Caffeine intake increases plasma ketones: an acute metabolic study in humans

Abstract

Brain glucose uptake declines during aging and is significantly impaired in Alzheimer’s disease. Ketones are the main alternative brain fuel to glucose so they represent a potential approach to compensate for the brain glucose reduction. Caffeine is of interest as a potential ketogenic agent owing to its actions on lipolysis and lipid oxidation but whether it is ketogenic in humans is unknown. This study aimed to evaluate the acute ketogenic effect of 2 doses of caffeine (2.5; 5.0 mg/kg) in 10 healthy adults. Caffeine given at breakfast significantly stimulated ketone production in a dose-dependent manner (+88%; +116%) and also raised plasma free fatty acids. Whether caffeine has long-term ketogenic effects or could enhance the ketogenic effect of medium chain triglycerides remains to be determined.

Full text

1
Caffeine intake increases plasma ketones: an acute metabolic study in humans
Camille Vandenberghe
1, 2
, Valérie St-Pierre
1, 2
, Alexandre Courchesne-Loyer
1, 2
, Marie
Hennebelle
1
, Christian-Alexandre Castellano
1
, Stephen C Cunnane
1, 2,3
1
Research Center on Aging, Sherbrooke, CIUSSS de l’Estrie – CHUS, QC, Canada (CV, VSP,
ACL, MH, CAC, SCC)
2
Department of Pharmacology & Physiology, Université de Sherbrooke, Sherbrooke, QC,
Canada (CV, VSP, ACL, SCC)
3
Department of Medicine, Université de Sherbrooke, Sherbrooke, QC, Canada (SCC)
Author for correspondence: Stephen Cunnane
Research Center on Aging, 1036 Belvedere St. South, Sherbrooke, QC, Canada J1H 4C4
Tel: 1 819 780-2220, ext 45670;
Stephen.Cunnane@USherbrooke.ca
Page 1 of 14
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Université de Sherbrooke on 11/28/16
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

ABSTRACT
Brain glucose uptake declines during aging and is significantly impaired in Alzheimer’s disease. Ketones
are the main alternative brain fuel to glucose so they represent a potential approach to compensate for the
brain glucose reduction. Caffeine is of interest as a potential ketogenic agent owing to its actions on
lipolysis/ lipid oxidation but whether it is ketogenic in humans is unknown. This study aimed to evaluate
the acute ketogenic effect of two doses of caffeine in healthy adults (2.5; 5.0 mg/kg) during a 4-hour
metabolic study period. Caffeine given at breakfast significantly stimulated ketone production in a dose-
dependent manner (+88%; +116%) and also raised plasma free fatty acids. Whether caffeine has long-
term ketogenic effects or could enhance the ketogenic effect of medium chain triglycerides remains to be
determined.
Key words: Ketones; Ketonemia; Caffeine; Free fatty acids; Medium chain triglycerides; Lipolysis;
Alzheimer’s disease.
RÉSUMÉ
La consommation cérébrale de glucose diminue avec l’âge et, tout particulièrement, avec la maladie
d’Alzheimer. L’élaboration de différentes stratégies nutritionnelles pour optimiser la production de cétones,
le principal carburant alternatif cérébral, est nécessaire afin de soutenir les besoins énergétiques du
cerveau vieillissant. La caféine est une molécule d’intérêt en raison de son action sur le métabolisme
lipidique. L’effet aigu de différentes doses de caféine (2.5; 5.0 mg/kg) sur la production de cétones était
évalué chez dix sujets. Nos résultats ont montré que la caféine ajoutée à un repas stimule
significativement la cétonémie à des concentrations comparables à un jeûne de 12h et cette réponse est
dose-dépendante (+88 à +116%). Ainsi, la prise de caféine combinée avec une source alimentaire
cétogène comme les triglycérides à chaine moyenne dans le but de maximiser la cétonémie constitue une
piste prometteuse d’intervention en concomitance avec d’autres traitements thérapeutiques dans un
contexte de maladies neurodégénératives.
Page 2 of 14
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Université de Sherbrooke on 11/28/16
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

INTRODUCTION
1
2
Caffeine upregulates metabolic rate (Miller et al. 1974), and stimulates energy expenditure. It is an
3
adenosine receptor antagonist that increases sympathetic activity (Bellet et al. 1969) and inhibits cyclic
4
nucleotide phosphodiesterase, which is responsible for catalyzing the conversion of cyclic adenosine
5
monophosphate (cAMP) to AMP (Butcher et al. 1968; Quan et al. 2013). As a result, higher tissue
6
concentrations of cAMP activate hormone-sensitive lipase and promote lipolysis (Acheson et al. 2004;
7
Butcher et al. 1968). Free fatty acids (FFA) are the product of lipolysis and can be used as an immediate
8
source of energy by many organs. They can also be converted by the liver into ketones (acetoacetate
9
[AcAc], β-hydroxybutyrate [β-HB] and acetone). Most organs use glucose and FFA as energy substrates.
10
However, the brain is unable to use FFA for energy, and requires ketones as the principal alternative fuel
11
to glucose (Cunnane et al. 2016). Plasma ketones are highly positively correlated to their utilization by the
12
brain (Cunnane et al. 2016; Mitchell et al. 1995) and can provide up to 70% of brain’s total energy during
13
period of hypoglycaemia as, for example, during fasting (Owen et al. 1967).
14
15
Brain glucose uptake is 10-15% lower during normal aging (Nugent et al. 2014), and can be up to 35%
16
lower in certain brain regions in neurodegenerative diseases such as Alzheimer’s disease (AD)
17
(Castellano et al. 2015). Several studies suggest that brain glucose hypometabolism potentially
18
contributes to the onset and/or progression of AD (Cunnane et al. 2016; Mosconi et al. 2005; Nugent et al.
19
2014; Reiman et al. 2004; Schöll et al. 2011). A ketogenic supplement could therefore potentially help
20
support the brain’s energy needs during aging. Hence, the primary aim of this study was to evaluate
21
whether the lipolytic effect of caffeine acutely increases plasma ketones in healthy adults during a four-
22
hour metabolic study period. The secondary aim was to confirm whether caffeine increases FFA as
23
previously reported (Acheson et al. 1980; Acheson et al. 2004).
24
25
PARTICIPANTS AND METHODS
26
27
Participants
28
Page 3 of 14
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Université de Sherbrooke on 11/28/16
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

4
Ethical approval for this study was obtained from the Research Ethics Committee of the Integrated
29
University Health and Social Services of the Eastern Townships – Sherbrooke University Hospital Center,
30
which oversees all human research done at the Research Center on Aging (Sherbrooke, QC, Canada). All
31
participants provided written informed consent prior to beginning the study. They underwent a screening
32
visit, including the analysis of a blood sample collected after a 12 h overnight fast. Exclusion criteria
33
included regular high consumption of caffeine (>300 mg/day), smoking, diabetes or glucose intolerance
34
(fasting glucose >6.1 mmol/L and glycosylated hemoglobin >6.0%), untreated hypertension, dyslipidemia,
35
abnormal renal, liver, heart or thyroid function. This project is registered on ClinicalTrials.gov (NCT
36
02694601).
37
38
Experimental design
39
The protocol involved three randomized four-hour metabolic study days: a baseline metabolic day (CTL)
40
and two days each with a different dose of caffeine (2.5 mg/kg [C-2.5] and 5.0 mg/kg [C-5.0]). On each
41
metabolic study day, the participants arrived at 8:00 a.m. after 12 h of fasting and 24 h without caffeine
42
intake. At the time of signing the consent form, participants were aware of the 12 h fast and to abstain
43
from consuming caffeine. They also received a reminder call 24 h before the metabolic study day. A
44
forearm venous catheter was installed and blood samples were taken every 30 min during 4 hours. After
45
installing the catheter and the first blood sample, participants received a standard breakfast comprised of
46
two pieces of toast with raspberry jam, a piece of cheese, applesauce and 100 ml of juice. The breakfast
47
contained 85 grams of carbohydrate, 9.5 g of fat and 14 g of protein. Commercially available caffeine
48
tablets (200 mg extra-strength Life Brand
™
, ON, Canada) were hand crushed to powder and two doses
49
were provided (2.5 mg/kg and 5.0 mg/kg) on separate test days. The low dose corresponding to 1½ cup of
50
coffee and the high dose to 3 cups of regular coffee, the highest quantity recommended by Heath
51
Canada. The caffeine dose to be given was mixed in 104 ml of applesauce and consumed during
52
breakfast. No caffeine was added to the breakfast for CTL. Water was available ad libitum throughout the
53
study day. Blood samples were centrifuged at 3500 rpm for 10 min at 4°C and plasma was stored at -80°C
54
until further analysis.
55
56
Page 4 of 14
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Université de Sherbrooke on 11/28/16
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

5
Plasma analyses
57
58
Caffeine
59
Plasma caffeine was measured using a complete ELISA Kits from Neogen (WI, USA), according to the
60
manufacturers’ protocol with the following modifications. Caffeine (Sigma-Aldrich
®
, St-Louis, Mo, USA)
61
was diluted with the Neogen kit buffer (EIA) at multiple dilutions ending with the standard curve dilutions
62
from 0 to 25 ng/ml. Plasma samples were then diluted with EIA buffer at a 1:50 000 dilution. Both
63
standards and samples were run in duplicate. The absorbance was then measured with a plate reader
64
(VICTOR, Perkin Elmer Inc, MA, USA) at 690 nm.
65
66
Metabolites
67
Plasma glucose, lactate, triglycerides, total cholesterol (Siemens Medical Solutions USA, Inc., Deerfield,
68
IL, USA) and free fatty acids (Randox Laboratories Ldt, West Virginia, USA) were measured using
69
commercial kits on a clinical biochemistry analyzer (Dimension Xpand Plus, Siemens Healthcare
70
Diagnosis Inc., Deerfield, IL, USA) as previously described (Courchesne-Loyer et al. 2013). Plasma β-HB
71
and AcAc were evaluated by an automated colorimetric assay as previously described (Courchesne-Loyer
72
et al. 2013).
73
74
Statistical analysis
75
All results are given as mean ± SEM. Ten participants were sufficient to meet the statistical power
76
(β=0.80) needed to observe a significant difference in plasma FFA with the caffeine supplementation
77
(Acheson et al. 1980). For lactate, metabolic study day values were normalized to baseline in order to
78
account for variability at the beginning of the study day. For post-caffeine ketone and FFA analysis, the
79
area under the curve (AUC) was calculated from 2 to 4-hour post-dose because that was when maximal
80
plasma caffeine was achieved. All statistical analyses were carried out using SPSS 23.0 software (SPSS
81
Inc., Chicago, IL, USA). Comparison of the three test conditions was done using the Friedman test, and
82
the effect of caffeine supplementation was determined in each group using a Wilcoxon’s signed rank test.
83
Page 5 of 14
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Université de Sherbrooke on 11/28/16
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

6
Differences were considered statistically significant at p≤0.05. Data were graphed using Prism version 6.0
84
(GraphPad Software Inc., San Diego, CA, USA).
85
86
RESULTS
87
Two men and eight women completed all three test conditions (Table 1). Participants were 33 ± 19 years
88
of age and had a body mass index of 24 ± 8 (n=10). The participant’s baseline biochemical parameters
89
corresponded to normal references values from the Sherbrooke University Hospital Center (Sherbrooke,
90
Qc). No significant side effects were reported following caffeine intake. Baseline plasma caffeine values
91
did not significantly differ from zero on any of the three study days (Fig.1). There was no difference in
92
plasma glucose, triglycerides, or cholesterol response across the three metabolic days (data not shown).
93
Plasma lactate differed across the three metabolic days (p=0.045), but after normalizing the data to
94
baseline, these differences disappeared (p=0.607).
95
96
A dose-response was observed for plasma caffeine across the three metabolic days (p<0.05; Fig. 1).
97
Plasma caffeine significantly increased during the first hour post-dose (p<0.05). C-2.5 increased plasma
98
caffeine to a maximum of 7.5 ± 1.5 mg/L at 2 h and C-5.0 increased plasma caffeine to a maximum of
99
10.0 ± 2.3 mg/L at 3 h (p<0.05). No difference in plasma AcAc levels was observed across the three test
100
days (p=0.497; Fig 2A, 2C). However, after normalizing the data to baseline, there was a significant group
101
difference between baseline and the two doses of caffeine at 3.5 h, at which time AcAc was significantly
102
increased (p<0.05; data not shown). A group difference was observed for the β-HB response from 2 to 4 h
103
post-dose (p<0.05; Fig. 2B and 2D). Caffeine increased plasma β-HB by 88% and 116% in a dose-
104
dependent manner (p<0.05). No significant difference in plasma FFA was observed during 0-2 h post-
105
dose (Fig. 3A). Globally, FFA decreased from 711 ± 398 µM to 91 ± 42 µM during this period (Fig. 3A).
106
Between 2 – 4 h after the breakfast, a dose-related increase of FFA was observed with the two doses of
107
caffeine (p<0.005; Fig. 3B). C-2.5 raised plasma FFA concentrations to 548 ± 276 µM after 4 h whereas
108
C-5.0 raised plasma FFA to 695 ± 433 µM.
109
Page 6 of 14
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Université de Sherbrooke on 11/28/16
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

7
110
DISCUSSION
111
112
This short-term study showed that caffeine intake can stimulate ketogenesis by increasing β-HB
113
concentrations by 88-116% with a maximum within four hours post-dose. A dose-response was observed
114
for plasma β-HB (Fig. 2D) but not for AcAc (Fig. 2C), which could be explained by the larger inter-group
115
variation in AcAc. The increase in plasma ketones obtained with these doses of caffeine could at least
116
transiently contribute to 5-6% of brain energy needs (Cunnane et al. 2016).
117
The increased plasma FFA after caffeine seen in the present study confirms prior results (Acheson et al.
118
1980; Acheson et al. 2004; Bellet et al. 1968; Bellet et al. 1969). Caffeine competes for the adenosine
119
receptor, inhibits phosphodiesterase activity and increases plasma FFA. FFA entering the liver are beta-
120
oxidized and converted to ketones due to condensation of pairs of acetyl-CoA units as their availability
121
exceeds their utilization by the tricarboxylic acid cycle (Wang et al. 2014).
122
123
The increase in blood ketones shown here was equivalent to that observed after an overnight fast.
124
Another way of increasing blood ketones is to provide a source of medium-chain triglyceride (MCT)
125
(Courchesne-Loyer et al. 2013). Caffeine combined with an MCT supplement could potentially prolong
126
mild ketonemia. Such products are already available on the market although no reports are available on
127
the ketogenic effect of the combination of these products.
128
One limitation of this study design is that the metabolic study period was only 4 hours. However, this was
129
sufficient to observe an effect on plasma ketones and FFA within the period during which peak plasma
130
caffeine was observed. The half-life of caffeine is 4.5 hours, which suggests that its peak metabolic effect
131
would take place over 2-3 hours. Furthermore, the effect of each caffeine dose was only assessed once,
132
so a longer term study would be useful.
133
134
In conclusion, by enhancing lipolysis and increasing blood FFA levels, which in turn provide substrates for
135
ketogenesis, caffeine at doses of 2.5 and 5.0 mg/kg stimulated safe and mild ketonemia in healthy adults
136
to a ketone level twice that seen after an overnight fast. Several studies suggest that regular caffeine
137
Page 7 of 14
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Université de Sherbrooke on 11/28/16
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

8
consumption may be linked to the decreased risk of developing late-life cognitive decline (Panza et al.
138
2015). Further studies are needed to evaluate caffeine’s long term effect on ketonemia and its impact on
139
brain function during aging.
140
141
ACKNOWLEDGMENTS
142
We thank our research nurses, Conrad Filteau and Christine Brodeur-Dubreuil, for their assistance in
143
participant screening, blood sampling and care of the participants. SCC, CV and VSP designed the study.
144
CV, VSP, ACL, CAC and MH conducted the study. CV, VSP, CAC and SCC analyzed and interpreted the
145
data. All the authors contributed to the final article.
146
147
REFERENCES
Acheson, K. J., Zahorska-Markiewicz, B., Pittet, P., Anantharaman, K., and Jéquier, E. 1980. Caffeine and
coffee: their influence on metabolic rate and substrate utilization in normal weight and obese individuals.
Am. J. Clin. Nutr. 33(5): 989-997.
Acheson, K. J., Gremaud, G., Meirim, I., Montigon, F., Krebs, Y., Fay, L. B., et al. 2004. Metabolic effects
of caffeine in humans: lipid oxidation or futile cycling? Am. J. Clin. Nutr. 79(1): 40-46.
Bellet, S., Kershbaum, A., and Finck, E. M. 1968. Response of free fatty acids to coffee and caffeine.
Metabolism, 17(8): 702-707. doi:10.1016/0026-0495(68)90054-1.
Bellet, S., Roman, L., DeCastro, O., Kim, K. E., and Kershbaum, A. 1969. Effect of coffee ingestion on
catecholamine release. Metabolism, 18(4): 288-291.
Butcher, R. W., Baird, C. E., and Sutherland, E. W. 1968. Effects of lipolytic and antilipolytic substances
on adenosine 3',5'-monophosphate levels in isolated fat cells. J. Biol. Chem. 243(8): 1705-1712.
Castellano, C. A., Nugent, S., Paquet, N., Tremblay, S., Bocti, C., Lacombe, G., et al. 2015. Lower brain
18F-fluorodeoxyglucose uptake but normal 11C-acetoacetate metabolism in mild Alzheimer's disease
dementia. J. Alzheimers Dis. 43(4): 1343-1353. doi: 10.3233/JAD-141074.
Courchesne-Loyer, A., Fortier, M., Tremblay-Mercier, J., Chouinard-Watkins, R., Roy, M., Nugent, S., et
al. 2013. Stimulation of mild, sustained ketonemia by medium-chain triacylglycerols in healthy humans:
estimated potential contribution to brain energy metabolism. Nutrition, 29(4): 635-640. doi:
10.1016/j.nut.2012.09.009.
Cunnane, S. C., Courchesne-Loyer, A., St-Pierre, V., Vandenberghe, C., Pierotti, T., Fortier, M., et al.
2016. Can ketones compensate for deteriorating brain glucose uptake during aging? Implications for the
risk and treatment of Alzheimer's disease. Ann. N. Y. Acad. Sci. 1367(1): 12-20. doi: 10.1111/nyas.12999.
Miller, D. S., Stock, M. J., and Stuart, J. A. 1974. Proceedings: The effects of caffeine and carnitine on the
oxygen consumption of fed and fasted subjects. Proc. Nutr. Soc. 33(2): 28A-29A.
Page 8 of 14
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Université de Sherbrooke on 11/28/16
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

9
Mitchell, G. A., Kassovska-Bratinova, S., Boukaftane, Y., Robert, M. F., Wang, S. P., Ashmarina, L., et al.
1995. Medical aspects of ketone body metabolism. Clin. Invest. Med. 18(3): 193-216.
Mosconi, L., Herholz, K., Prohovnik, I., Nacmias, B., De Cristofaro, M. T., Fayyaz, M., et al. 2005.
Metabolic interaction between ApoE genotype and onset age in Alzheimer's disease: implications for brain
reserve. J. Neurol. Neurosurg. Psychiatry, 76(1): 15-23. doi: 10.1136/jnnp.2003.030882.
Nugent, S., Tremblay, S., Chen, K. W., Ayutyanont, N., Roontiva, A., Castellano, C. A., et al. 2014. Brain
glucose and acetoacetate metabolism: a comparison of young and older adults. Neurobiol. Aging, 35(6):
1386-1395. doi: 10.1016/j.neurobiolaging.2013.11.027.
Owen, O. E., Morgan, A. P., Kemp, H. G., Sullivan, J. M., Herrera, M. G., and Cahill, G. F. 1967. Brain
metabolism during fasting. J. Clin. Invest. 46(10): 1589-1595. doi: 10.1172/JCI105650.
Panza, F., Solfrizzi, V., Barulli, M. R., Bonfiglio, C., Guerra, V., Osella, A., et al. 2015. Coffee, tea, and
caffeine consumption and prevention of late-life cognitive decline and dementia: a systematic review. J.
Nutr. Health Aging, 19(3): 313-328. doi: 10.1007/s12603-014-0563-8.
Quan, H. Y., Kim, D. Y., and Chung, S. H. 2013. Caffeine attenuates lipid accumulation via activation of
AMP-activated protein kinase signaling pathway in HepG2 cells. BMB Rep. 46(4): 207-212. doi:
10.5483/BMBRep.2013.46.4.153.
Reiman, E. M., Chen, K., Alexander, G. E., Caselli, R. J., Bandy, D., Osborne, D., et al. 2004. Functional
brain abnormalities in young adults at genetic risk for late-onset Alzheimer's dementia. Proc. Natl. Acad.
Sci. U.S.A. 101(1): 284-289. doi: 10.1073/pnas.2635903100.
Schöll, M., Almkvist, O., Bogdanovic, N., Wall, A., Långström, B., Viitanen, M., et al. 2011. Time course of
glucose metabolism in relation to cognitive performance and postmortem neuropathology in Met146Val
PSEN1 mutation carriers. J. Alzheimers Dis. 24(3): 495-506. doi: 10.3233/JAD-2011-101563.
Wang, S. P., Yang, H., Wu, J. W., Gauthier, N., Fukao, T., and Mitchell, G. A. 2014. Metabolism as a tool
for understanding human brain evolution: lipid energy metabolism as an example. J. Hum. Evol. 77: 41-49.
doi: 10.1016/j.jhevol.2014.06.013.
Page 9 of 14
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Université de Sherbrooke on 11/28/16
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

10
Table 1. Baseline demographic and biochemical parameters of the participants
148
(Mean ± SEM)
149
150
Characteristics
Age (y) 33
± 19
Men/Women 2/8
Weight (kg) 65
± 14
Height (cm) 163
± 16
Body mass index (kg/m
2
) 24
± 8
Glucose (mmol/L) 4.2
± 0.4
Lactate (mmol/L) 1.86
± 1.0
Glycated hemoglobin (%) 5.3
± 0.3
Total cholesterol (mmol/L) 4.3
± 0.8
Triacylglycerol (µmol/L) 749
± 282
Free fatty acids (µmol/L) 711
± 392
Ketones (µmol/L) 175
± 65
151
Page 10 of 14
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Université de Sherbrooke on 11/28/16
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

11
Figure 1. Plasma caffeine concentrations during the control (CTL) metabolic study day (), after receiving
152
a 2.5 mg/kg (C-2.5) (☐) or 5.0 mg/kg dose of caffeine (C-5.0) (∇). Arrow indicates breakfast. Values are
153
presented as mean ± SEM (n = 10/point); * p<0.05 CTL vs C-2.5, † p<0.05 CTL vs C-5.0, # p<0.05 C-2.5
154
vs C-5.0.
155
156
Figure 2. Plasma acetoacetate [A] and β-hydroxybutyrate [B] concentrations during the control (CTL)
157
metabolic study day (), and after receiving a 2.5 mg/kg (C-2.5) (☐) or 5.0 mg/kg dose of caffeine (C-5.0)
158
(∇). Arrow indicates breakfast. The area under the curve was measured from 2 to 4-hour post-dose for
159
acetoacetate [C] and β-hydroxybutyrate [D]. Values are presented as mean ± SEM (n = 10/point);
160
* p<0.05 CTL vs C-2.5, † p<0.05 CTL vs C-5.0, # p<0.05 C-2.5 vs C-5.0.
161
162
Figure 3. Plasma free fatty acids (FFA) concentrations [A] during the control (CTL) metabolic study day
163
obtained before (), after receiving a 2.5 mg/kg dose (C-2.5) (☐) or 5.0 mg/kg dose of caffeine (C-5.0) (∇).
164
Arrow indicates breakfast. The area under the curve [B] was measured from 2 to 4-hour post-dose. Values
165
are presented as mean ± SEM (n = 10/point); * p<0.05 CTL vs C-2.5, † p<0.05 CTL vs C-5.0, # p<0.05 C-
166
2.5 vs C-5.0.
167
Page 11 of 14
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Université de Sherbrooke on 11/28/16
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

01234
0
2
4
6
8
10
12
14
Time (h)
Plasma caffeine [mg/L]
*
†
#
C-5.0C-2.5CTL
*
†
#
*
†
#
*
†
#
Page 12 of 14
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Université de Sherbrooke on 11/28/16
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

01234
20
40
60
80
100
Time (h)
Plasma acetoaceteate [µmol/L]
A
C-5.0
C-2.5
CTL
CTL C-2.5 C-5.0
0
200
400
600
800
*
†
#
D
ß-hydroxybutyrate [µmol*h/L]
234
+25
+50
+75
0
Time,(h)
Δ,Plasma,acetoacetate,vs,baseline,2,h,[µmol/L]
*
**
#
A
01234
100
150
200
250
300
Time (h)
Plasma ß-hydroxybutyrate [µmol/L]
*
†
†
#
*
†
B
C-5.0
C-2.5
CTL
CTL C-2.5 C-5.0
0
50
100
150
200
Acetoacetate [µmol*h/L]
C
Page 13 of 14
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Université de Sherbrooke on 11/28/16
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

01234
200
400
600
800
1000
Time (h)
Plasma free fatty acids [µmol/L]
*
†
*
†
A
CTL
C-2.5
C-5.0
*
†
#
CTL C-2.5 C-5.0
0
500
1000
1500
2000
*
†
#
B
Free fatty acids [µmol*h/L]
Page 14 of 14
Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Université de Sherbrooke on 11/28/16
For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.