Plant Regeneration from Friable Embryogenic Callus and Cell Suspension Cultures of Zea mays L.

Research article

Mild experimental ketosis increases brain uptake of 11C-acetoacetate and 18 F-fluorodeoxyglucose: a dual-tracer PET imaging study in rats Fabien Pifferi 1,4, Sébastien Tremblay1,2, Etienne Croteau2, Mélanie Fortier1, Jennifer Tremblay-Mercier 1, Roger Lecomte 2, Stephen C Cunnane 1,3 1

Research Center on Aging, Sherbrooke University Geriatric Institute, Departments of 2Nuclear Medicine and Radiobiology and 3Medicine, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada, 4UMR CNRS-MNHN 7179, Brunoy, France Brain glucose and ketone uptake was investigated in Fisher rats subjected to mild experimental ketonemia induced by a ketogenic diet (KD) or by 48 hours fasting (F). Two tracers were used, 11C-acetoacetate (11C-AcAc) for ketones and 18F-fluorodeoxyglucose for glucose, in a dual-tracer format for each animal. Thus, each animal was its own control, starting first on the normal diet, then undergoing 48 hours F, followed by 2 weeks on the KD. In separate rats on the same diet conditions, expression of the transporters of glucose and ketones (glucose transporter 1 (GLUT1) and monocarboxylic acid transporter (MCT1)) was measured in brain microvessel preparations. Compared to controls, uptake of 11C-AcAc increased more than 2-fold while on the KD or after 48 hours F (P < 0.05). Similar trends were observed for 18FDG uptake with a 1.9–2.6 times increase on the KD and F, respectively (P < 0.05). Compared to controls, MCT1 expression increased 2-fold on the KD (P < 0.05) but did not change during F. No significant difference was observed across groups for GLUT1 expression. Significant differences across the three groups were observed for plasma beta-hydroxybutyrate (beta-HB), AcAc, glucose, triglycerides, glycerol, and cholesterol (P < 0.05), but no significant differences were observed for free fatty acids, insulin, or lactate. Although the mechanism by which mild ketonemia increases brain glucose uptake remains unclear, the KD clearly increased both the blood–brain barrier expression of MCT1 and stimulated brain 11C-AcAc uptake. The present dual-tracer positron emission tomography approach may be particularly interesting in neurodegenerative pathologies such as Alzheimer’s disease where brain energy supply appears to decline critically. Keywords: Ketone bodies, MCT1, GLUT1, PET,

18

F-FDG, Acetoacetate

Introduction Under normal conditions, the brain relies primarily on glucose as fuel, but ketone bodies (ketones: acetoacetate (AcAc) and beta-hydroxybutyrate (beta-HB)) become an important alternative source of energy for the brain when blood glucose declines over a period of hours to days. During starvation, ketones can supply up to 70% of the human brain’s energy requirements.1 Neuroprotective effects of ketones have also been reported in isolated neurons,2,3 in epilepsy4 and in models of ischemic stroke.5 During the neonatal Correspondence to: Sébastien Tremblay, Research Center on Aging, Health and Social Services Centre – Sherbrooke University Geriatric Institute, 1036 Belvedere Street South, Sherbrooke (Québec), Canada J1H 4C4. Email: [email protected]

© W.S. Maney & Son Ltd. 2011 DOI 10.1179/1476830510Y.0000000001

period, ketones are also precursors for the synthesis of lipids (especially cholesterol) and amino acids.6,7 Recent studies demonstrate that raising blood ketones induces short-term improvement in cognitive function both in Alzheimer’s disease (AD)8,9 and in experimental hypoglycemia induced in type 1 diabetics.10 Neuronal activity is tightly coupled to glucose utilization and thus to the transfer of glucose from blood to neurons. Glucose is first transported across the endothelium of the blood–brain barrier by the 55-kDa glucose transporter 1 (GLUT1). From there, it is transported into astrocytes by the 45-kDa GLUT1 isoform and then into neurons by GLUT3.11 Glucose uptake depends on three factors: GLUT transporter activity, the number of GLUT transporters present at the

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blood–brain barrier, and, most importantly, the driving force created by the glucose gradient across the blood– brain barrier. It is now generally accepted that glucose transport itself is not rate limiting. The first rate-limiting step is encountered farther downstream at the catalytic step of glucose phosphorylation by hexokinase.12 When neurons are activated, brain glucose utilization is increased, thereby stimulating glucose uptake from the blood. Fasting (F) and very high-fat ketogenic diets (KDs) raise plasma ketones and breath acetone.13,14 In humans, the cerebral metabolic rate of ketones varies directly with the concentration of ketones in blood,15–17 an observation first made in small rodents.18–20 Cerebral ketone metabolism is also regulated by the permeability of the blood–brain barrier to ketones, which depends on the abundance of monocarboxylic acid transporters (MCT1). Ketone transport into astrocytes occurs via MCT1, whereas their transport into neurons requires MCT2.21 Using positron emission tomography (PET) and 18 F-fluorodeoxyglucose (18F-FDG), lower brain uptake of glucose has been reported in elderly people, and more significantly in patients with AD.22–24 Brain hypometabolism appears to be the earliest known defect in AD, occurring up to four decades before the onset of clinical signs of cognitive decline.25,26 Therefore, developing a way to correct or bypass this AD-associated deterioration in brain glucose uptake is warranted. Mild ketonemia clearly seems to be a promising way of providing energy to the brain when blood glucose declines, but exploiting this opportunity depends on a better understanding of brain ketone uptake and utilization. The aim of the present study was thus to investigate the impact of mild experimental ketosis induced by a KD or 48 hours F, on brain uptake of ketones and glucose. Brain uptake of these two energy substrates was assessed by dual-tracer PET imaging using 11 C-AcAc for ketone uptake and 18F-FDG for glucose uptake. Expression of the transporters MCT1 and GLUT1 in brain microvessels was also measured in each of the three dietary conditions. The expression of these transporters was determined specifically in brain microvessels where the blood– brain barrier is located. We also report the impact of these dietary treatments on blood fatty acid composition with particular attention to polyunsaturates, since these fatty acids are potential ketogenic substrates (alpha-linolenate) and may stimulate ketogenesis (eicosapentaenoate).27

Material and methods Study design For the PET study, six male Fisher rats were used, each animal being its own control for the three stages of the

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experiment. The first dual-tracer PET scans were done while the rats were on a standard control diet (Table 1). After 1 week of rest, the same rats were fasted for 48 hours but with ad libitum access to water and the second dual-tracer PET scans were performed. Immediately afterwards, the same rats were given the very high-fat KD (Table 1) for 2 weeks, at which point the third dual-tracer PET scan was performed. Brain expression of transporters and blood metabolites was assessed on separate rats and was treated identically as in the PET study, i.e. they were given the control diet, fasted for 48 hours, or fasted for 48 hours and then fed the KD for 2 weeks.

PET measurements Before each double-tracer PET scan, the rats were anesthetized with 1–2% isoflurane and a blood sample obtained from the tail vein was analyzed for glucose and ketone concentration. 11C-AcAc was synthesized as previously described28 and 18F-FDG was prepared with a TRACERlab FDG-MX synthesis unit (GE HealthCare, Waukesha, WI, USA). The PET data were acquired in list mode using a LabPET™-4 scanner equipped with avalanche photodiode detectors (Gamma Medica, Northridge, CA, USA). The head of the animal was held in a frame with a bite bar on the teeth, and a transverse bar passing over the head was fixed in the frame. The frame was tightly fixed to minimize head motion during the experiment. Both bars were filled with a

Table 1 Composition of the diets Macronutrient Protein Casein L-cystine Carbohydrate Corn starch Sucrose Dextrin Fiber Cellulose Vitamins AIN-93G-VX** Minerals AIN-93G-MX** Fat Soybean oil*** Flaxseed oil Butter Ratio (fat:protein and carbohydrate)

Control diet*

KD*

87 3.0

154 5.4

466 138 165

— — 33

41

72

0.33

0.59

27

48

70 — — 1:12.1

124 56 508 3.5:1.0

*Values are given as g/kg with an accuracy of ∼2%. Both control and KDs contained 0.014 g/kg t-butylhydroquinone, an antioxidant, to improve storage. **AIN-93G vitamin and mineral mixes contain no carbohydrates48. ***Soybean oil in the KD came from the diet premix, in which it serves as a carrier for fat-soluble vitamins. The caloric energy of the diet was 3781 and 6903 kcal/kg for the control and the KD, respectively; data are calculated from information provided by Dyets Inc.

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precise amount of 18F-FDG that served as landmarks and external standards for count efficiency. Injection of 11C-AcAc was performed through the tail vein with an average injected dose of 84 ± 12 MBq (range: 71–108 MBq) in order to evaluate brain ketone uptake. This injection was followed by an injection of 72 ± 16 MBq 18F-FDG (range: 43–100 MBq) to evaluate brain glucose uptake. The precision for the dose calibrator CRC 35R was ±1% of the measured dose. All injections used a volume of 300 μl at 1 ml/minute followed by a flush with 300 μl of 0.9% sodium chloride solution at 1 ml/minute and were completed within 45 seconds. For 11C-AcAc injection, brain PET scans were acquired over 20 minutes. For 18F-FDG, PET scans were acquired over 30 minutes. Each 18F-FDG scan was preceded by a 30-second data acquisition before tracer injection to evaluate the residual radioactivity from the 11 C-AcAc injection. The mean radioactivity estimated from this frame was removed from all the decay-corrected subsequent frames. The images were then reconstructed with the following sequence: 1 × 30; 6 × 5; 2 × 15, 2 × 30, 6 × 60; 6 × 120, n × 300 seconds, where n = 0 for the 20-minute scans and n = 2 for the 30-minute scans. We were unable to obtain blood samples during PET image acquisition, thereby preventing us from determining the arterial input function of both tracers needed to quantify their metabolism. Thus, our data are provided in the form of standardized uptake values (SUVs) of activity observed on normalized injected dose ((MBq/ml)/(MBq/g)).

Brain GLUT1 and MCT1 expression Microvessel preparation Brain microvessels were isolated as described previously with modifications.29 Briefly, the cerebral cortex of each rat was dissected free of meninges and homogenized in isotonic phosphate-buffered saline containing 1% bovine serum albumin at pH 7.4. Microvessels were separated from the homogenate by centrifugation at 4000 × g for 15 minutes in 17.5% dextran buffer (MW 73200; Sigma, St Louis, MO, USA) before storage at −80 °C in isotonic phosphatebuffered saline containing an antiprotease cocktail.29 Western blots Protein concentration was determined using BCA protein Assay kit (23227, Pierce, Rockford, IL, USA). Four micrograms of total brain microvessel protein were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis in denaturant conditions and electrotransferred to a polyvinylidene fluoride membrane during 3 hours at 40 mA. Membranes were incubated with Tris-buffered saline containing 0.1% Tween 20 (v:v; TBST) and 5% milk for 16 hours at 4 °C, rinsed in TBST, and incubated

Brain uptake of ketones and glucose in mild ketosis

with a rabbit anti-GLUT1 polyclonal IgG (ab15309, Abcam, Cambridge, MA, USA) diluted 1:3000 in TBST for 3 hours at 4 °C. The membranes were washed several times in TBST and incubated with peroxidase-conjugated goat anti-rabbit IgG secondary antibody (111-035-045, Jackson ImmunoResearch, Baltimore Pike, PA, USA) diluted 1:10 000, for 2 hours at room temperature. Immunopositive areas were detected by enhanced chemiluminescence with the ECL Plus Western blotting detection system (RPN2132, GE Healthcare, Baie d’Urfé, QC, Canada). Staining intensity was measured with Image J (National Institutes of Health, Public Domain). The procedure was repeated with 8 μg of protein with rabbit anti-MCT1 polyclonal IgG diluted 1:500 (AB3540P, Millipore, Billerica, MA, USA) with the same secondary antibody. The densitometry of GLUT1 and MCT1 was normalized with reference to beta-actin blotted on each membrane. Beta-actin was assayed with a mouse anti-beta-actin monoclonal antibody (MAB1501R, Millipore) diluted 1:10 000 and a peroxidase-conjugated goat anti-mouse IgG secondary antibody (115-035-062, Jackson ImmunoResearch) using the same procedure as for GLUT1. Blood metabolite analysis From each rat used to measure brain transporter protein expression, about 5 ml of blood sample was drawn at sacrifice. The blood was held on ice in EDTA tubes and centrifuged for plasma recovery. The plasma was kept at −80 °C until analysis. Metabolite analysis was performed with a clinical chemistry analyzer (Dimension Xpand Plus, Siemens Healthcare Diagnostics Inc, Deerfield, IL, USA) with kits for glucose (DF40), triglycerides (DF69A), cholesterol (DF27), and lactic acid (DF16). The kit for free fatty acids (HR series NEFA-HR2) was purchased from Wako (Wako Ltd, Richmond, VA, USA). For each test, quality control and duplicate were performed. Glycerol was measured with a spectrophotometric method by the EnzyChrom Glycerol assay kit (EGLY-200; BioAssay Systems, Hayward, CA, USA). Plasma insulin concentration was obtained by enzyme-linked immunosorbent assay (kit 80INSHU-E01, Alpco, Salem, NH, USA). The ketone assays were based on previous methods30,31 and automated on an open channel of the Dimension Xpand Plus analyzer. Due to low stability of AcAc in plasma, this assay was performed within 48 hours of blood sampling. Briefly, 25 μl of plasma was used with 330 μl of fresh reagent for AcAc measurement (Tris buffer, pH 7.0, 100 mM; sodium oxamate 20 mM; NADH 0.15 mM; betahydroxybutyrate dehydrogenase [BHBDH]; 1 U/ml) or beta-HB measurement (Tris buffer, pH 9.0;

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sodium oxamate 20 mM; NAD 1 mM; BHBDH 1 U/ml). The stock solution of 200 U/ml of BHBDH was stable at 4 °C in 75 mM potassium phosphate buffer. The change in absorbance at 340 nM between 15 and 120 seconds was automatically measured. The assay was calibrated with freshly diluted standards from frozen aliquots of a 10 mM standard from lithium AcAc or DL-β-HB, sodium salt, stable 2 and 6 mo, respectively, at −20 °C. Fatty acid composition was analysed on 400 μl of plasma as previously described.32 Data expression and statistical analyses PET tracer uptake data are expressed as mean ± SEM of five independent determinations while on the control diet and six in the fasted and KD states. From the PET scans, SUVs were obtained in the whole brain, excluding the cerebellum. Blood metabolite analyses and body weights are expressed as mean ± SD (n = 12 independent determinations in each groups). Fatty acid analyses are expressed as mean ± SD (n = 5 independent determinations in control group and n = 7 determinations in fasted and KD groups). Transporter protein expression data are expressed as mean ± SEM (n = 5 independent determinations in the control group, n = 7 independent determinations in the fasted group and n = 6 determinations in the KD group). Transporter expression in fasted or KD rats was normalized to that of the control rats on the standard diet. Statistical comparisons between groups were performed using one-way analysis of variance with Bonferroni post-test with significance level set at P ≤ 0.05. Statistical analyses were performed using Origin 8 software (OriginLab, Northampton, MA, USA) for PET tracers uptake and Prism 5 software (Graphpad Software, La Jolla, CA, USA) for other analyses.

Results Plasma metabolites Compared to the control diet, body weight did not differ significantly when the rats were fasted or were on the KD (Table 2). However, once on the KD, they weighed significantly more than while F (P ≤ 0.05), but the rats on the KD were 2 weeks older, so this difference was more than a normal growth effect rather than a diet effect.33 On the KD, plasma AcAc and β-HB were increased 4.3-fold and 4.7-fold, respectively, compared to control state (P ≤ 0.05). F also induced a 6.0-fold and 4.3-fold increase, respectively, in blood ketones, which was higher than that induced by the KD (Table 2). Both the KD and F induced significant hypoglycaemia compared to control rats (43 and 22% lower blood glucose, respectively; P ≤ 0.05), but with significantly less pronounced hypoglycaemia on the KD. Compared to controls, neither F nor the KD changed plasma free fatty acids, cholesterol, or

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Table 2 Biological parameters of control, fasted, and KD-fed rats

Weight (g) AcAc (mmol/l) Beta-HB (mmol/l) Glucose (mmol/l) Free fatty acids (mmol/l) Triglycerides (mmol/l) Cholesterol (mmol/l) Lactate (mmol/l) Glycerol (mmol/l) Insulin (μU/ml)

Control

48 hours F

KD

228 ± 36 0.08 ± 0.03 0.26 ± 0.08 14.8 ± 2.6 0.9 ± 0.9

198 ± 26 0.48 ± 0.19** 1.46 ± 0.63** 8.5 ± 1.8** 0.6 ± 0.5

268 ± 54* 0.34 ± 0.14*,** 1.22 ± 0.54** 11.7 ± 3.7*,** 1.0 ± 0.5

2.7 ± 1.8

0.5 ± 0.1**

4.9 ± 2.0*,**

1.6 ± 0.2

1.4 ± 0.1

1.2 ± 0.1

2.8 ± 0.4 0.20 ± 0.30 3.8 ± 1.9

1.6 ± 0.4** 0.04 ± 0.00** 2.1 ± 1.3

2.1 ± 1.1 0.60 ± 0.20*,** 4.9 ± 2.8

Data are expressed as mean ± SD (n = 12 independent determinations in each groups). *Significant difference between 48 hours F and ketogenic dietfed rats. **Significant difference between treatments (either 48 hours F or KD) and controls.

insulin. Plasma triglycerides were 80% lower in the fasted rats (P ≤ 0.05) but 80% higher on the KD (P ≤ 0.05). Plasma glycerol was 80% lower during F (P ≤ 0.05), but three times higher on the KD compared to the control diet (P ≤ 0.05). Compared to the control period, lactate was 42% lower during F (P ≤ 0.05) but remained unchanged on the KD (Table 2).

Plasma fatty acids Compared to control diet, the sum of plasma saturated fatty acids remained unchanged but 18:0 was 60% higher during F (P ≤ 0.05; Table 3). Other F-induced changes were markedly lower 18:1n-9 and a 117% increase in 20:4n-6. Total saturates in plasma were 25% higher on the KD compared to controls, mainly due to higher 14:0 and 18:0. The KD raised plasma 18:3n-3 7-fold to 4.9%, 14:0 6.6-fold and 18:0 1.5-fold compared to controls.

Ketone and glucose transporter expression in brain MCT1 and GLUT1 expression in the brain of the three groups are reported in Fig. 1. F had no significant effect on the expression of the two transporters compared to controls. However, MCT1 expression was doubled (P ≤ 0.05) in rats on the KD compared to controls or fasted rats. GLUT1 expression was statistically unchanged on the KD.

Brain uptake of 11C-AcAc and

18

F-FDG

At the plateau, brain uptake of 11C-AcAc was 160% higher than control in the fasted rats and 123% higher while on the KD (Fig. 2A).F and the KD also increased the uptake of 18F-FDG (+277 and +123%, respectively; P ≤ 0.05). Uptake of 18F-FDG was 70% higher at the plateau (P ≤ 0.05) during F compared to the KD. Brain 11C-AcAc uptake was positively correlated to

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Table 3 Plasma fatty acids of control, fasted, and KD-fed rats Fatty acids 12:0 14:0 16:0 18:0 24:0 12:1 14:1 n-5 16:1 n-7 18:1 n-12 18:1 n-9 18:1 n-7 18:2 n-6 20:3 n-6 20:4 n-6 18:3 n-3 20:5 n-3 22:5 n-3 22:6 n-3

Control

48 hours fasted

KD

0.5 ± 0.0 (1.3) 0.7 ± 0.1 (1.7) 23.5 ± 2.0 (59.2) 8.2 ± 0.4 (20.7) 0.5 ± 0.1 (1.2) 0.4 ± 0.0 (0.8) 0.4 ± 0.0 (0.9) 4.4 ± 0.2 (11.1) 16.6 ± 0.5 (18.5) 11.3 ± 6.6 (27.2) 4.2 ± 0.3 (9.8) 24.6 ± 1.2 (62.5) 0.6 ± 0.0 (1.5) 12.2 ± 1.3 (30.6) 0.7 ± 0.0 (1.7) 1.0 ± 0.0 (2.0) 1.0 ± 0.3 (2.5) 3.1 ± 0.7 (8.0)

nd nd 21.7 ± 0.7 (26.1) 13.3 ± 1.2* (15.9) nd 0.7 ± 0.00* (0.9) nd 3.0 ± 1.0* (3.6) 10.3 ± 0.7* (13.0) 4.3 ± 4.0* (4.9) 2.0 ± 0.2* (2.2) 18.8 ± 0.7* (22.7) nd 26.8 ± 1.6* (32.4) nd nd nd 4.0 ± 0.4 (4.9)

0.8 ± 0.3* (3.6) 4.6 ± 1.0* (21.1) 23.5 ± 1.0 (101.6) 12.5 ± 1.0* (52.6) 0.2 ± 0.1* (1.0) nd 0.4 ± 0.1 (1.9) 1.4 ± 0.5*,** (6.1) 11.5 ± 1.0* (37.6) 10.0 ± 1.2** (58.0) 0.5 ± 0.5*,** (3.1) 20.9 ± 1.0 (89.1) 0.4 ± 0.2 (1.3) 7.0 ± 2.3*,** (27.0) 4.9 ± 1.0* (22.4) 0.7 ± 0.3 (2.1) 0.6 ± 0.2 (2.5) 1.0 ± 0.4*,** (3.6)

Values are expressed as % of total fatty acids (mean ± SD; control: n = 5; 48 hours fasted: n = 7; KD: n = 7). Values in parentheses are expressed in mg/dl and obtained from addition of internal standard 17:0. nd: not detected. *Represents a significant difference between treatments (either 48 hours F or KD) and control rats. **Represents a significant difference between 48 hours F and KD-fed rats.

blood total ketones (AcAc + beta-HB) (P = 0.0406, r 2 = 0.2666; Fig. 3). Conversely, brain 18F-FDG uptake was negatively correlated to blood glucose concentration (P = 0.0033, r 2 = 0.45; Fig. 3).

Discussion In the present study, we investigated the impact of two mildly ketogenic conditions (48 hours F and a very high-fat KD) on brain transport and utilization of AcAc and glucose. This dual-tracer PET approach was made possible using the new PET tracer 11 C-AcAc,27 the utilization of which was coupled sequentially to that of 18F-FDG so as to avoid some of the biological variation due to repeated PET scans. Biodistribution and utilization of 11C-AcAc metabolism has been previously reported in rodents.34 Using PET and autoradiography, we have

reported increased brain uptake of 11C-AcAc in rats on a KD35,36 but these previous studies did not assess brain 18F-FDG uptake or GLUT1 or MCT1 expression in the brain. The mild ketonemia associated with hypoglycaemia during both ketogenic treatments was expected and has previously been described.37 AcAc and beta-HB levels in control and 48 hours fasted animals were very close to those described by Bates et al.38 In the present study, F and the KD both induced a significant and comparable increase in brain 11C-AcAc uptake of about +150%. Both treatments also significantly increased 18F-FDG uptake, with a significantly greater effect in fasted rats (+100%) compared to the KD (+50%). Increased brain uptake of 11C-AcAc was accompanied by higher MCT1 expression in brain microvessels, a result in accordance with

Figure 1 MCT1 and GLUT1 Western immunoblots of brain microvessels of a control (CTL), fasted (F) or ketogenic-diet-fed (KD) rat (A). MCT1 and GLUT1 relative expression in control (n = 5), fasted (n = 7), or KD-fed (n = 6) animals (B). *Significant difference between treatments (either 48 hours F or KD) and control rats. $Significant difference between 48 hours F and KD-fed rats.

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Figure 2 SUVs (mean ± SEM) of 11C-AcAc (A), and 18F-FDG (B) obtained by PET imaging in the brain of control (•, n = 5), fasted (▴, n = 6), and KD-fed rats (▪, n = 6). SUVs are expressed as measured activity/normalized dose. *Significant difference between treatments (either 48 hours F or KD) and control rats. $Significant difference between 48 hours F and KD-fed rats.

several other studies.39–41 Vannucci and Simpson39 described maximal MCT1 mRNA and protein expression in the blood–brain barrier during suckling when ketone bodies are the predominant fuel for the brain, and a decline with maturation, coincident with the switch to glucose as the main cerebral fuel. Leino et al.40 reported an 8-fold increased level of MCT1 measured by electron microscopy in brain endothelial cells of rats fed with a high-fat KD. More recently, 40% of brain capillaries stained positive for MCT1 in rats on a standard diet, an amount that doubled in rats on a KD for 6 weeks.41 In our rats, mild ketonemia induced by 48 hours F led to higher 11C-AcAc uptake (Fig. 2) but, unlike with the KD, this was not accompanied by a higher expression of MCT1 (Fig. 1). In agreement with our present results, Matsuyama et al.42 also reported that F did not significantly affect MCT1 mRNA expression in any areas of the rat brain they examined. As observed previously for rats infused with ketone bodies,19 fasted for 48 hours, or treated with alloxan,38,20 regulation of brain AcAc uptake is predominantly determined by blood ketone concentration.

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Figure 3 Correlations between plasma AcAc + beta-HB and 11 C-AcAc uptake (A), and between plasma glucose and 18 F-FDG uptake (B) of control (•, n = 5), fasted (▴, n = 6), and KD-fed rats (▪, n = 6).

Furthermore, 11C-AcAc uptake was not significantly different between our 48 hours fasted versus KD groups, whereas MCT1 expression was doubled (P ≤ 0.05) on the KD. These results suggest that MCT1 expression does not limit AcAc entry into the brain. Plasma insulin did not differ significantly between groups, but a decreasing trend was observed in the fasted state, and an increasing trend in the KD group. An insulin change between fasted and control animals was observed by Hawkins et al.43 This suggests that insulin does not play a major role in the regulation of brain MCT expression or transport activity, but may be more implicated in ketone synthesis. Compared to controls, both ketogenic treatments reduced plasma glucose, and both led to a significantly higher brain 18F-FDG uptake (Fig. 2). However, blood glucose was significantly lower in the fasted rats compared to those on the KD. A comparable difference was found for brain 18F-FDG uptake, which was higher during F compared to the KD. Neither dietary treatment significantly affected the brain expression of glucose transporters, which is different from the previous observations of Puchowicz et al.41, who reported an 8-fold increased density of GLUT1 protein expression in isolated brain microvessels from rats on a KD for

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6 weeks. However, their level of ketosis was more severe than in the present study (beta-HB at 4.8 versus 1.2 mM in the present study). Consistent with our results, GLUT1 mRNA levels and the blood–brain barrier GLUT1 protein did not change in rats with hypoglycaemia induced by 8 days of daily subcutaneous injection of intermediate-acting insulin.44 According to Leybaert12 and Leybaert et al.45 under basal conditions, brain glucose shuttling via GLUT1 normally operates at about one-third of maximal transport capacity. When more glucose is needed within brain cells, this can be achieved by increasing the driving force on glucose through the blood–brain barrier. Our hypothesis is that, in rats in mild ketosis, the driving force of glucose through the blood–brain barrier is still high due to a relatively low brain glucose concentration. Indeed, blood glucose is lower in ketosis, so according to Leybaert12 and Leybaert et al.45 brain glucose level would be expected to be even lower, a point confirmed by the inverse correlation between blood glucose and 18 F-FDG uptake (Fig. 3). Higher plasma glucose on the KD compared to F can be at least partially explained by the much higher plasma glycerol on the KD (600 versus 40 ± 0.0 μmol/l) because glycerol is a good substrate for gluconeogenesis. Nehlig et al.46 recently reported the impact of a KD on brain glucose and ketones transporters of the genetic absence epilepsy rat from Strasbourg. They observed that despite a decrease in plasma glucose and a large increase in beta-HB, the KD did not affect the level of expression of GLUT1 or MCT1 at the blood–brain barrier. These results are consistent with ours concerning GLUT1 but not MCT1 expression. However, in their study the rats were on the KD for 3 weeks and the difference in blood glucose was small (7.95 mmol/l in controls versus 7.20 mmol/l on the KD), and beta-HB rose only modestly to 0.92 mmol/l. AcAc data were not reported. Astrocytes play a critical role in the regulation of brain metabolic responses to activation. One mechanism proposed to describe the role of astrocytes is the astrocyte–neuron lactate shuttle hypothesis.47 This hypothesis suggests the existence of a coupling mechanism between neuronal activity and glucose utilization that involves an activation of aerobic glycolysis in astrocytes and lactate consumption by neurons. Since lactate transport in the brain occurs via transporters of the MCT family expressed in endothelial cells, astrocytes, and neurons, we speculate that during mild ketonemia, the increased brain expression of MCT tranporters could be also beneficial to the utilization of other alternative substrates such as lactate. However, we did not measure the expression of MCT in astrocytes and neurons in the present study. In the present study, we assessed the impact of mild ketonemia on blood fatty acid composition and more

Brain uptake of ketones and glucose in mild ketosis

particularly to polyunsaturates, since they are potentially ketogenic substrates (18:3n-3, alpha-linolenate) and may stimulate ketogenesis (20:5n-3, eicosapentaenoate).26 The marked rise in plasma 18:3n-3 on the KD can be easily explained by the presence of flaxseed oil in these diets which contains very high levels of 18:3n-3 (∼50% of total fatty acids). Since 18:3n-3 was undetected in the plasma of fasted animals in which ketonemia was more pronounced, it seems that its presence in the diet is not necessary to induce ketosis. The rise in plasma 18:0 and 14:0 could be attributed to their notable presence in butter and the high content of butter in the KD diet.48 However, the 6-fold elevation of 14:0 in the plasma of our KD-fed animals may contribute to them attaining higher ketonemia.

Conclusion The present study demonstrates that mild experimental ketosis, whether induced by F or by a KD, stimulates brain uptake not only of ketones (AcAc) but also of glucose (in the form of 18F-FDG). Mild ketonemia increased brain glucose uptake but did not increase brain microvessel GLUT expression, so the mechanism by which glucose uptake increased remains unclear. However, the KD clearly increased both the blood–brain barrier expression of MCT1 and stimulated brain 11 C-AcAc uptake. Mechanisms governing brain uptake of these two energy substrates should be considered independently because, unlike for glucose, it is the plasma concentration of AcAc that seems to drive the entry of AcAc into the brain. Brain glucose uptake was stimulated by both forms of mild ketosis (F and KD), an effect that seems to be due to ketonemia-induced changes in glycaemia. The dual-tracer approach with PET imaging was made possible by the use of the short-lived 11C-AcAc as a ketone tracer, a characteristic making it suitable for use in human studies. The present dual-tracer approach is very promising in the comparative investigation of mechanisms that could help supply fuel to the aging brain, particularly in AD, where brain energy supply becomes critically low.26

Acknowledgements This study was supported by CFI, CIHR, the Canada Research Chair secretariat (SCC), and by a post-doctoral fellowship to FP from the Department of Medicine, Université de Sherbrooke. The authors thank Jean-François Beaudoin and Mélanie Archambault for their excellent technical assistance.

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