196 E
21
mM; K 50.079 min . For 30-day-old rats, glucose
random noise with the same Gaussian distribution as was
D
transport parameters were assumed to be equal to adult observed in the original data. Each of the simulated sets
values of the same species measured in a previous study was analyzed using the same fitting procedure as was
[24] where V CMR
55.8 and K 513.9 mM. Lactate performed with the original data, and the 2000 sets of fitted
max GLU
m
transport kinetic parameters assumed for the 30-day-old parameters were recorded. The Monte-Carlo procedure
cortex were based on those reported for adult rats by provided an estimate of the uncertainties in the fitted
21 21
Pardridge [39] V 50.12 mmol g
min ; K 51.9 mM;
parameters if the experiment were repeated 2000 times. To
max m
21
evaluate the significance of differences in the fitted param- K 50.028 min
.
D
eters between the 10- and 30-day-old groups, the 2000-row arrays of Monte-Carlo generated parameters were sub-
2.8. Calculation of fluxes in brain slices tracted one from the other, generating a 2000-row differ-
ence array. The P-value was calculated as the fraction of Glycolytic and TCA cycle fluxes were determined using
the elements that lay above or below zero for each fitted the same steady state metabolic model but modified to
parameter. The sensitivity of the calculated value of V
TCA
permit free communication between perfusate and the to assumed parameters is given in Appendix A.
extracellular space due to the absence of a blood–brain The statistical significance of comparisons between
barrier. Therefore glucose and lactate transport terms were cortical metabolic rates in vivo and in slices were evalu-
not included in the analysis. Injured cells at the surface of ated by a pair-wise Z-test.
the slice may have impaired their ability to aerobically
14
metabolize glucose. However, studies using C radio-
isotope labeled substrates [15], have estimated that these
3. Results
cells make up less than 2 of the glutamate pool which turns over slowly; therefore, this pool would be expected
13
3.1. Effects of [ C]glucose infusion on blood glucose to have a negligible contribution to glucose metabolism.
and lactic acid levels and their enrichments in 10- and
The TCA cycle flux V was determined from an
TCA
30-day-old rats in vivo analysis of the time courses of the enrichment of
glutamate-C as shown in Fig. 1B. In the construction of
13 4
The intravenous infusion of [1- C]glucose increased the model we made the following assumptions: intracellu-
total plasma glucose concentrations and fractional isotopic lar glucose-C labeling reaches a steady state enrichment
1
enrichments to similar and nearly constant levels in of 99 at t50 i.e. step function and lactic acid leaving
animals of both age groups. Average plasma glucose levels the tissue V
to the medium does not return, i.e.
efflux
measured over the 60–90 min of the glucose infusion were V
50. Because no labeling was detected in glutamine,
influx
9.261.4 mM in 10-day-old and 10.461.2 mM in 30-day- V
was set equal to zero. The values of V and the
gln x
old rats. The fractional enrichment of plasma glucose-C
1
concentration of total glutamate were assumed to be equal was 0.4560.03 and 0.4160.02 in 10- and 30-day-old rats,
to the values determined in vivo for the respective postnat- respectively.
al ages. The total glucose utilization was determined as the Average plasma lactic acid was lower in the 10-day-old
sum of the rates of glucose oxidation calculated from the 1.9560.46 mM compared to 30-day-old rats 2.7260.18
TCA cycle flux and 1 2 times the rate of lactate appear- mM but remained relatively constant throughout the
ance in the perfusion medium. Lactate in the perfusion labeled glucose infusion in each group. Blood lactate-C
21 3
medium was converted to mmol g wet weight based on
13
was labeled rapidly from [1- C]glucose with average measured protein and wet tissue weights determined for
fractional enrichment values over the course of the infu-
21
both age groups 8069 mg g n56, mean6S.D. and
sion of 0.09660.033 and 0.06860.007 in the 10- and
21
106614 mg g n56 wet weight for 10- and 30-day-old
30-day-old rats, respectively. cortex, respectively. Total protein weights in brain slices
were 3.561.2 mg n517, mean6S.D. and 4.460.7 mg 3.2. Rates of the TCA cycle and glucose oxidation in
n519 of protein for the 10- and 30-day-old tissue, the
10- and 30-day-old cortex in vivo respectively.
13 13
[1- C]Glucose is metabolized to pyruvate- C which
3
2.9. Error analysis enters the mitochondrial TCA cycle at the level of citrate
13
and quickly flows to a-ketoglutarate- C Fig. 1. Be-
4
A Monte-Carlo scatter analysis was used to determine cause a-ketoglutarate is in exchange with the much larger
the standard deviations of metabolic rates or other fitted cytosolic pool of glutamate, the label is trapped initially as
13
parameters derived from each group of data [24–26], and glutamate- C and with time other positions C , C , and
4 2
3
those standard deviations were used to evaluate statistical C will be labeled in subsequent turns of the TCA cycle.
1
significance between groups. Briefly, 2000 simulated noisy As shown in Fig. 2, the rate of cortical glutamate
data sets were created from the least-squares fit by adding labeling, which is determined by the TCA cycle flux, was
E .J. Novotny et al. Brain Research 888 2001 193 –202
197
strongly age-dependent.
Brain glutamate-C
labeling
4
reached a constant level within 15 min FE511.062.3 in the 30-day-old cortex, whereas a comparable enrichment
11.561.2 was not observed until 90 min in the 10-day- old brain, despite similar plasma enrichments of glucose-
C for rats in both age groups.
1 13
The rate of C labeling of glutamate-C
was slower
3
than glutamate-C for rats in both age groups, as expected
4
from previous studies in adult rats [13,27], but this too was age-dependent and considerably slower in 10-day-old as
compared to the 30-day-old cortex. The metabolic rates were determined by the best fit of
the model depicted in Fig. 1A to the isotopic turnover of
13
glutamate-C and C
during the [1- C]glucose infusion
4 3
Fig. 2. The TCA cycle flux V , which was calculated
TCA
from the rate of glutamate-C turnover, increased 4.4-fold
4 21
P50.001 from 0.4660.12 to 2.0160.54 mmol g
21
min , in the 10- and 30-day-old cortex, respectively
Table 1. The calculated rate of cortical glucose oxidation, CMR
, increased in nearly equal proportion |4.1-
GLU Ox
fold, P50.001 to the TCA cycle flux over the same
21 21
period, from |0.16 to |0.66 mmol g min
, respective- ly. At isotopic steady state, the flow of unlabeled carbon
through acetyl-CoA and into the TCA cycle is reflected as a dilution of the glutamate-C
enrichment. The dilution
4
flux V , when expressed as a percentage of V , was
dil TCA
not significantly different between 10-day-old 28611 and 30-day-old 3466 rat cortex under the conditions of
the glucose infusion. The most likely potential sources of the dilution of cortical glutamate labeling in vivo was
oxidation of plasma lactic acid and ketone bodies. In
13
contrast to the rapid C labeling of plasma lactic acid,
ketone bodies were not labeled by the glucose infusion.
13
The C labeling of glutamate involves the movement of
13
Fig. 2. C isotopic labeling of neocortical glutamate during an intraven-
the label from mitochondrial a-ketoglutarate to cytosolic
13
ous infusion of [1- C]glucose in 10- and 30-day-old rats. The ordinate
13
glutamate through isotopic exchanges mediated by en-
represents the concentration of neocortical glutamate- C upper panel
4 13
zymes and transporters associated with the malate-aspar-
and glutamate- C lower panel at discrete times during the intravenous
3 13
[1- C]glucose infusion for each age group. The continuous lines repre-
tate shuttle. The rate of isotopic label exchange between
sent the best fit of the metabolic model Fig. 1A to the time course data.
a-ketoglutarate and glutamate V , which is derived from
x
Table 1 Metabolic rates in 10- and 30-day-old cortices
b
Glutamate V
V CMR
CMR
TCA dil
GLU Ox GLU Tot
21 21
21 a
21 21
21 21
mmol g mmol g
min V
mmol g min
mmol g min
TCA [
[
10-day-old In vivo
8.761.1 0.4660.12
28611 0.16
c
Slice 8.7
0.1760.03 2964
0.06 0.09
[[ [[
30-day-old In vivo
13.161.2 2.0160.54
3466 0.66
c
Slice 13.1
0.3460.02 2762
0.12 0.16
a
V was not significantly different for either age group.
dil b
CMR 5CMR
11 2 DLac Dt.
GLU Tot GLU Ox
c
The concentration of glutamate in the brain slices was assumed to equal the concentration measured in vivo for the respective age group. Significantly different from 10-day-old slice, P,0.0005.
Significantly different from 10-day-old cortex in vivo, P50.001.
[
Significantly different from 10-day-old slice P50.029, pair-wise Z-test.
[[
Significantly different from 30-day-old slice P50.002, pair-wise Z-test.
198 E
a simultaneous fit of the model to the glutamate-C and C Metabolic fluxes were determined by fitting the metabol-
4 3
data, differed greatly between the two age groups. Whereas ic model depicted in Fig. 1B to the glutamate-C enrich-
4
the value of V in the 30-day-old cortex was fast, .200 ment time course Fig. 3. TCA cycle flux as derived from
x 13
21 21
the C isotopic turnover of glutamate Table 1, was
mmol g min
, and similar to previous findings in adult 2-fold greater P,0.0005 in 30-day-old slices 0.3460.02
rats [27], V was much less in the 10-day-old cortex, only
x 21
21 21
21
mmol g
min compared
to 10-day-old
slices 3.161.6 mmol g
min . The slower a-ketoglutarate
21 21
0.1760.03 mmol g min
. The percentage increase in glutamate exchange flux in the more immature brain may
glucose oxidation 200 was similar to the increase in reflect reduced activity of the dicarboxylate carriers associ-
V between the two age groups. Labeling of glutamate-
ated with the malate-aspartate shuttle, which transfers
TCA
C was minimal over the 60–90-min perfusion period and redox equivalents from NADH in the cytosol to the
3
the sensitivity was not high enough for accurate quantita- mitochondria during respiration.
tion. The low enrichment in glutamate-C is consistent
3
with the lower TCA cycle flux in the slice compared to the 3.3. Rates of the TCA cycle and glucose utilization in
cortex in vivo. 10- and 30-day-old brain slices
3.4. Comparison of metabolic fluxes in the cortex in
13 13
C label from [1- C]glucose was incorporated into vivo and in brain slices in vitro
lactate-C and glutamate-C
in both 10- and 30-day-old
3 4
cortical slices. Rates of lactate production measured in the TCA cycle rate was significantly different 6-fold great-
perfusates were linear for 10- and 30-day-old slices over er in 30-day-old cortex of rats in vivo compared to the
the measured 60–90-min interval with average values of slice preparation in vitro P50.002, pair-wise Z-test.
21 21
0.065 and 0.075 mmol g min
, respectively. In contrast Although the TCA cycle rate was significantly different
to lactate labeling, the time courses of glutamate-C
4
between the 10-day-old cortex in vivo and the slice in vitro labeling were hyperbolic for both age groups Fig. 3. The
P50.029, the difference between them was less 2.7-fold fractional enrichment of glutamate-C reached 0.3260.04
4
greater in vivo than in vitro than at 30 days. and 0.3360.04 in the 10- and 30-day-old slices by 120
min, respectively, which was not significantly different from the enrichments at 90 min in either age group
P50.09 and P50.90, respectively; two-tailed t-test. Thus
4. Discussion
