University of Groningen
Quantitative on-line monitoring of cellular and cerebral energy metabolism
Leegsma-Vogt, Gepke Henriëtta
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Leegsma-Vogt, G. H. (2004). Quantitative on-line monitoring of cellular and cerebral energy metabolism
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4
DIFFERENTIAL GLUCOSE AND
LACTATE METABOLISM OF
PERFUSED CULTURED ASTROCYTES
ASSESSED WITH QUANTITATIVE ONLINE MONITORING
ABSTRACT
Both glucose and lactate are important substrates in brain metabolism. Astrocytes are
known to release lactate, and are suggested to have a feeding role for neurons. In this study
we investigated glucose and lactate metabolism in primary astrocytic cultures, using a
novel in vitro on-line monitoring unit. By placing cells in a specially designed flow-through
cell holder, we measured glucose use and lactate uptake/release every minute, using an online flow injection analysis (FIA) system with biosensors for glucose and lactate. Primary
astrocytic cultures were perfused with medium containing different concentrations of
glucose, lactate or a combination of both energy substrates. We measured a glucose use of
approximately 3.5 femtomoles/cell/minute, and found that glucose was the most preferred
substrate. Furthermore, the present experiments suggest the existence of large glycogen
stores in primary astrocytes, that are broken down and released as lactate. Although lactate
is taken up from the medium, medium supplemented with lactate did not alter glucose
metabolism, suggesting a separate lactate metabolic compartment in astrocytes.
CHAPTER 4
INTRODUCTION
Astrocytes, strategically positioned between the capillaries and the neurons, are thought to
play a role in neuronal energy metabolism (Pellerin and Magistretti, 2003;Forsyth et al.,
1996). According to the astrocyte-neuron-lactate-shuttle-hypothesis, cerebral glucose
uptake takes place in astrocytes, whereas the astrocytically formed and released lactate is
consequently metabolized by neurons (Magistretti et al., 1999;Pellerin et al., 1998). Indeed,
in vitro, substantial amounts of lactate are released by astrocytes (Walz and Mukerji, 1988),
which can be formed from both glucose and glycogen. Glycogen is localized in the brain
almost exclusively in astrocytes (Gruetter, 2003;Tsacopoulos and Magistretti, 1996;Peters
et al., 1991). In vitro astrocytic glucose and lactate metabolism is mainly studied using the
batch-wise approach, with relatively low temporal resolution. Quantification of metabolism
in terms of utilization per minute cannot be achieved using the batch-wise approach.
Recently, we described on-line monitoring techniques for brain slices and non-neural cells
with a high temporal resolution (Gramsbergen et al., 2003;Leegsma-Vogt et al., 2003a),
that are capable of quantifying substrate utilization per minute.
In the present study, we monitored glucose and lactate metabolism in primary astrocytic
cultures, using a modification of our previously described in vitro on-line monitoring unit
(Gramsbergen et al., 2003). We measured glucose use and lactate uptake/release by placing
cells in a specially designed flow-through cell holder, connected to an on-line flow
injection analysis (FIA) system equipped with biosensors for glucose and lactate. With our
on-line system we can estimate glucose and lactate consumption per cell per minute. Here
we show the energy substrate utilization of cultured astrocytes perfused with varying lactate
and glucose concentrations. This new method revealed indications for glycogen breakdown
from large glycogen stores in astrocyte metabolism, and evidence that lactate and glucose
are metabolized in non-interexchangeable pools.
MATERIALS AND METHODS
Cell cultures and cell chamber
The incubation of cell cultures and the cell chamber are essentially the same as described
earlier (Gramsbergen et al., 2003;Leegsma-Vogt et al., 2003a). In short, primary cells were
derived from mouse cortex (embryonic day 16-18). Cells were plated on poly-D-lysine
coated cell culture inserts (0.3x10exp6 cells/insert, Sigma Chemical Co, St. Louis, MO,
inserts: Millicell-CM, 12 mm, pore size 0.4 µm, Millipore Corporation, Bedford, USA) for
the monitoring of metabolism, and coated glass cover slips (0.15x10exp5 cells/glass,
Menzel, Germany) for immunocytochemistry. 6-12 inserts were plated per animal sacrifice.
Astrocytes were cultured serum free (Dulbecco´s modified Eagle medium (DMEM),
supplemented with penicilline (100 U/ml)/streptomycine (100 µg/ml), 1 mM Na-pyruvate,
0.5 mM glutamine, B27 (1x) (All Gibco, Invitrogen Corporation, Auckland, New Zealand),
or were cultured in DMEM with 10% fetal calf serum (FCS). The cells were incubated in
either high glucose (pre-incubation in 25mM for one week, thereafter incubation in 5 mM
glucose) or low glucose (incubation in 5mM) medium, at 37 °C and 95% O2, 5% CO2. On
the monitoring day, the culture insert was placed inside the cell chamber (figure 4-1) and
2
Chapter 4
GLUCOSE AND LACTATE METABOLISM OF PERFUSED CULTURED ASTROCYTES
connected to the flow injection analysis system. The cell chamber has minimal dead space,
with approximately 10 µL volume area on both sides of the insert membrane.
Flow injection analysis (FIA) system and biosensors
The FIA system with biosensors used here is a minor modification of our previously
described system (Gramsbergen et al., 2003). In short, the cell chamber is connected to the
analytical part of the set-up by an intercalated valve with a 20 nl internal loop (Vici-Valco
Instruments, Houston, USA). The valve injects a cell chamber sample every minute. By
applying underpressure, oxygenized (95% O2, 5% CO2) DMEM with 5 mM glucose is
pulled through the cell chamber towards the FIA with a Harvard 22-syringe pump (Harvard
Apparatus, South Natick, MA, USA).
Glucose and lactate are measured using glucose oxidase and lactate oxidase respectively,
placed in separate enzyme reactors together with horseradish peroxidase (all enzymes from
Roche, Mannheim, Germany). The cell chamber and the enzyme reactors are kept at 37 0C.
The current is measured with wall-jet-type electrochemical flowcells (VT-03, Antec
Leyden B.V., Zoetermeer, The Netherlands) and two potentiostates (Decade: Antec Leyden
B.V., Zoetermeer, The Netherlands; Amor: Spark Holland, Emmen, The Netherlands).
Figure 4-1: A solid drawing (A)
and a schematic diagram (B) of the
cell chamber. The central part of
the cell chamber is the insert (2).
Oxygenized medium is transported
through fused silica tubing (1), and
passes the insert. Arrows indicate
flow direction. The insert is placed
inside a holder (4) with two spacers
(3). The assembled cell chamber
has outer dimensions of 3.5 cm by
2.2 cm, with an inner diameter of
7.6 mm. The inner pieces of the
cell chamber are slightly convex
shaped (3%).
Before the experiment, an in vitro calibrationcurve is run on the FIA (glucose: 0, 2.5, 5, 10,
20 mM; lactate: 0, 1.25, 2.5, 5 and 10 mM). The glucose and lactate concentration is
measured every minute and the currents (in nano-amperes) are recorded on a double-pens
recorder (BD 112, Kipp en Zonen, Zoetermeer, The Netherlands) and by a data acquisition
program (Chromeleon, Dionex Corporation, Sunnyville, CA, USA).
3
Chapter 4
CHAPTER 4
Immunocytochemistry
The cell cultures on glass cover slips were fixated for 10 minutes in 4% para-formaldehyde,
19 ± 9 days after plating. After fixation, the cover slips were rinsed and stored in phoshatebuffered saline (PBS) until further processing.
The cover slips were used for glial fibrillary acidic protein (GFAP) and neuronal nuclei
(NeuN) staining: GFAP and NeuN stainings from the same culturing day with identical
incubation conditions were compared to analyze the percentage GFAP-positive and NeuNpositive cells. The cover slips were collected in 0.02M phosphate buffered saline (PBS, pH
7.4) and rinsed 3x10 minutes. For GFAP staining, the cover slips were incubated with a
goat polyclonal antibody raised against GFAP (Santa Cruz Biotechnology, 1:500 in 0.02M
PBS-Triton 0.25%, 2% normal rabbit serum) overnight at 4°C. For NeuN staining, a
blocking step was performed for 1 hour (0.02M PBS-Triton 0.25%, 3% normal sheep
serum). The cover slips were incubated with an mouse polyclonal antibody raised against
NeuN (Chemicon international, USA, 1:500 in 0.02M PBS-Triton 0.25%, 3% normal sheep
serum) overnight at 4°C. Subsequently, the sections were washed in 0.02M PBS (6x5 min,
pH 7.4) and incubated for 2 hours at room temperature with biotinylated rabbit anti goat
IgG (GFAP staining, Vector, 1:500 in 0.02M PBS, 2% normal rabbit serum) or biotinylated
sheep anti mouse IgG (NeuN staining, Amersham, 1:250 in 0.02M PBS, 3% normal sheep
serum). After rinsing with 0.02M PBS (3x10 min, pH 7.4) the immunoreactivity was
visualized with a standard ABC method (Vectastain ABCkit, Vector, 1 drop A + 1 drop B)/
10ml PBS for 2hrs). After washing with PBS 0.02M (3x10 min, pH 7.4) the peroxidase
reaction was developed with a DAB-nickel solution and 0.3% H2O2 (20mg DAB, 1.5g
NAS, 0.8203g NaAC in 50ml H2O). To stop the reaction the cover slips were washed with
0.02 M PBS (3x10 min, pH 7.4) and mounted on slides (with the cells between the slide
and the cover slip) with aquamount.
Experimental procedure and calculations
The “standard” medium consisted of oxygenized DMEM with 5 mM glucose. In this
standard medium the basal glucose consumption was calculated. Lactate uptake was studied
with medium containing lactate as the sole substrate (5 or 10 mM lactate in DMEM), and
with lactate-supplemented glucose medium (5 mM glucose medium with either 2.5 mM, 5
mM, or 10 mM lactate). DMEM without glucose was used to study glycogen breakdown.
All experiments were performed at a flowrate of 0.5 µl/min.
In some experiments, there was lactate output during lactate-medium incubation (with and
without glucose), which interfered with the calculation on lactate use. Therefore a
correction was applied, by subtracting the decrease in lactate output found in the zero
glucose experiments (1.2 ± 0.1 mM/hour), from the measured lactate concentration,
assuming that the same decrease in lactate efflux takes place in the lactate experiments
(without glucose). In the experiments where both glucose and lactate were added to the
medium, we assumed that the lactate output during the mixed medium was equal to that
during the preceding glucose medium, which was subtracted from the total measured lactate
concentration.
4
Chapter 4
GLUCOSE AND LACTATE METABOLISM OF PERFUSED CULTURED ASTROCYTES
Glucose use, lactate use, lactate output and the aerobic ratio, a possible indicator of aerobic
glucose metabolism, were calculated as follows (Gramsbergen et al., 2003):
Glucose use (nmoles/min) = flow (µl/min) * (glucose in medium before perfusion (mM) –
glucose detected after perfusion (mM)).
Lactate use (nmoles/min) = flow (µl/min) * (lactate in medium (without glucose) before
perfusion (mM) – lactate detected after perfusion (mM)).
Lactate output (nmoles/min) = flow (µl/min) * lactate detected (after perfusion with
glucose) (mM).
“Aerobic ratio” (%) = 100% * (glucose used – lactate output/2)/glucose used.
The relative change of substrate use was calculated as follows:
Relative glucose use: glucose use during experimental condition / glucose use during basal
condition * 100%
Relative lactate use: (lactate use during experimental condition/ 2) / glucose use during
basal condition *100%
Relative total substrate use: % glucose use + % lactate use
As the aerobic ratio and the relative substrate use are calculated within experiments, they
are independent of cell quantity.
To calculate glucose use and glycogen content per cell, we extrapolated the correlated data
between glucose use and incubation time to zero (to the plating day), to calculate glucose
use per cell using the known plated cell number (0.3x10exp6 cells).
Differences in substrate use were calculated with ANOVA with post-hoc Dunnett T3. The
relative glucose use during 10 mM glucose was compared with the relative 5 mM glucose
use (100%) using the one sample t-test. Correlations were calculated between the aerobic
ratio and incubation conditions, and between glucose use, lactate output, or aerobic ratio
and incubation time. The effect of glucose content during the cultivation of the astrocytes
on glucose use, lactate output and aerobic ratio was studied with the independent samples ttest.
RESULTS
Astrocytic cultures with FCS serum were 95% pure, serum-free cultures consisted of
astrocytes (>75%) with occasional neuronal contamination (<20%), as assessed by
comparing GFAP- and NeuN positive cells. There was no effect of the neuronal
contamination on glucose use, lactate output and aerobic ratio. Because there were no
significant differences between the astrocyte cultures with or without neuronal
contamination, we pooled all the experiments.
There was no correlation between incubation time and glucose use. The average glucose
use during perfusion with standard medium containing 5 mM glucose was 1.11 ± 0.56
nmoles/min (n=31). Using the plated cell number (i.e. 0.3 x 10exp6 per insert), the glucose
use per cell is 3.7 femtomoles/cell/min.
5
Chapter 4
CHAPTER 4
Both the lactate output and the aerobic ratio appeared to be dependent on the glucose
content of the culture medium during cultivation of the astrocytes. When astrocytes were
pre-incubated in 25 mM glucose, the lactate output was much higher (25mM: 3.11±0,85
nmoles/min, 5mM: 1.84±0.88 nmoles/min, t-test, p=0.006, t=-2.988, n=31) and the aerobic
ratio was lower (negative)
(25mM: -81.14±81.43 nmoles/min, 5mM: 12.54±53.33
nmoles/min, t-test, p=0.005, t=3,060, n=30) as compared to cultures grown in 5 mM
glucose.
Primary astrocyte culture
3,5
3,0
Glucose
Lactate
2,5
2,0
1,5
0,5
0,0
0 mM glucose
1,0
5 mM glucose
Measured glucose, lactate conc (mM)
4,0
1:00
2:00
3:00
4:00
5:00
6:00
7:00
Time (hrs)
Figure 4-2: Effect of glucose deprivation on lactate efflux. During basal
conditions (perfusion with medium containing 5 mM glucose), 3 mM glucose and
2.5 mM lactate is detected in the efflux medium, which means that 1 nanomoles of
glucose/min (flow 0.5 µl/min*glucose concentration change (2mM)) is taken up.
When the cells are subjected to glucose deprivation, glucose concentrations
rapidly decrease to zero, whereas the lactate efflux from the cells decreases slowly
to zero in 6-7 hours, indicating an intracellular source of lactate (probably
glycogen).
In all zero-glucose experiments (n=5, example figure 4-2), we found a substantial but
gradually decreasing lactate output for several hours in the absence of glucose. There is no
correlation between the decrease in lactate and the cultivation time. The average lactate
decrease in the absence of glucose is 0.97 ± 0.34 mM per hour, with a zero glucose
treatment time between 1.5 and 16 hours, and lactate output levels approaching zero (n=3)
after 5.5-10 hours. The long duration of lactate release without an energy substrate present
in the medium indicates an intracellular source of lactate, probably glycogen. The total
amount of lactate released in the absence of glucose ranged from 39 to 262 nanomoles (or
20-130 nanomoles in glucose equivalents, average 55 ± 39 nanomoles). Assuming that all
glycogen is consumed anaerobically (i.e. converted to lactate and not further metabolized)
in astrocytes, we estimate a minimal average glycogen content (in glucosyl equivalents) of
6
Chapter 4
GLUCOSE AND LACTATE METABOLISM OF PERFUSED CULTURED ASTROCYTES
55 nanomoles per 0.3 x 10exp6 cells per insert, which yields 183 femtomoles glycogen per
astroglial cell.
In four experiments, astrocytes were perfused with 5mM glucose after zero glucose
treatment. These cells still showed viability after 1 (n=1), 1.5 (n=2) or 13 (n=1) hours of
zero glucose treatment as assessed by glucose consumption and lactate production.
The relative glucose consumption is significantly higher during perfusion with 10 mM
glucose compared to 5 mM glucose (figure 4-3, p=0.045, t=2.870, n=5, one sample t-test),
while lactate efflux is not significantly changed. Addition of lactate to the glucose
containing medium does not affect glucose consumption (figure 4-3). The relative lactate
use tends to increase with higher lactate concentrations; this effect is, however, not
significant. The total substrate use (in glucose equivalents) is lower when lactate is the sole
substrate for the cell cultures (figure 4-3, 5 lact vs 5 gluc: p=0.061, 5 lact vs 10 gluc:
p=0.008, 5 lact vs 5 gluc 2.5 lact: p=0.049, 5 lact vs 5 gluc 5 lact: p=0.036).
% glucose use
% lactate use
300
280
+
Total substrate use (%)
260
240
#
220
~
*
@
200
180
160
140
120
100
80
60
40
20
0
5 gluc
10 gluc
n=5
5 gluc
2.5 lact
n=6
5 gluc
5 lact
n=5
5 gluc
10 lact
n=5
5 lact
n=6
10 lact
n=6
Figure 4-3: Glucose and lactate consumption in astrocytes perfused with
different glucose and lactate concentrations in the medium. Data are
expressed as relative changes of substrate use as compared to basal
conditions (medium with 5 mM glucose). The total substrate use is
subdivided in relative glucose use (gray bars) and relative lactate use
(hatched bars). Substrate use is higher during 10 mM glucose compared
to 5 mM (@: p=0.045, one sample t-test). The presence of lactate in the
medium has no effect on the glucose use. The total substrate use is lower
when lactate is the sole energy substrate: #: p=0.061, *: p=0.008, +:
p=0.049, ~: p=0.036, ANOVA. Data are means ± SD of 5-6 independent
experiments.
7
Chapter 4
CHAPTER 4
DISCUSSION
The on-line system used in this study, has due to the small-volume tubing and low dead
volumes of the connections, low lag-times (approximately 5 minutes) and high temporal
resolution: the simultaneous minute-to-minute measurement of glucose and lactate results
in almost 1.000 measurements in an 8-hour experiment.
Calculations on relative substrate use indicate that glucose is the preferred energy substrate
in astrocytes, and that glucose consumption does not change when lactate is added to the
perfusion medium, although lactate uptake does occur. The extra substrate use in the form
of lactate indicates that there might be a separate pool for lactate, as glucose use would
become lower if lactate were used as a replacement for glucose. Similar results were also
seen in our previous in vivo study, in which lactate entered the brain but did not replace
glucose as an energy source (Leegsma-Vogt et al., 2003b). There is a substantial lactate
release from astrocytes, which, contrary to the astrocyte-neuron-lactate-shuttle-hypothesis
(Chih and Roberts, 2003;Pellerin and Magistretti, 2003), is not dependent on glutamate, as
glutamate was not present in the perfusion medium. Although our results do not exclude
that under glutamergic acitvation a substantial amount of lactate is released from astroglia
cells, the present results emphasize that such a coupling may not exclusively depend on the
release of glutamate. Moreover, our present in vitro and previous in vivo studies suggest
that lactate formed by braincells may diffuse out of the brain via astrocytes.
The aerobic ratio was calculated under assumption that almost all of the glucose taken up
by the cells is metabolized via glycolysis and subsequently metabolized aerobically via the
Krebs cycle or converted anaerobically to lactate (Gramsbergen et al., 2003). However, the
glucose taken up from the medium may also be metabolized via other pathways (e.g.
pentose phosphate pathway (Sanchez-Abarca et al., 2001;Bonarius et al., 2001), amino acid
synthesis (Attwell and Laughlin, 2001)), which, if this metabolism contributes significantly
to the measured glucose uptake, would lead to an underestimation of the “aerobic ratio”. In
addition, the production of lactate from intracellular sources, like glycogen, would decrease
and thereby underestimate the aerobic ratio. Negative aerobic ratios are a clear indication of
an intracellular energy source, as the amount of lactate released is higher than can be
expected on the basis of the amount of glucose taken up from the medium. In this study we
have found various negative aerobic ratios, lower (or negative) aerobic ratios when the cell
cultures were pre-incubated in high glucose levels (25 mM glucose), and sustained lactate
release, probably indicating large glycogen stores. Similar effects of glucose deprivation
were previously reported in organotypic hippocampal slice cultures (Gramsbergen et al.,
2003) and may explain that organotypic hippocampal cultures can survive hypoglycemia
for at least 24 hours (Cater et al., 2001). These findings are also in line with literature on
glycogen in astrocyte cultures: glycogen content in astrocytes is dependent on the glucose
concentration in the medium (Dringen and Hamprecht, 1992;Cummins et al., 1983),
glycogen can be a source of lactate (Dringen et al., 1993), glycogen provides energy for
long periods of time (Choi et al., 2003;Gruetter, 2003), and glycogen is mainly found in
astrocytes (Cruz and Dienel, 2002;Eyre et al., 1994). The profound effect of hyperglycemic
culture conditions on lactate output by astrocytes indicates that extrapolation of conclusions
from in vitro studies claiming a lactate shuttle between astrocytes and neurons to the in
vivo situation should be critically evaluated.
8
Chapter 4
GLUCOSE AND LACTATE METABOLISM OF PERFUSED CULTURED ASTROCYTES
The glycogen concentration per cell can also be calculated from data in the literature:
cultured astrocytes contain approximately 50 nanomoles glycogen (in glucosyl
equivalents)/ mg protein (Waagepetersen et al., 2000;Swanson and Choi, 1993;Swanson et
al., 1990). With a 6% protein content per wet weight, this calculates to 3 µmol glucosyl
equivalents per gram (wet weight) cell culture. Assuming an average cell count in
organotypic hippocampal slices of 1 x 10exp5, and a slice wet weight of 0.8 mg, the
glycogen content per cell is 24 femtomoles/cell. Glycogen content in the rat brain is
approximately 12.5 µmol/g (Cruz and Dienel, 2002)), and, assuming 20 times more glia
than neurons in brain (thus 1.85 x 10exp9 glia per gram), this would result in a glycogen
content of 6.8 femtomoles glycogen/glia. The in vivo calculated data are lower than the
estimated in vitro results, possibly partly caused by the difficult analysis of glycogen
content (Cruz and Dienel, 2002).
The on-line system also allows us to calculate substrate use per cell per minute. We have
found a glucose use of 3.7 femtomoles/astroglia/minute, which might even be an
underestimation of total substrate use as we found glycogen breakdown. Substrate use per
cell can also be calculated from literature: assuming a glucose uptake of 0.64 nmoles/min
by an organotypic hippocampal slice, as found in Gramsbergen et al (2003), and an average
cell count in hippocampal slices of 1 x 10exp5, a calculated glucose use of 6.4
femtomoles/cell/min is found, which is in the same order of magnitude as found in the
present study. In vivo, the brain uses approximately 1.3 µmol/g/min glucose (Attwell and
Laughlin, 2001), and, with a cell number of 9.2 x 10exp7 neurons per gram in rats, and 20
times more glia than neurons in brain (thus 1.85 x 10exp9 glia per gram) (Attwell and
Laughlin, 2001;Braitenberg and Schüz, 1998), and the notion that astrocytes only use 5% of
brain energy (Attwell and Laughlin, 2001), this leads to an substrate use of approximately
0.035 femtomoles/astroglia/minute, which is at least twenty times lower than substrate use
found in the present study. Although this large difference between in vitro and in vivo
substrate use may be partly caused by the assumptions made on cell count/g in the
calculations on substrate use, these results might suggest that primary astrocyte cultures
have very large energy stores, and use more energy than astrocytes in vivo. Such
observation can have significant implications in the consideration of significance of in vitro
results to understand in vivo observations and hypotheses.
ACKNOWLEDGEMENTS:
We would like to thank M. Schipper and A.M. Huls for their assistance. This study is
supported by the Dutch Technology Foundation (STW), grant number GGN 4680.
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