1 Uppsala University PET Centre, University Hospital Uppsala, S-75185 Uppsala; Departments of 2 Endocrinology and Physiology, 4 Clinical Neuroscience, and 6 Clinical Physiology, and 5 Karolinska Pharmacy, Karolinska Hospital, S-17176 Stockholm; and 3 Section Endocrinology, Department of Medicine, University Hospital of Trondheim, N-7006, Norway
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ABSTRACT |
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Using
R--[1-11C]hydroxybutyrate and positron emission
tomography, we studied the effect of acute hyperketonemia (range
0.7-1.7 µmol/ml) on cerebral ketone body utilization in six
nondiabetic subjects and six insulin-dependent diabetes mellitus (IDDM)
patients with average metabolic control (HbA1c = 8.1 ± 1.7%). An infusion of unlabeled R-
-hydroxybutyrate was
started 1 h before the bolus injection of
R-
-[1-11C]hydroxybutyrate. The time course of the
radioactivity in the brain was measured during 10 min. For both groups,
the utilization rate of ketone bodies was found to increase nearly
proportionally with the plasma concentration of ketone bodies (1.0 ± 0.3 µmol/ml for nondiabetic subjects and 1.3 ± 0.3 µmol/ml
for IDDM patients). No transport of ketone bodies from the brain could
be detected. This result, together with a recent study of the tissue
concentration of R-
-hydroxybutyrate in the brain by magnetic
resonance spectroscopy, indicate that, also at acute
hyperketonemia, the rate-limiting step for ketone body utilization
is the transport into the brain. No significant difference in transport
and utilization of ketone bodies could be detected between the
nondiabetic subjects and the IDDM patients.
-hydroxybutyrate; blood-brain barrier; positron emission
tomography
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INTRODUCTION |
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KETONE BODIES SUPPLEMENT GLUCOSE as a fuel of the brain. The role of ketone bodies becomes important during special physiological conditions, such as long-term fasting. The resulting hyperketonemia is coupled to increased uptake and oxidation of ketone bodies in the brain. A parallel decrease in glucose oxidation maintains the total energy balance (8).
Type 1 diabetes in the poorly treated state is also characterized by hyperglycemia and hyperketonemia caused by insulinopenia. However, most type 1 diabetic patients also have 24-h levels of plasma ketone bodies during normal insulin treatment that are somewhat higher than those in nondiabetic subjects (7). Hyperketonemia will increase the uptake of ketone bodies in the brain, resulting in an increased oxidation of ketones. Such an increase would be additive to the excessive glucose uptake due to hyperglycemia and, unless counteracted by other regulation, would exacerbate the excess energy being delivered to the brain. Chronic hyperglycemia is known, at least in animals, to downregulate glucose transporters in the brain (13), thereby decreasing the glucose load to the brain. Analogously, ketone uptake and oxidation in the brain could also be subject to regulation in diabetes, although, to our knowledge, this has not previously been tested in subjects with type 1 diabetes.
A method has previously been developed for measuring regional cerebral
utilization of ketone bodies in humans with positron emission
tomography (PET) using R--[1-11C]hydroxybutyrate
(
-[11C]HB) as tracer (1). The method was
applied in studies of healthy male subjects at normoketonemia. The
plasma concentration of R-
-hydroxybutyrate (
-HB) was in the range
of 0.02-0.09 µmol/ml. Three main features of ketone body
utilization were observed. First, ketone body utilization was found to
increase almost linearly with increasing concentration of ketone bodies
in arterial plasma. Second, the uptake of ketone bodies could be well
described by a model with a single rate constant, indicating that the
uptake is essentially irreversible. Third, the tissue concentration of
ketone bodies was found to be very low, suggesting that the transport
across the blood-brain barrier (BBB) is the rate-limiting step for
ketone body utilization.
As early as 1971, Daniel et al. (4) reported that the transport of ketone bodies across the BBB in rat is essentially irreversible. Together with other studies on rats, this led to the hypothesis (2) that the carrier for ketone bodies is reversibly used for brain-blood transport of pyruvate and lactate.
The aim of the present study was to compare the ketone body utilization in nondiabetic subjects and subjects with insulin-dependent diabetes mellitus (IDDM) at hyperketonemia and to investigate whether the features of ketone body utilization previously observed at normoketonemia also persist at hyperketonemia.
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MATERIALS AND METHODS |
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Subjects. The experimental procedure was approved by the Ethics and Radiation Safety Committees of the Karolinska Hospital and Institute. Before the subjects agreed to participate, they received written and oral information about the nature, purpose, and possible risks of the experiment. Six healthy male subjects served as a control group. Their mean age was 30.0 ± 7.4 yr (range 23-44 yr), and their body weight was 81 ± 10 kg. Six male patients with type 1 diabetes mellitus participated in the study. Their mean age was 32.0 ± 5.1 yr (range 23-38 yr), their body mass index was 24.5 ± 2.9 kg/m2, and their body weight was 78 ± 10 kg. The age at onset of the disease was 15.2 ± 10.3 yr, and the duration was 16.3 ± 6.9 yr. All of them were treated with multiple insulin injections except one patient, who was treated with continuous subcutaneous insulin infusion. Their usual daily dose of insulin was 37.0 ± 4.5 U of short-acting and 19.2 ± 4.8 U of long-acting insulin. The level of glycosylated hemoglobin (HbA1c) was 8.1 ± (SD) 1.7%. (The upper level of normal HbA1c was 5.6%.). None of the patients had recently experienced a serious hypoglycemic episode. Minimal signs of background retinopathy were present in four patients. One patient had macroalbuminuria but no other signs of nephropathy. Another patient was treated with antihypertensive drugs and a low dose of prednisolone because of glomerulonephritis due to systemic lupus erythematosus. Blood pressure was normal in all patients (<140/90 mmHg).
Experimental procedures.
To obtain ~1 mmol/l -HB in the plasma at steady state, a primed
infusion of a racemic mixture of unlabeled
-HB was started 1 h
before the PET scan in all of the subjects. The proportion between the
R and S forms was close to 1:1. The priming dose, given during the
first 20 min (19), was twice the subsequent continuous
infusion dose, which lasted to the end of the PET scan. The priming
dose was 6 mg · kg
1 · min
1
except in two cases (IDDM patients), when 3 and 4.5 mg · kg
1 · min
1,
respectively, were administered.
Data processing. The images of the radioactivity concentration in the form of matrices with 128 × 128 pixels for each of 47 slices, with a center-to-center distance of 3.125 mm, were reconstructed by standard software provided by the manufacturer.
To calculate the rate of uptake ofKinetic analysis.
Because of the low uptake of ketone bodies in the brain, determination
of regional ketone body utilization was not attempted in this study.
Identification of brain regions in PET studies using
-[11C]HB requires auxiliary measurement with magnetic
resonance imagery or some PET tracer with high uptake in the brain.
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RESULTS |
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Figure 1 shows the average
[-HB]plas as a function of time after start of the
infusion for the nondiabetic subjects and the IDDM patients. As can be
seen from Fig. 1, the start of the infusion (loading dose) caused an
immediate, rapid rise of [
-HB]plas, but after 30 min
the rise was much less pronounced, and the concentration approached a constant level. As the error bars indicate, at a given
time point [
-HB]plas varied considerably between the
experiments. In contrast, for each separate experiment, the time
variation of [
-HB]plas was small during the PET
scan (60-70 min). In this interval, the difference between the
lowest and highest value of [
-HB]plas within each
particular experiment was 3.3 ± (SD) 2.7% for the nondiabetic
subjects and 6.9 ± 2.5% and for the IDDM patients. In the same
time interval, [
-HB]plas was found to be somewhat
higher for the IDDM patients than for the nondiabetic subjects
(1.28 ± 0.31 and 0.98 ± 0.33 µmol/ml, respectively). In
comparison, during the previous study, at normoketonemia
[
-HB]plas was 0.04 ± 0.03 µmol/ml. Thus, in
the present study, [
-HB]plas was, on the average, more
than 20 times higher than in the previous study at normoketonemia.
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Figure 2, A-D,
shows examples of uptake curves and model fits for one control subject
(Fig. 2, A and C) and one IDDM patient (Fig. 2,
B and D). The results of applying the 1k model
and the Gjedde-Patlak analysis to the data are displayed. Figure 2
illustrates that both models give satisfactory fits of the data over
the whole time interval for both control subjects and IDDM patients.
For the healthy control subjects, Kket was found
to be 0.093 min1 with the 1k model, whereas for the IDDM
patients Kket was found to be 0.091 min
1 with the same model. When the 3k model was applied
(not shown), the compartment of unmetabolized
-[11C]HB
was always found to be small compared with the compartment of
metabolized
-[11C]HB. Therefore, the fit is close to
the one obtained with the 1k model, and consequently the corresponding
values of Kket are also close to each other.
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Figure 2, C and D, illustrates that the data in
the Gjedde-Patlak plot are well described by a straight line over the
whole measuring interval and that the intercept DVapp is
close to zero for all subjects. For many other tracers, it is possible
to distinguish an initial phase in the Gjedde-Patlak plot, before the
reversible part has reached equilibrium with the input function, but
clearly such a phase cannot be distinguished in this study. For the two distributions displayed in Fig. 2, C and D,
DVket was found to be 0.0011 and 0.0017, whereas
Kket was found to be 0.093 and 0.089, respectively. The initially large scatter in the data points is due to
difficulties in describing the dominant blood peak accurately in the
model. The radioactivity concentration is measured in arterial blood.
However, ~80% of the blood in the brain is venous, in which the
tracer has a different time course than in arterial blood.
The F-test and the Akaike information criterion (AIC) were applied to discriminate between the models. When testing the 1k model against the 3k model, i.e., when testing whether the two extra parameters are needed, significance was reached in 4 experiments (2 control subjects and 2 IDDM patients) out of 12 (level of significance 0.05) when the F-test was applied, and the 3k model was better in 7 experiments (4 controls and 3 IDDM patients) according to the AIC. It should be kept in mind that the F-test is strictly applicable (i.e., provides correct levels of significance) only for hypotheses that are linear in the parameters to be fitted. Clearly, the statistical analysis gives no preference for any of the models. Visually, good fits are obtained in all experiments for all models. For each experiment, nearly the same values of Kket were obtained with the different models. Unless otherwise stated, only Kket and the corresponding CMRket obtained with the 1k model are used in the following summary.
A summary of measured and fitted quantities (using the three models
discussed) is presented in Table 1. For
purposes of comparison, the corresponding quantities obtained in the
previous study at normoketonemia by use of the same tracer
(1) are also presented. Sample averages and standard
deviations are given. The values are averages over the brain, because
only average uptake curves have been used. It should be noted that the
presented values of concentrations in the plasma, such as [-HB],
in Table 1 are averages over the two or three measurements made during
the PET scan. In fact, most of the quantities stayed relatively
constant over the whole time interval (70 min) with the exceptions of
[
-HB], [glucose], and [glycerol]. [Glucose] fell 3-10%
in the nondiabetic subjects and 15-35% in the IDDM patients in
the time period before the PET scan, but it then stayed constant within
a few percentage points during the PET scan. The concentration of
glycerol fell rapidly during the first 30 min, from 0.044 ± 0.019 (average ± SD) and 0.031 ± 0.009 µmol/ml for nondiabetic
subjects and IDDM patients, respectively. The averages reached in the
time interval of 60-70 min are presented in Table 1. Attempts to
fit the 3k model to the data often terminated with
k2 and/or k3 at the
allowed upper limit for these parameters (50 min
1), and
therefore only upper bounds of DVket [=
k1/(k2+k3)]
can be given.
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Figure 3 shows CMRket vs.
[-HB]plas for each individual in the two groups and
also the corresponding data obtained in the previous study of
nondiabetic subjects at normoketonemia. The displayed line is the
result of a linear regression analysis of all data in the present and
the previous studies. The slope, intercept, and R value were
found to be 7.9 ± 0.5, 0.39 ± 0.54, and 0.97, respectively.
With data only from the present study, the three parameters became
6.9 ± 1.3, 1.7 ± 1.6, and 0.85, respectively. In the
previous study, the same parameters were found to be 10.7 ± 0.8, 0.032 ± 0.040, and 0.99, respectively. Thus there is a weak
indication that the slope decreases with increasing
[
-HB]plas, which means that the relationship between
[
-HB]plas and CMRket is not perfectly
linear over the range of [
-HB]plas in the present and
previous studies (0.02-1.74 µmol/ml). The regression
analysis of CMRket vs.
[
-HB]plas reveals no significant difference between the two experimental groups of the present study. For the nondiabetic subjects, the slope 7.1 ± 2.1 and intercept 0.9 ± 2.2 are
obtained, whereas for the IDDM patients the slope 4.6 ± 1.9 and
intercept 5.1 ± 2.6 are obtained.
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The relationship between plasma concentration and utilization is more
clearly seen in Fig. 4, which shows
Kket as a function of [-HB]plas
for all experiments in the present and the previous studies.
Kket and [
-HB]plas
are measured independently of each other, and therefore, from a
statistical point of view, the data in Fig. 4 are easier to handle than
the data in Fig. 3, where the x- and y-variables
have a factor ([
-HB]plas) in common. Clearly Kket has a tendency to decrease with increasing
[
-HB]plas. Linear regression gives the slope
0.0025 ± 0.0006, intercept 0.0114 ± 0.0006, and R
value 0.76 for all experiments. The slope is significantly different from zero (P < 0.0005). For this regression,
all models gave consistent results. Figures 3 and 4 show that it is
difficult to distinguish any difference between the nondiabetic
subjects and the IDDM patients at hyperketonemia. When data from only
the present study are used, the slope
0.0023 ± 0.0021 and
intercept 0.011 ± 0.002 are obtained for the nondiabetic
subjects, whereas the slope
0.0033 ± 0.0017 and intercept
0.013 ± 0.002 are obtained for the IDDM patients. With the
present statistics, these regression lines are not significantly
different from each other or from the regression line obtained
from the combined data in the previous and present studies.
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DISCUSSION |
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Tissue concentration of -HB
choice of model.
The 1k model gives a good fit of the uptake data for both nondiabetic
subjects and IDDM patients, indicating that the uptake of tracer across
the BBB is essentially irreversible. One parameter, Kket, describes the uptake in the tissue well
(Fig. 2, A and B). The successful fit of the
uptake data with this model implies that attempts to fit the data with
more complex models, such as the 3k model, result in unreliable
parameter estimates. This situation is a general feature of tracer
studies that use PET, because with this technique only the total
radioactivity concentration can be measured, which implies that the
compartmental structure that follows a nearly irreversible transfer is
very difficult to resolve. In particular, the PET experiment
alone gives unreliable information about DVket and the
related [
-HB]tiss. In fact, as good fits as with the
1k and 3k models are obtained with a constrained 3k model that forces
DVket to be large.
Metabolic rate of -HB
comparison between control subjects and
IDDM patients.
The data in Table 1 show that the values of the rate constant for net
utilization Kket, estimated with different
models, are close to each other. The good fits using the 1k model and the Gjedde-Patlak analysis show that, also at acute hyperketonemia, the
rate of utilization is very close to the unidirectional influx of
-HB across the BBB. Therefore, in this case, influx, uptake, and
utilization are synonymous.
Conclusions.
With plasma concentration of -HB in the range 0.02-1.74
µmol/ml, the brain tissue was found to react to increased
availability of ketone bodies by an increased net utilization of these
compounds. There was no sign of saturation of this process. This holds
true for both nondiabetic subjects and type 1 diabetic patients with average metabolic control. The transport of ketone bodies across the
BBB is found to be essentially irreversible. Together with a recent
study with MR spectroscopy showing low tissue concentration of
-HB
(15), the findings of this study indicate that, also at
acute hyperketonemia, the transfer from blood to brain is the rate-limiting step in ketone body utilization.
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APPENDIX |
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Kinetic Models Used
In the 1k model, it is assumed that the transfer of
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(A1) |
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(A2) |
In the 3k model, with one reversible and one irrreversible tissue
compartment and with three rate constants,
Ctiss(T) is expressed as
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(A3) |
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In the Gjedde-Patlak analysis, the operational equation is
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(A4) |
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ACKNOWLEDGEMENTS |
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This study was supported by grants from the Swedish Medical Research Council, MFR, project nos. K98-04F-12394-01, K97-04P-11319-03A, 04540, and 8276, and the Swedish Diabetes Association.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. Blomqvist, Uppsala Univ. PET Centre, UAS, 75185 Uppsala, Sweden (E-mail: gunnar.blomqvist{at}pet.uu.se).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 26, 2002;10.1152/ajpendo.00294.2001
Received 5 July 2001; accepted in final form 22 February 2002.
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