(Received for publication, September 6, 1995; and in revised form, October 30, 1995)
From the
Treatment of the mouse fibrosarcoma cell line L929 with tumor
necrosis factor (TNF) induces necrotic cell death. A crucial step in
the cytotoxic action mechanism of TNF involves perturbation of
mitochondrial functions leading to the formation of reactive oxygen
intermediates (ROI). L929 cells have energy requirements adapted to a
high proliferation rate. Glutamine (Gln) is utilized as a major energy
source and drives mitochondrial ATP formation, while glucose is mainly
converted to lactate through glycolysis. We investigated the role of
the bioenergetic pathways involved in substrate utilization on the
cytotoxic action of TNF and established a link between Gln oxidation
and TNF-induced mitochondrial distress. Omission of Gln from the medium
desensitizes the cells to TNF cytotoxicity, while the lack of glucose
in the medium does not alter the TNF response. Sudden depletion of Gln
from the culture medium results in a sharp decline in mitochondrial
respiration in the cells, which might explain the decreased TNF
responsiveness. However, when L929 cells are adapted to long term
growth under conditions without Gln, these so-called
L929/Gln cells have restored respiration, but they
still display a decreased sensitivity to TNF cytotoxicity. Thus the TNF
responsiveness of L929 cells depends on bioenergetic reactions that are
specifically involved in the oxidation of Gln. This is further
confirmed by the desensitizing effect of specific inhibitors of these
Gln-linked enzyme reactions on TNF cytotoxicity in the parental cells,
but not in the L929/Gln
cells. Analysis of the
induction of mitochondrial ROI formation by TNF in parental and
L929/Gln
cells suggests that the effect of Gln on the
sensitivity to TNF cytotoxicity involves a mechanism that renders the
mitochondria more susceptible to TNF-induced mediators, resulting in
enhanced ROI production and accelerated cytotoxicity.
Tumor necrosis factor (TNF) ()is a pleiotropic
cytokine mainly produced by activated macrophages. Besides its role in
the host defense against microorganisms and bacterial pathogens, TNF is
involved in the pathology of various diseases, such as the systemic
inflammatory response syndrome(1, 2, 3) .
Furthermore, TNF is specifically cytotoxic for many types of
transformed cells, especially in the presence of interferon. Most
TNF-mediated activities, including cytotoxicity, are initiated by
ligand-induced cross-linking of the p55 TNF receptor; only in
T-lymphocytes has a role of the p75 TNF receptor in cell proliferation
and in cytotoxicity been unambiguously demonstrated(4) .
Cross-linking of the receptors initiates signal transduction, possibly
through association of the intracellular death domain of the p55
receptor (5) .
In the mouse L929 fibrosarcoma cell line, TNF signaling leads to necrotic cell death(6, 7) . A major step in the cytotoxic mechanism is the formation of ROI in the mitochondria. Their crucial role was demonstrated by the interference of specific inhibitors of the electron transport chain with necrotic cell response (8) and by the correlation between sensitivity to TNF cytotoxicity and mitochondrial activity in the cell(9) . More recently, we followed directly the TNF-induced mitochondrial ROI and correlated it with cytotoxicity(10) . Hence the change from a sensitive to a resistant phenotype observed under hypoxic growth conditions (11, 12) can be explained by the inability to generate ROI in the mitochondria.
Besides oxygen, another
important parameter that affects mitochondrial functionality is the
availability of energy substrates. Normal and malignant cells often
exhibit different metabolic requirements (13, 14, 15, 16, 17) ; the
exuberant proliferation rate of transformed cells demands an adapted
energy metabolism. Therefore, the amino acid Gln, instead of the
Glc-derived pyruvate, is used preferentially as a substrate for ATP
production by oxidative phosphorylation. The underlying adaptations of
the intermediary metabolism include (i) an increased activity of the
mitochondrial matrix enzyme glutaminase, converting Gln to glutamate,
(ii) formation in the mitochondria of the citric acid cycle
intermediate -ketoglutarate via transamination of glutamate, and
(iii) the presence of malic enzyme, generating intramitochondrial
pyruvate(14, 18, 19) .
Here we describe the result of the above changes in mitochondrial enzyme composition and substrate utilization to TNF cytotoxicity. We show that L929 cells use Gln and not Glc as the major energy substrate and that this particular energy metabolism promotes the cytotoxic response of the cells to TNF. Our results also demonstrate that the dependence of TNF cytotoxicity on Gln is not due to the overall rate of mitochondrial respiration per se. Enzymatic pathways specifically utilized in mitochondrial oxidation of Gln appear to sensitize the mitochondria to TNF-induced perturbation of their activity and thereby amplify the resulting production of cytocidal ROI.
As shown in Fig. 1, the presence of both Glc and Gln, i.e. normal growth conditions, supports a metabolism that combines a high rate of respiration with a high production of lactate. A high lactate release indicates that a considerable part of the pyruvate generated from Glc by glycolysis does not enter the citric acid cycle but is reduced to lactate. Nevertheless, the cells show a high rate of respiration. Apparently, the substrate that fuels this high respiration is Gln, since cells maintained exclusively with Gln showed an equal or even slightly increased respiration compared with the control condition. As expected, lactate production in these cells dropped to background levels due to the absence of substrate suitable for glycolysis. When Gln was omitted, Glc being the only metabolic substrate available to the cells, both the rates of lactate release and of oxygen consumption dropped below 50% of the control value. Apparently, under this culture condition, part of the pyruvate that otherwise is reduced to lactate now enters the citric acid cycle to drive respiration. Finally, when both Gln and Glc were omitted from the medium, all cells died within 48 h (results not shown), indicating that no other energy-providing substrate was available to the cells under this condition. These results clearly indicate that in L929 cells Gln is metabolized in the mitochondria through the citric acid cycle and hereby fuels oxidative phosphorylation, whereas the metabolism of Glc is limited to glycolysis, resulting in a high lactate production. These characteristics are as expected for a tumor cell-type energy metabolism as described before for other cell lines(14, 15) .
Figure 1:
Analysis of the energetic pathways used
by L929 cells. For lactate production (open bars), 2
10
cells were seeded in 6-well dishes in the respective
media for 18 h. Then the medium was refreshed in all wells, and the
lactate released in the medium was measured after 10 h of incubation.
The 100% value of lactate equals a concentration of 5.2
mM/10
cells. For oxygen consumption (filled
bars), cells were cultured in suspension cultures for 18 h in the
respective media, collected by centrifugation, and resuspended in a
small volume of O
-saturated medium to measure oxygen
consumption with a Clark-type oxygen electrode at 37 °C. The 100%
value of oxygen consumption equals 115 ng oxygen/min/10
cells. ITU refers to Ino, dThd, and
Urd.
Figure 2:
Comparison of TNF-induced cell death in
parental L929 and L929/Gln cells. TNF-induced cell
death in parental L929 cells (
), L929/Gln
cells (
), and L929/Gln
cells that subsequently
had been cultured in the presence of Gln for over 2 weeks (
) is
shown. Cells were grown in suspension cultures 18 h before TNF
treatment (1000 IU/ml); cell death was detected by PI uptake and flow
cytometry.
Figure 3:
Analysis of TNF-induced ROI response and
cell death. Analysis was performed in parental L929 cells () and
L929/Gln
cells (
) in the absence (full
line) or presence (dashed line) of the radical scavenger
butylated hydroxyanisole. A, percentage of TNF-responsive
viable cells (DHR123 profiles of TNF-treated cells minus DHR123
profiles of untreated cells at the corresponding time points), showing
an increased DHR123-derived R123 fluorescence intensity after TNF
treatment. B, TNF-induced increment in ROI formation: mean
R123 fluorescence intensity of TNF-responsive viable cells compared
with untreated control cells. C, TNF-induced cell death:
percentage of PI-positive cells in TNF-treated cultures as compared
with untreated cultures. The arrows indicate the time of
addition of butylated hydroxyanisole to the cell
cultures.
Binding of TNF to its p55 receptor in malignant, nonlymphoid
cells triggers a complex signal transduction mechanism leading to
cytostasis, necrosis, or apoptosis, depending on the cell type and the
physiological condition. In this report we analyzed the necrotic death
induced by TNF in the mouse fibrosarcoma cell line L929 and show that
one of the factors that modulates the responsiveness of the cells to
the cytotoxic activity of TNF is the availability of energy substrate
and, more specifically, the species of respiratory substrate used by
the cell at the time of TNF treatment. Neoplastic cells have been
reported to have a changed metabolic behavior characterized by the
preferential use of Gln, instead of Glc, as the major energy source.
Gln is oxidized at a high rate in tumor mitochondria, while Glc is
primarily converted to lactate. We established that L929 cells grown in
culture indeed use Gln preferentially as a substrate for oxidative
metabolism and as a corollary produce high amounts of lactate from Glc.
Furthermore, we demonstrated that modulating the cellular energy
metabolism by altering substrate availability in the culture medium
resulted in significant changes in TNF response. More specifically,
omission of Gln from the medium shortly before TNF treatment
desensitized the cells to TNF cytotoxicity. This treatment led to a
sharp decrease in electron flow through the mitochondrial electron
transport chain. Since several lines of evidence previously established
that TNF cytotoxicity in L929 cells requires functional mitochondria (8, 9) , apparently as a source of cytocidal
ROI(10) , this loss of mitochondrial activity might explain the
reduced TNF responsiveness of Gln-deprived L929 cells. Therefore, we
adapted L929 cells to grow without Gln, thus enforcing the use of Glc
as major respiratory substrate. The resulting stable
L929/Gln population had the appearance of
differentiated fibroblasts, as opposed to the dedifferentiated,
scattered morphology of parental L929 cells; moreover, it showed a
reduced proliferation rate and in fact nearly behaved like
untransformed fibroblasts. Also the metabolic parameters, namely low
lactate release and high oxygen consumption, corresponded to those of
normal cells. These parameters indicate that Glc supports in
L929/Gln
cells a restored electron flow through the
mitochondrial electron transport chain. Nevertheless, these adapted
cells still displayed the TNF-resistant phenotype observed after short
term depletion of Gln. We therefore conclude that a factor responsible
for sensitivity of the TNF-cytotoxic response in the presence of Gln
resides in the activity of enzyme systems linked to the oxidative Gln
metabolism and not in the overall mitochondrial electron flow. This
conclusion was confirmed by the mimicking effect observed with
inhibitors that block, at various steps, the enzymatic oxidation of Gln
through the citric acid cycle in the mitochondria. Thus the use of Gln
as an energy substrate facilitates TNF signal transduction leading to
necrotic cell death.
Previous reports from this laboratory have shown that TNF induces excess ROI production in the mitochondria of L929 cells and that these ROI are directly cytocidal and/or necessary for downstream events leading to cell death. In the absence of Gln, this ROI response was markedly attenuated, whereby both the absolute levels of ROI generated as well as the rate of appearance of cells exhibiting increased ROI levels were abated. Apparently, the oxidation of Gln creates a metabolic condition in the mitochondria that facilitates mitochondrial production of excess ROI upon TNF stimulation and that enhances the cytotoxic response of the cells to TNF. This facilitation is not based on the overall rate of electron flow in the mitochondria, since L929 cells using Glc or Gln as a respiratory substrate showed similar rates of oxygen consumption. Possibly, the kind of substrate used affects the formation of multienzyme complexes of a higher order (14) that are differentially sensitive to regulatory mechanisms (in)activated by TNF. Alternatively, by-products of Gln oxidation through the citric acid cycle, such as citrate(14) , a precursor of fatty acid and cholesterol biosynthesis, may affect upstream signaling events and amplify the activation signal delivered to the mitochondria. Further experiments will be needed to clarify this mechanism.
Since both the use of Gln as an oxidative substrate and sensitivity to the cytotoxic action of TNF are tumor cell-specific features (the first facilitating the latter), the validity of a causal link between tumor cell bioenergetics and tumor cell responsiveness to TNF cytotoxicity has to be considered. However, as expected for a very pleiotropic cytokine like TNF, there is no evidence for a strict correlation between the use of Gln as a respiratory substrate and the sensitivity to TNF cytotoxicity. For example, the TNF sensitivity of cells that die in an apoptotic mode, was not diminished after Gln omission (not shown). This may be related to the early inactivation of mitochondrial activity observed during programmed cell death(26) , rendering an active contribution of mitochondria impossible. In contrast, when mitochondria actively contribute to the cytotoxic process, such as in L929 or WEHI 164 cl 13 cells (not shown), Gln enhanced the TNF-cytotoxic response. Hence we may conclude that the tumor cell-characteristic use of Gln as an oxidative substrate contributes to the TNF responsiveness of those tumor cells in which mitochondria play an active role in the cytotoxic process. Since Gln is abundantly present in body fluids and since its concentration may alter during the progression of neoplastic disease, maintaining high concentrations in circulation may, for certain tumors, positively affect the therapeutic value of TNF.