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Original Studies |
Neurobiology Research Unit, Department of Neurology, and the PET and Cyclotron Unit, University Hospital, Rigshospitalet (S.G.H., G.M.K., O.B.P.), DK-2100 Copenhagen, Denmark; and Institute of Internal Medicine and Metabolic Diseases, Universita Degli Studi Di Napoli Federico II (B.C., A.P.), 80131 Naples, Italy
Address all correspondence and requests for reprints to: Steen G. Hasselbalch, M.D., Neurobiology Research Unit, Building 9201, National University Hospital, Rigshospitalet, 9 Blegdamsvej, DK-2100 Copenhagen Ø, Denmark. E-mail: sgh{at}pet.rh.dk
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Abstract |
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The reduction in K1* was compatible with Michaelis-Menten kinetics for facilitated transport. Our findings indicate no major adaptational changes in the maximal transport velocity or affinity to the blood-brain barrier glucose transporter. Finally, hyperglycemia did not change global CBF or CMRglc.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using the iv double indicator method, we studied BBB glucose transport during acute hyperglycemia. Furthermore, we studied the regional cerebral glucose metabolism (rCMRglc) before and during acute hyperglycemia using positron emission tomography with [18F]fluorodeoxyglucose as the tracer (PET-FDG). The questions to be addressed were the following. Does BBB glucose transport change during acute hyperglycemia? Is CMRglc affected by marked acute elevations in blood glucose levels?
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Under local analgesia, catheters were inserted percutaneously low on the neck into the internal jugular vein, in the radial artery, and in two antecubital veins as previously described (3). BBB transport of glucose was studied twice during hyperglycemia using the double indicator method; one measurement was performed approximately 15 min after a constant level of hyperglycemia had been obtained, and the other was performed after 2 h of steady state hyperglycemia. Due to technical difficulties, BBB permeability could not be determined in one subject. BBB permeability during hyperglycemia was compared with BBB permeability studied in a normoglycemic control group of age-matched subjects (3). Regional CMRglc was studied twice with PET-FDG in the same subject on 2 separate days: in a normoglycemic control condition and in a hyperglycemic condition.
Hyperglycemic clamp
The hyperglycemic, normoinsulinemic clamp was induced by a constant iv infusion of somatostatin (0.6 mg/kg·h) and insulin (0.15 mIU/kg·min), dissolved in isotonic saline, and infused in an antecubital vein by two separate pumps at the rate of 10 mL/h. The desired plasma glucose level of approximately 15 mmol/L was reached within 45–85 min by a variable infusion of 20% glucose, and plasma glucose was clamped at this level.
In the control condition, somatostatin and insulin were infused at the same rate as during the hyperglycemic condition, and plasma glucose was kept constant by a variable infusion of 20% glucose.
BBB permeability measurements
The iv double indicator method was used for estimation of BBB transfer variables. The iv approach was developed in our laboratory and has been described in detail previously (14). In brief, a 5- to 10-mL bolus containing the test substance [7 megabecquerels (MBq) [3H]glucose] and three BBB-impermeable reference substances [7 MBq 24Na+, 40 MBq [99mTc]diethylenetriamine pentaacetic acid ([99mTc]DTPA), and 0.4 MBq 36Cl-] was injected iv through an antecubital catheter. Starting 2–3 s before injection, 1-mL blood samples were continuously collected from the radial artery and jugular vein for 50 s by means of a sampling machine (Ole Dich Instrumentmakers, Hvidovre, Denmark) at a fixed interval of 1.3 s. Blood samples were centrifuged, and after at least 3 weeks 3 mL scintillation fluid (Picofluor 40, Packard, Downers Grove, IL) was added to 300-µL plasma samples, and ß-emission was counted (Packard PA 800-CA) with spillover and quench corrections by external standardization.
To correct for differences in the brain input of test and reference substances due to iv injection, a five-parameter Dirac impulse response for passage through the cerebrovascular bed was computed from the input and output of the reference substance. This response was then combined with the single membrane (well mixed) model of the brain (14) and convolved with the arterial input curve of the test substance to yield a theoretical test output curve, which was iteratively compared with the actual test output curve. When cerebral blood flow (CBF) was known, the model variables could be obtained by minimizing the sum of square of the differences between the theoretical and the measured outflow test curve by means of the simplex method. CBF was measured using the Kety-Schmidt technique for measurement of global CBF as previously described (15).
Estimates for the following parameters were obtained: PS1, the permeability surface area product from the blood to the brain, and E, the average unidirectional extraction. PS2/Ve, the PS product from the brain to the blood divided by the tracer distribution space was usually also obtained, but in hyperglycemia, PS2 approached zero, and values are not reported here. The unidirectional clearance, K1, was calculated from E x CBF. In application of the iv double indicator technique, it is assumed that the iv injected bolus mixes completely with blood before arrival at the brain capillaries, so that systemically measured arterial blood substrate concentrations equal those of the brain capillaries. This assumption seems acceptable because the bolus must pass through the venous system to the heart and the lungs before arriving at the carotid artery, and during this long passage it is likely that the bolus completely mixes with systemic blood. In a previous study this has also been experimentally verified (3).
Determination of CMRglc by PET-FDG
We used a PC4096+pet camera (General Electric Medical Systems,
Milwaukee, WI) yielding 15 consecutive slices with a slice thickness of
6.7 mm and a spatial resolution in the image plane of 6.7 mm. Slices
were placed parallel to the canthomeatal line (CM line: a line through
the lateral canthus of the eye and the external meatus of the ear) with
midslice planes from approximately 10–103 mm above the CM line. After
placement of the subject in the scanner, a transmission scan was
performed immediately before the activity scan for attenuation
correction. At the start of the scanning, 185–210 MBq FDG in 10 mL
saline were injected as a bolus over 20 s through an antecubital
catheter followed by 5–10 mL saline at the same infusion rate.
One-milliliter blood samples were drawn simultaneously from the jugular
vein and the radial artery at 10-s intervals from 0–3 min, at 20-s
intervals from 3–5 min, at 1-min intervals from 5–10 min, at 2-min
intervals from 10–20 min, and at 5-min intervals for the rest of the
scanning period. FDG blood samples were immediately placed one ice and
centrifuged, and 500 µL plasma were taken for 
-counting (COBRA
5003, Packard Instruments, Downers Grove, IL). Dynamic scanning was
started at time zero with the following scan sequence: 10 6-s
scans (0–1 min), 3 20-s scans (1–2 min), 8 1-min scans (2–10 min), 5
2-min scans (10–20 min), and 8 5-min scans (20–60 min). Regional
CMRglc was calculated pixel by pixel from the
time-activity curves in brain and blood using the Patlak Plot method
supplied with the standard GE 4096 software. The absolute time interval
from 10–45 min was used to calculate the net clearance of FDG (K1).
The transfer coefficients for FDG BBB transport
(K11 and k21, inward and
outward transports, respectively) and phosphorylation
(k31) were estimated as described by Sokoloff and
co-workers (16). CMRglc was calculated from
CMRglc = (Cp/LC) x
K1, where Cp is the mean plasma glucose
concentration during the scan period, LC is the FDG lumped constant,
and K1 is the slope of the Patlak plot described above. LC was
calculated as LC =
(k31/k3) +
((K11/K1) -
(k31/k3))
(K1/K11) (17), where the transport
coefficient (K11/K1) was set at 1.48, and the phosphorylation
coefficient (k31/k3) was
set at 0.39 (15, 18).
Mean CMRglc values for several cortical and subcortical regions were determined using a computerized brain atlas (19). With this software, the last scan in the dynamic sequence (55–60 min) was resliced by linear and nonlinear transformation into standard brain slices, and slices from approximately 50–90 mm above the CM line were used for the regional analysis. Global CMRglc was calculated from whole slice regions of interest weighted with their area corrected for 2.5% cerebrospinal fluid space (20).
Determination of CBF
Global CBF was measured by the Kety-Schmidt technique (21) in the desaturation mode, using 133Xe as the flow tracer. Cerebral venous blood and arterial blood were sampled from the internal jugular vein and the radial artery, respectively, as previously described in detail (22). In brief, the brain was saturated by an iv infusion of 133Xe dissolved in saline at a constant rate of approximately 15 MBq/min for 30 min. Blood samples were obtained at -2, -1, 0, 0.5, 1, 2, 3, 4, 6, 8, and 10 min, where 0 denotes the time when the infusion was terminated and placed in sealed vials for counting in a well counter (COBRA 5003, Packard Instrument). The measured CBF values were corrected for the systematic overestimation of flow values due to incomplete tracer washout at the end of the measurement period (22). Assuming a constant rate of cerebral oxygen metabolism during the experiment, CBF was corrected to the time of the BBB measurements as previously described (18).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During hyperglycemia, glucose was infused at a mean rate of 125
mL/h (range, 54–275 mL/h), and the plasma glucose concentration
increased to a mean steady state level of 15.5 ± 0.7 mmol/L. CBF
measured during acute hyperglycemia was 47.3 ± 5.8 mL/100
g·min. No significant differences in any of the measured BBB
parameters or in the unidirectional influx were observed between the
first and second BBB measurements (Table 1
). The mean unidirectional clearance of
FDG (K11) obtained by dynamic PET decreased by
45% from 0.110 ± 0.013 in normoglycemia to 0.061 ± 0.006
in hyperglycemia (P < 0.0002, by paired t
test; Table 2).
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Regional CMRglc was measured in several
cortical and subcortical regions (Table 2
).
CMRglc was significantly increased in white
matter in centrum semiovale, whereas glucose metabolism in cortical,
and subcortical gray matter regions remained unchanged. Despite the
changes in white matter glucose metabolism, global
CMRglc was constant in hyperglycemia (Table 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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No significant differences were found between the two BBB permeability studies performed at the start and at the end of the hyperglycemic clamp, suggesting that BBB glucose transport capacity does not change within hours of acute hyperglycemia. It should be noted that the acute hyperglycemic condition used in the present study was unphysiological in the sense that insulin secretion was suppressed to control for other variables during the study. Because of the small number of subjects and the variation in the data, the possibility of a type 2 error must also be considered. Further, variation in the data could have been induced by the use of a separate control group. On the other hand, the absolute value for PS1 during acute hyperglycemia was fully compatible with that obtained from literature values for Michaelis-Menten parameters. Using mean values from Ref. 23 for Tmax (maximal transport velocity) and Kt (the half-saturation constant), and assuming Kd to be 0.01 mL/100 g·min, the excepted value for PS1 calculated on the basis of Tmax and Kt was not significantly different from the determined PS1 at 2 h of hyperglycemia (0.042 ± 0.001 vs. 0.059 ± 0.032 mL/100 g·min; P > 0.05, by paired t test).
BBB transport of FDG obtained with PET (K11) decreased by 45% compared with normoglycemia. Because BBB hexose transport follows Michaelis-Menten kinetics for facilitated diffusion, increases in blood glucose induce a decrease in K11 because of the competitive inhibition of the glucose transporter. The PS1 obtained during hyperglycemia by the double indicator method in the present study was, however, not significantly different from PS1 values previously obtained in normoglycemia [0.076 ± 0.010 mL/100 g·min (3)]. We ascribe the discrepancy in the unchanged PS1 values and the decrease in K11 to the fact that the sample size was small. Although Michaelis-Menten parameters for glucose BBB transport were not determined in this study, the decrease in K11 could be fully explained by the increase in blood glucose without implying changes in the Michaelis-Menten parameters. We acknowledge, however, that this negative conclusion is inferred from previous data and should be corroborated in future studies, in which other methods may allow for repeated measurements, i.e. functional magnetic resonance (24). No other studies in humans have to date confirmed our findings in acute hyperglycemia. Studies performed in diabetic subjects have not shown changes in BBB glucose transport: With [methyl-11C]glucose and PET, Brooks and co-workers (10) studied glucose transport in four diabetic subjects and found no changes in BBB parameters during normoglycemia and chronic hyperglycemia compared with normal controls. In accordance with these results, using [11C]glucose and PET, Gutniak and co-workers (11) demonstrated no differences in unidirectional glucose clearances from blood to brain in six insulin-dependent diabetic subjects or in control subjects. Finally, Fanelli and co-workers found BBB transport unchanged in poorly controlled diabetic subjects compared with that in normal subjects (12). In two experimental studies in rats, BBB glucose transport was evaluated during acute hyperglycemia and was unchanged (8, 9). Thus, although experimental data for glucose transport during acute hyperglycemia are scarce, the findings are in agreement with the present results. However, the possibility that the negative conclusion of the present study was due to a small number of subjects and variation in the data must be borne in mind.
The unidirectional influx of glucose increased during hyperglycemia (Jin), but as the net uptake of glucose remained unchanged, the glucose efflux from the brain must have increased as well. The physiological significance of this presumed increase in glucose flux across the BBB remains unclear.
Cerebral glucose metabolism
CMRglc measured by PET-FDG was unchanged during acute hyperglycemia. The estimation of CMRglc by PET-FDG depends on the lumped constant, LC, which is the conversion factor between FDG and glucose net uptake. LC decreases during hyperglycemia because of changes in the brain distribution volumes of glucose and FDG (25, 26). In line with this observation, we found a 30% decrease in LC during hyperglycemia. We calculated LC directly from the FDG transfer coefficients and found a value of 0.81 in normoglycemia, which is higher than the standard value of 0.52 normally applied in human PET-FDG (27). Direct estimation of LC from global net uptake of FDG and glucose suggests, however, that this value is considerably underestimated, and the value of 0.81 agrees with previously obtained values for LC in our laboratory (15). The calculation of LC depends on the assumption that the transport and phosphorylation coefficients are constant with changes in blood glucose concentration and, further, that they are uniform throughout the brain tissue. Both assumptions have been verified for the transport coefficient (18), and it is reasonable to assume that they are also valid for the phosphorylation coefficient, as previously argued by Sokoloff and co-workers (16). Thus, we conclude that CMRglc did not change during acute hyperglycemia, and experimental studies support this conclusion; using the deoxyglucose method in rats, Orzi and co-workers (28) and Brøndsted and Gjedde (29) found no change in CMRglc during acute hyperglycemia. Likewise, using labeled glucose in rats, Duckrow and co-workers could not demonstrate changes in CMRglc during acute hyperglycemia (8). No studies of brain glucose metabolism during acute hyperglycemia have been performed in humans. In poorly controlled diabetic subjects, CMRglc has been found to be unchanged, suggesting that chronic elevated blood glucose levels do not change CMRglc (12), in line with our observations in acute hyperglycemia.
The regional analysis of CMRglc surprisingly showed that white matter CMRglc increased during hyperglycemia. At present, we have no explanation for this finding, which should be corroborated in future studies.
Cerebral blood flow
In experimental studies both acute and chronic hyperglycemia have been found to be associated with a decrease in CBF (30, 31, 32). This flow reduction could not be explained by changes in CMRglc (30). The decreased CBF has been suggested to have significant pathophysiological consequences in experimental hyperglycemia, and if these findings apply to humans, they may further increase the ischemic brain damage in hyperglycemic stroke patients. In the present study the mean CBF value of 47.3 ± 5.8 mL/100 g·min was identical to previously obtained values in normoglycemia (15, 22). Studies in humans also found no change in CBF in poorly controlled diabetes (12), suggesting that neither acute nor chronic hyperglycemia per se induces major changes in CBF.
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Acknowledgments |
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Footnotes |
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Received November 6, 2000.
Revised January 18, 2001.
Accepted February 5, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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of the lumped constant of the
deoxyglucose method in rat brain: determination of lambda and lumped
constant from tissue glucose concentration or equilibrium brain/plasma
distribution ratio for methylglucose. J Cereb Blood Flow Metab. 11:25–34.[Medline]
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