet with 4 tablespoons of MCT oil twice
daily [96]. An attempt to increase his dosage to 5 table-
spoons twice daily was halted by severe GI intolerance.
Henderson et al. observed that 20 % of patients re-
ported GI side effects with a 20 g dose of ketogenic
agent AC-1202 in a double blind trial in mild to moderate
Alzheimers patients [24]. We visually observed similar
gastrointestinal side effects (loose stools) in the rats
treated with MCT oil in our study. Rats were closely mon-
itored to avoid dehydration, and gastric motility returned
to normal between 1224 h. Interestingly, the BMS +
MCT supplement elevated βHB similarly to MCT oil
alone, without causing the adverse gastrointestinal effects
seen in MCT-supplemented rats. However, this could be
due to the fact in a 10 g/kg dose of BMS + MCT, only 5 g/
kg is MCT alone, which is less than the 10 g/kg dose that
elicits the GI side effects. This suggests that this novel
combination may provide a more useful therapeutic
option than MCT oil alone, which is limited in its ability
to elevate ketones in humans.
Exogenously delivered ketone supplements signifi-
cantly altered rat weight gain for the duration of the
study (Fig. 6). However, rats did not lose weight and
maintained a healthy range for their age. Rats have been
shown to effectively balance their caloric intake to prevent
weight loss/gain [9799]. Due to the caloric density of the
exogenous ketone supplements (Table 1) it is possible for
the rats to eat less of the standard rodent chow and there-
fore less carbohydrates while maintaining their caloric
intake. Food intake was not measured for this study.
However, if there was a significant carbohydrate restric-
tion there would be a signifcant change in basal blood ke-
tone and blood glucose levels. As the hallmark to the KD,
carbohydrate restriction increases blood ketone levels and
reduces blood glucose levels. Neither an increase in basal
blood ketone levels nor a decrease in basal blood glucose
levels was observed in this study (Fig. 7). Additionally, if
there were an overall blood glucose decrease due to a
change in food intake, this would not explain the
rapid reduction (within 30 min) in blood glucose cor-
related with an elevation of blood ketone levels after
an intragastric bolus of ketone supplement (Figs. 2, 3
and 4).
Several studies have investigated the safety and efficacy
of ketone supplements for disease states such as AD and
Parkinsons disease, and well as for parenteral nutrition
[40, 4850, 100103]. Our research demonstrates that
several forms of dietary ketone supplementation can ef-
fectively elevate blood ketone levels and achieve deleted:
therapeutic nutritional ketosis without the need for diet-
ary carbohydrate restriction. We also demonstrated that
ketosis achieved with exogenous ketone supplementation
can reduce blood glucose, and this is inversely associated
with the blood ketone levels. Although preliminary results
are encouraging, further studies are needed to determine
if oral ketone supplementation can produce the same
therapeutic benefits as the classic KD in the broad-
spectrum of KD-responsive disease states . Additionally,
further experiments need to be conducted to see if the ex-
ogenous ketone supplementation affects the same physio-
logical features as the KD (i.e. ROS, inflammation, ATP
production). Ketone supplementation could be used as an
alternative method for inducing ketosis in patients
uninterested in attempting the KD or those who have pre-
viously had difficulty implementing the KD because of
palatability issues, gall bladder removal, liver abnormal-
ities, or intolerance to fat. Additional experiments should
be conducted to see if ketone supplementation could be
used in conjunction with the KD to assist and ease the
transition to nutrition ketosis and enhance the speed of
keto-adaptation. In this study we have demonstrated the
ability of several ketone supplements to elevate blood
ketone levels, providing multiple options to induce thera-
peutic ketosis based on patient need. Though additional
studies are needed to determine the therapeutic potential
of ketone supplementation, many patients that previously
Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 12 of 15
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
were unable to benefit from the KD may now have an
alternate method of achieving therapeutic ketosis. Ketone
supplementation may also represent a means to further
augment ketonemia in those responsive to therapeutic
ketosis, especially in those individuals where maintaining
low glucose is important.
AcAc: acetoacetate; ALS: amyotrophic lateral sclerosis; AD: Alzheimers;
βHB: beta-hydroxybutyrate; BMS: sodium/potassium βHB mineral salt; BMS +
MCT: BMS + MCT 1:1 mixture; BD: 1,3-butanediol; CNS: central nervous
system; HDL: high density lipoprotein; HDACs: histone deacetylases; LDL: low
density lipoprotein; KD: ketogenic diet; KE: 1, 3-butanediol acetoacetate dies-
ter/ketone ester; MCT: medium chain triglyceride oil; PCOS: polycystic ovary
syndrome; ROS: reactive oxygen species; SAD: standard American diet;
TBI: traumatic brain injury; T2D: type-2 diabetes.
Competing interests
International Patent # PCT/US2014/031237, University of South Florida, D.P.
DAgostino, S. Kesl, P. Arnold, Compositions and Methods for Producing
Elevated and Sustained Ketosis. P. Arnold (Savind) has received financial
support (ONR N000140610105 and N000140910244) from D.P. DAgostino
(USF) to synthesize ketone esters. The remaining authors have no conflicts of
Conceived and designed the experiments: SK, AP, NW, TF, DP. Performed the
experiments: SK, AP, NW, TF, CA, JS, AVP. Analyzed the data: SK, AP, DP.
Contributed reagents/materials/analysis tools: PA. Helped draft the
manuscript: SK, AP, NW, CA, DP. All authors read and approved the final
The authors would like to thank Savind Inc for manufacturing some of the
ketone supplements, Jay Dean for the use of his Hyperbaric Research
Laboratory, and the ONR and Scivation Inc. for funding the project.
This study was supported by the Office of Naval Research (ONR) Grant
N000140610105 (DPD); and a Morsani College of Medicine Department of
Molecular Pharmacology and Physiology departmental grant.
Author details
Department of Molecular Pharmacology and Physiology, Morsani College of
Medicine, University of South Florida, 12901 Bruce B. Downs Blvd. MDC8,
Tampa, FL 33612, USA.
Savind Inc, 205 South Main Street, Seymore, IL
61875, USA.
Received: 10 September 2015 Accepted: 28 January 2016
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... The ketone ester, R,S-1,3-butanediol diacetoacetate (BD-AcAc 2 ), increases circulating ketone concentrations in rodents from 0.5 to 1.0 mM (Poff et al., 2014;Ari et al., 2016;Kesl et al., 2016;Davis et al., 2019). Studies from our laboratory show that BD-AcAc 2 decreases body weight and adiposity in obese mice on a highfat diet (HFD) (Davis et al., 2019) and attenuates weight gain in lean mice on a low-fat diet (LFD) (Deemer et al., 2019). ...
... BD-AcAc 2 reduces (Ciarlone et al., 2016;Davis et al., 2019) or lowers the accretion of (Poff et al., 2014;Kesl et al., 2016;Deemer et al., 2019;Deemer et al., 2020b) body weight and adiposity. A more recent publication from our group showed that BD-AcAc 2 decreased hepatic steatosis, fibrosis, and inflammatory markers compared to ad libitum HFD-fed obese controls (Moore et al., 2021). ...
... In addition, current hand-held technology does not detect S-βHB and AcAc concentrations from whole blood, and more-sophisticated approaches using expensive tandem mass spectrometry procedures were outside the scope of the current investigation. This was also not a primary outcome measure for the study, as it has been demonstrated that the ketone ester increases βHB and AcAc concentrations in rodents (Poff et al., 2014;Ari et al., 2016;Ciarlone et al., 2016;Kesl et al., 2016;Davis et al., 2019;Deemer et al., 2020b). ...
Full-text available
Objective: The ketone diester, R,S-1,3-butanediol diacetoacetate (BD-AcAc2), attenuates the accretion of adiposity and reduces hepatic steatosis in high-fat diet-induced obese mice when carbohydrate energy is removed from the diet to accommodate energy from the ester. Reducing carbohydrate energy is a potential confounder due to the well-known effects of carbohydrate restriction on components of energy balance and metabolism. Therefore, the current investigation was designed to determine whether the addition of BD-AcAc2 to a high-fat, high-sugar diet (with no reduction in carbohydrate energy) would attenuate the accretion of adiposity and markers of hepatic steatosis and inflammation. Methods: Sixteen 11-week-old male C57BL/6J mice were randomized to one of two groups for 9 weeks (n = 8 per group): 1) Control (CON, HFHS diet) or 2) Ketone ester (KE, HFHS diet + BD-AcAc2, 25% by kcals). Results: Body weight increased by 56% in CON (27.8 ± 2.5 to 43.4 ± 3.7 g, p < 0.001) and by 13% in KE (28.0 ± 0.8 to 31.7 ± 3.1 g, p = 0.001). Non-alcoholic fatty liver disease activity scores (NAS) for hepatic steatosis, inflammation, and ballooning were lower in the KE group compared to CON (p < 0.001 for all). Markers of hepatic inflammation [Tnfα (p = 0.036); Mcp1 (p < 0.001)], macrophage content [(Cd68 (p = 0.012)], and collagen deposition and hepatic stellate cell activation [(αSma (p = 0.004); Col1A1 (p < 0.001)] were significantly lower in the KE group compared to CON. Conclusion: These findings extend those of our previous work and show that BD-AcAc2 attenuates the accretion of adiposity and reduces markers of liver steatosis, inflammation, ballooning, and fibrosis in lean mice placed on a HFHS diet where carbohydrate energy was not removed to accommodate energy from addition of the diester.
... There are indications that combining ketone salts and MCTs could be potentially useful. In a rodent study (61), tested the effects of a 28-day course of an exogenous ketone supplement made up of an Na/K β-hydroxybutyrate (βHB) mineral salt (BMS) in a 1:1 ratio with an MCT oil comprised of approximately 65% caprylic triglyceride and 45% capric triglyceride. When compared with a MCT monotherapy, the combined BMS + MCT supplement was shown to elevate βHB to a similar extent to MCT without causing the GI side-effects observed in the MCT cohort. ...
Full-text available
In recent times, advances in the field of metabolomics have shed greater light on the role of metabolic disturbances in neuropsychiatric conditions. The following review explores the role of ketone bodies and ketosis in both the diagnosis and treatment of three major psychiatric disorders: major depressive disorder, anxiety disorders, and schizophrenia. Distinction is made between the potential therapeutic effects of the ketogenic diet and exogenous ketone preparations, as exogenous ketones in particular offer a standardized, reproducible manner for inducing ketosis. Compelling associations between symptoms of mental distress and dysregulation in central nervous system ketone metabolism have been demonstrated in preclinical studies with putative neuroprotective effects of ketone bodies being elucidated, including effects on inflammasomes and the promotion of neurogenesis in the central nervous system. Despite emerging pre-clinical data, clinical research on ketone body effectiveness as a treatment option for psychiatric disorders remains lacking. This gap in understanding warrants further investigating, especially considering that safe and acceptable ways of inducing ketosis are readily available.
... [28] Exogenous ketone supplements have been shown to be successful in a variety of animal studies, and they may offer patients with KD intolerance an alternative method for achieving therapeutic ketosis. [29] Although there is a lack of clinical evidence for exogenous ketone supplements, the results of the limited available research suggest that they may help with aging, neurodegenerative disorders, mental illnesses, and other conditions. [26,30] Medium-chain triglycerides oils, 1,3-butanediol, 3-butanediol-acetoacetate diester, K-β-hydroxybutyric acid, and others are examples of exogenous ketone supplements currently available. ...
The ketogenic diet (KD) is a potential nutritional therapy that is frequently utilized in various conditions. More and more studies are being done on KD in recent years. However, as far as we know, few studies have made an effort to offer a thorough synthesis and assessment of this topic. This paper aims to do a rigorous and thorough evaluation of the knowledge structure, development trend, and research hotspot of scientific outputs connected to KD. The bibliographic records connected to KD from January 1, 2001 to April 22, 2022 were collected using the core collection database of Web of Science. The complex data input, that consisted of the amount of publications, journals, authors, institutions, countries, keywords and cited references, was generated and analyzed visually using CiteSpace. A total of 2676 literatures on the KD were published between 2001 and 2022. The most KD-related publications were found in Epilepsia and Epilepsia Research. The authors with the most KD-related papers are Kossoff EH and Rho J. The United States is the country with the most publications, and Johns Hopkins University, Johns Hopkins University Hospital, and Johns Hopkins Medical Institutions are the institutions with the most articles. The high frequency keywords are "KD," "ketone body," "children," "efficacy," "weight loss," "low carbohydrate diet," "metabolism," "epilepsy," "beta hydroxybutyrate," and "modified atkins diet." The 2018 study by Kossoff EH on epilepsia and the 2017 study by Puchalska P on ketone body metabolism earned 127 and 114 citations, respectively. The results of this bibliometric analysis provide information on the state and trends in KD and may be used by researchers to pinpoint hot issues and discover new areas of study.
... When a similar dose of 4 g/kg MCT was used to supplement a fructose-rich diet, 12 weeks of such feeding has been reported to exacerbate the liver damage associated with a high-fructose diet in mice [41]. An even larger dose of 10 g/kg/day of MCT added to standard feed for 28 days has been reported to increase liver size and reduce HDL cholesterol in rats [57]. These values agree well with our findings in the present study, where 28 days of 6 g/kg/day MCT supplementation protocol resulted in increased liver weight. ...
Full-text available
Medium-chain triglycerides (MCT) possess neuroprotective properties. However, the long-term metabolic consequences of supplementing a regular diet with cognition-enhancing doses of MCT are largely unknown. We studied the effects of chronic (28 days) supplementation of regular diet with different doses of MCT oil (1, 3, or 6 g/kg/day) or water (control) on working memory (Y-maze), behavior in the Open Field, spatial learning (Morris water maze), and weight of internal organs in male Wistar 2.5-m.o. Rats. In a separate experiment, we evaluated acute (single gavage) and chronic (28 days) effects of MCT or lard supplementation (3 g/kg) on blood biochemical parameters. MCT-1 and MCT-3 doses improved working memory in YM. In MWM, MCT-6 treatment improved spatial memory. Chronic MCT-1 or MCT-3 treatment did not affect internal organ weight, while MCT-6 dose increased liver weight and the brown/white adipose tissue ratio. Acutely, MCT administration elevated blood β-hydroxybutyrate and malondialdehyde levels. Chronic MCT administration (3 g/kg) did not affect the blood levels of glucose, lactate, pyruvate, acetoacetate, β-hydroxybutyrate, total and HDL cholesterol, triglycerides, malondialdehyde, and aspartate transaminase and alanine transaminase activities. Therefore, daily supplementation of standard feed with MCT resulted in mild intermittent ketosis. It improved working memory at lower concentrations without significant adverse side effects. At higher concentrations, it improved long-term spatial memory but also resulted in organ weight changes and is likely unsafe. These results highlight the importance of monitoring the metabolic effects of MCT supplementation alongside cognitive assessment in future studies of MCT's neuroprotective properties.
... The known signalling activities by BA played a significant motivational role in our design of experimentation that could suggest the synergistic application of BHB with BA in the context of fatty acid oxidation, ketone synthesis and the facilitation of ketosis. It is well established that exogenous ketone supplementation can play a role in ketosis and therapy [23,24]. However, it is important to explore how endogenously activated ketogenesis could be induced by signalling activity independent of dietary changes to support the metabolic activity needed to help activate and maintain endogenously generated ketosis. ...
... Ketone esters have been Abbreviations: BD-AcAc 2 , R,S-1,3, butanediol diacetoacetate; KE, ketone ester; LBM, lean body mass; FM, fat mass; KD, ketogenic diet; Tnc, tenascin-C; ER, endoplasmic reticulum; Gadd45a, growth arrest and DNA damage inducible alpha; Runx1, family Transcription Factor 1; Chasm, calponin homology-associated smooth muscle protein; CnA, calcineurin A; PGC-1α, peroxisome proliferator-activated receptorgamma coactivator; MEF2, myocyte enhancer factor-2; NFAT, nuclear factor of activated T-cells; Atrogin-1/MAFbx, Muscle Atrophy F-box gene; Pim1, pim-1 oncogene protein; Mecom, MDS1 And EVI1 Complex Locus Protein EV1; Camk2b, Calcium/Calmodulin Dependent Protein Kinase II Beta; P38α MAPK, p38 mitogen-activated protein kinases; Irak2, Interleukin-1 receptor-associated kinase-like 2; MuRF1, Muscle-specific RING finger protein 1; Cops9, COP9 singalosome; Dvl1, disheveled segment polarity protein 2 (Dvl2); Traf7, TNF receptor associated factor 7; Hdac6, histone deacetylase 6; CryZ, crystallin zeta; Bax, bcl-2-associated X protein; VDAC1, voltage-dependent anion channel 1. used in many forms such as R,S-1,3-butanediol diacetoacetate (BD-AcAc 2 , ketone diester), D-β-hydroxybutyrate-(R)-1,3 butanediol (ketone monoester), and more recently Bis-hexanoyl (R)-1,3-butanediol (BH-BD) (11)(12)(13). Studies in animal models show that oral BD-AcAc 2 consumption results in body weight loss or maintenance with moderate increases in circulating ketones (0.5-1.0 mM) (14)(15)(16). Recently, we examined concentration-dependent effects of BD-AcAc 2 on body weight, adiposity, energy intake, and energy expenditure in lean mice showing that on an ad libitum basis, mice consuming a 25% (by kcals) KE diet consumed the same amount of food as an ad libitum fed control group, but had a significant difference in body weight (BW) and fat mass (FM), and maintained lean body mass (LBM) (17). ...
Full-text available
Exogenous ketone ester supplementation provides a means to increase circulating ketone concentrations without the dietary challenges imposed by ketogenic diets. Our group has shown that oral R,S-1,3, butanediol diacetoacetate (BD-AcAc2) consumption results in body weight loss or maintenance with moderate increases in circulating ketones. We have previously shown a diet consisting of 25% BD-AcAc2 can maintain lean body mass (LBM) and induce fat mass (FM) loss in young, healthy male mice, but the underlying mechanisms are still unknown. Therefore, the purpose of this study was to determine if a diet consisting of 25% BD-AcAc2 (ketone ester, KE) would alter body composition, transcriptional regulation, the proteome, and the lipidome of skeletal muscle in aged mice. We hypothesized that the KE group would remain weight stable with improvements in body composition compared to controls, resulting in a healthy aging phenotype. Male C57BL/6J mice (n = 16) were purchased from Jackson Laboratories at 72 weeks of age. After 1 week of acclimation, mice were weighed and randomly assigned to one of two groups (n = 8 per group): control (CON) or KE. A significant group by time interaction was observed for body weight (P < 0.001), with KE fed mice weighing significantly less than CON. FM increased over time in the control group but was unchanged in the KE group. Furthermore, LBM was not different between CON and KE mice despite KE mice weighing less than CON mice. Transcriptional analysis of skeletal muscle identified 6 genes that were significantly higher and 21 genes that were significantly lower in the KE group compared to CON. Lipidomic analysis of skeletal muscle identified no differences between groups for any lipid species, except for fatty acyl chains in triacylglycerol which was 46% lower in the KE group. Proteomics analysis identified 44 proteins that were different between groups, of which 11 were lower and 33 were higher in the KE group compared to CON. In conclusion, 72-week-old male mice consuming the exogenous KE, BD-AcAc2, had lower age-related gains in body weight and FM compared to CON mice. Furthermore, transcriptional and proteomics data suggest a signature in skeletal muscle of KE-treated mice consistent with markers of improved skeletal muscle regeneration, improved electron transport chain utilization, and increased insulin sensitivity.
As common complication of prediabetes, type I and type II diabetes, diabetic peripheral neuropathy (DPN) includes a series of sensory and motor changes associated with slow nerve conduction, nerve degeneration, gate disturbances, pain, and loss of sensation. Although proper glycemic control can prevent DPN progression, these complications remain difficult to clinically treat. Current pharmacological medications have limited effectiveness, creating the need for additional clinical options. Lifestyle interventions hold great promise as the broad spectrum of improvements derived from certain lifestyle changes appears promising to improve diabetes management and DPN. In this chapter, we highlight research that illustrates the consequences of poor diet on DPN and discuss the benefits of lifestyle changes associated with dietary change and/or exercise. Reversal of dietary changes appears to have positive impact on DPN, and we highlight new studies in which a low-carbohydrate/high-fat diet has been used to prevent and/or reverse DPN. In addition, a growing number of basic and clinical studies are revealing how exercise can improve symptoms of DPN. These interventions affect a broad range of cellular and metabolic changes that can lead to improvements in DPN symptoms. These interventions likely involve overlapping cellular pathways but could also improve DPN through unique mechanisms. As approaches using personalized medicine increase, clinical treatments for DPN will need to determine the most impactful interventions that are relevant to specific symptoms in patients suffering from DPN. Lifestyle and dietary interventions should play an important role in these treatment plans and the convergence of shared mechanisms should be a focus of preclinical and clinical research.KeywordsDiabetesKetogenic dietMediterranean dietExercisePhysical activity
Significance: Diabetic peripheral neuropathy (DPN), a complication of metabolic syndrome, type I, and type II diabetes, leads to sensory changes that include slow nerve conduction, nerve degeneration, loss of sensation, pain, and gate disturbances. These complications remain largely untreatable, although tight glycemic control can prevent neuropathy progression. Nonpharmacological approaches remain the most impactful to date, but additional advances in treatment approaches are needed. Recent advances: This review highlights several emerging interventions, including a focus on dietary interventions and physical activity that continue to show promise for treating DPN. We provide an overview of our current understanding of how exercise can improve aspects of DPN. We also highlight new studies in which a ketogenic diet has been used as an intervention to prevent and reverse DPN. Critical issues: Both exercise and consuming a ketogenic diet induce systemic and cellular changes that collectively improve complications associated with DPN. Both interventions may involve similar signaling pathways and benefits but also impact DPN through unique mechanisms. Future directions: These lifestyle interventions are critically important as personalized medicine approaches will likely be needed to identify specific subsets of neuropathy symptoms and deficits in patients and determine the most impactful treatment. Overall, these two interventions have the potential to provide meaningful relief for patients with DPN and provide new avenues to identify new therapeutic targets.
Full-text available
Ketogenic diets and orally administered exogenous ketone supplements are strategies to increase serum ketone bodies serving as an alternative energy fuel for high energy demanding tissues, such as the brain, muscles, and the heart. The ketogenic diet is a low-carbohydrate and fat-rich diet, whereas ketone supplements are usually supplied as esters or salts. Nutritional ketosis, defined as serum ketone concentrations of ≥ 0.5 mmol/L, has a fasting-like effect and results in all sorts of metabolic shifts and thereby enhancing the health status. In this review, we thus discuss the different interventions to reach nutritional ketosis, and summarize the effects on heart diseases, epilepsy, mitochondrial diseases, and neurodegenerative disorders. Interest in the proposed therapeutic benefits of nutritional ketosis has been growing the past recent years. The implication of this nutritional intervention is becoming more evident and has shown interesting potential. Mechanistic insights explaining the overall health effects of the ketogenic state, will lead to precision nutrition for the latter diseases.
Ketogenic diets have been used to treat epilepsy for nearly a century. Alongside enduring clinical success with a ketogenic diet, metabolism’s critical role in health and in diseases in the central nervous system and throughout the body is increasingly appreciated. Furthermore, metabolism-based strategies have been proven equal or even superior to pharmacological treatments in specific cases and for specific diseases. Rather than causing unwanted off-target pharmacological side effects, addressing metabolic dysfunction can improve overall health simultaneously. Enduring interest in the ketogenic diet’s proven efficacy in stopping seizures and emerging efficacy in other disorders has fueled renewed efforts to determine key mechanisms and diverse applications of metabolic therapies. In parallel, multiple strategies are being developed to mobilize similar metabolic benefits without reliance on such a strict diet. Research interest in metabolic therapies has spread into laboratories and clinics of every discipline, and could yield entirely new classes of drugs and treatment regimens. This work is the first comprehensive scientific resource on the ketogenic diet, covering the latest research into the mechanisms, established and emerging applications, metabolic alternatives, and implications for health and disease. Experts in clinical and basic research share their research into mechanisms spanning from ion channels to epigenetics, their insights based on decades of experience with the ketogenic diet in epilepsy, and their evidence for emerging applications ranging from autism to Alzheimer’s disease to brain cancer.
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the ketogenic diet (KD) has become a widely used nutritional approach for weight loss. Some of the KD's positive effects on metabolism and cardiovascular risk factors are similar to those seen after n-3 polyunsaturated fatty acids (ω-3) supplementation. We hypothesized that a ketogenic Mediterranean diet with phytoextracts combined with ω-3 supplementation may have increased positive effects on cardiovascular risk factors and inflammation. We analyzed 34 male overweight subjects; aged between 25 and 65 years who were overall healthy apart from overweight. The subjects followed a ketogenic diet protocol for four weeks; with (KDO3) or without (KD) ω-3 supplementation. All subjects experienced a significant loss of body weight and body fat and there was no significant differences between treatment (body weight: KD-4.7 kg, KDO3-4.03 kg, body fat KD-5.41 kg, KDO3-5.86 kg). There were also significant decreases in total cholesterol, LDL-c, and glucose levels. Triglycerides and insulin levels decreased more in KDO3 vs. KD subjects, with a significant difference. All the investigated inflammatory cytokines (IL-1β, IL-6, TNF-α) decreased significantly in KDO3 subjects whilst only TNF-α showed a significant decrease in KD subjects over the 12 month study period. No significant changes were observed in anti-inflammatory cytokines (IL-10 and IL-1Ra), creatinine, urea and uric acid. Adiponectin increased significantly only in the KDO3 group. ω-3 supplementation improved the positive effects of a ketogenic Mediterranean diet with phytoextracts on some cardiovascular/metabolic risk factors and inflammatory state.
Much scientific and anecdotal data demonstrate favorable metabolic responses to very-low-carbohydrate diets. We believe that very-low-carbohydrate diets merit further study for weight loss, and that criticisms of these diets lack scientific evidence.
Long-term outcomes of the ketogenic diet in the treatment of epilepsy have not previously been reported. A retrospective chart review of children treated with the ketogenic diet for more than 6 years at the Johns Hopkins Hospital was performed. The response was documented at clinic visits and by telephone contacts; laboratory studies were obtained approximately every 6 to 12 months. Satisfaction and tolerability were assessed by means of a brief parental telephone questionnaire. In all, 28 patients (15 males, 13 females), currently aged 7 to 23 years, were identified. The median baseline seizure frequency per week at diet onset was 630 (range 1–1400). Diet duration ranged from 6 to 12 years; 19 remain on the diet currently. After 6 years or more, 24 children experienced a more than 90% decrease in seizures, and 22 parents reported satisfaction with the diet's efficacy. Ten children were at less than the 10th centile for height at diet initiation; this number increased to 23 at the most recent follow-up (p=0.001). Kidney stones occurred in seven children and skeletal fractures in six. After 6 years or more the mean cholesterol level was 201mg/dl, high-density lipoprotein was 54mg/dl, low-density lipoprotein was 129mg/dl, and triglycerides were 97mg/dl. Efficacy and overall tolerability for children are maintained after prolonged use of the ketogenic diet. However, side effects, such as slowed growth, kidney stones, and fractures, should be monitored closely.
Context: Ketone bodies are substrates during fasting and when on a ketogenic diet not the least for the brain and implicated in the management of epileptic seizures and dementia. Moreover, D-β-hydroxybutyrate (HOB) is suggested to reduce blood glucose and fatty acid levels. Objectives: The objectives of this study were to quantitate systemic, cerebral, and skeletal muscle HOB utilization and its effect on energy metabolism. Design: Single trial. Setting: Hospital. Participant: Healthy post-absorptive males (n = 6). Interventions: Subjects were studied under basal condition and three consecutive 1-hour periods with a 3-, 6-, and 12-fold increased HOB concentration via HOB infusion. Main outcome measures: Systemic, cerebral, and skeletal muscle HOB kinetics, oxidation, glucose turnover, and lipolysis via arterial, jugular, and femoral venous differences in combination with stable isotopically labeled HOB, glucose, and glycerol, infusion. Results: An increase in HOB from the basal 160-450 μmol/L elicited 14 ± 2% reduction (P = .03) in glucose appearance and 37 ± 4% decrease (P = .03) in lipolytic rate while insulin and glucagon were unchanged. Endogenous HOB appearance was reduced in a dose-dependent manner with complete inhibition at the highest HOB concentration (1.7 mmol/L). Cerebral HOB uptake and subsequent oxidation was linearly related to the arterial HOB concentration. Resting skeletal muscle HOB uptake showed saturation kinetics. Conclusion: A small increase in the HOB concentration decreases glucose production and lipolysis in post-absorptive healthy males. Moreover, cerebral HOB uptake and oxidation rates are linearly related to the arterial HOB concentration of importance for modifying brain energy utilization, potentially of relevance for patients with epileptic seizures and dementia.