Download The Crabtree Effect: A Review

CANCER RESEARCH
VOLUME 21
AUGUST
1961
NUMBER 7
The Crabtree Effect" A Review
KENNETH H . IBSEN
(Departments of Surgery and Physiological Chemistry, University of California Medical Center, Los Angeles, Calif.)
Recently much interest has been shown in the
inhibition of respiration induced by glucose addition to cells. This inhibition is often called the
reversed or inverted Pasteur effect or the Crabtree effect. In this paper the term Crabtree effect
has been utilized to describe the inhibition observed after the addition of any hexose or hexose
analog capable of inhibiting respiration in any
tissue. Unfortunately, the studies on the Crabtree
effect are scattered throughout a large variety of
journals and often appear incidental to other
work. This review is an attempt to bring many of
the publications which deal with the Crabtree
effect together so that it might be easier to visualize the scope, mechanism, and possibly the meaning of this competition between catabolic pathways. Apparently, no previous attempt has been
made to review this field of interest.
T H E C H A R A C T E R I S T I C S OF T H E
E F F E C T I N E H R L I C H ASCITES
T U M O R CELLS
Although work had been done since 1929, when
Crabtree (~4) first reported the effect, modern
workers were stimulated by the publications of
El'tsina and Seitz (31) and Kun et al. (56). Both
of these groups of investigators employed the
Ehrlich ascites tumor cell, and since then the
majority of recent studies on the Crabtree effect
have been made with this cell. Therefore, it is convenient to refer to results obtained with the
Ehrlich cells as a standard of comparison.
Respiratory inhibition can be induced by glucose, fructose, mannose (1~, 21, 56, 80, 88) and
'2-deoxyglucose (48, 76). The course of inhibition
Received for publication March ~0, 1961.
with glucose is quite constant and has been
studied in bicarbonate (31, 37, 88), phosphate
(Pi) (10, 12, ~1, 43, 56, 76, 88, 95), and Tris buffer
systems (10, 50). There is an initial stimulatory
period lasting from 20 to 120 seconds (15, 76).
This stimulatory period is followed by an inhibitory period, which, after equilibration, is
constant and equal to about 80 per cent of the
endogenous rate and lasting until all the glucose is
utilized (31, 48). About ~5 seconds 1 after the glucose disappears the cells will be released from
inhibition and will consume oxygen at a rate which
can be as great as the initial pre-inhibitory rate
(48). The earlier literature, in which manometric
technics were utilized, reported that low levels of
glucose stimulated rather than inhibited respiration (71, 80). However, the use of oxygen electrode technics has shown that extremely minute
quantities of glucose can cause inhibition (19, 50).
The earlier workers overlooked this inhibition,
since it was released, owing to the full utilization
of glucose, before the first manometric reading
had been made. The reported stimulation was
probably caused by the availability of extra
lactate, formed from the glucose, which will prevent the slight decline in the endogenous oxygen
consumption rate (50). The glucose-induced inhibition is released by uncouplers of oxidative
phosphorylation such as s dinitrophenol (DNP)
(3, 19, 56, 80, 85, 97), dicoumarol (15, 19), dibromophenol (4~), n-butyl-S,5-diiodo-4-hydroxybenzoate (19), and dinitrocresol (21). The D N P
studies had been carried out in media with phosphate concentration varying from zero to 55 mM.
K v a m m e (58) reported that D N P did not release
1R. W. McKee, private communication.
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the inhibition when the Pi level of the media is less
than 20 raM. This observation may have been in
error because of the lack of control of pH change
(50). Methylene blue also releases the inhibition
(87, 56, 80, 95). It has been shown by Racker (80)
and Wenner et al. (96) that the methylene blue
effect may be due to the extramitochondrial
stimulation of glucose oxidation.
An important point about which there is controversy is the effect of inhibitors of triose dehydrogenase on the Crabtree effect. Several investigators have found that additions of iodoacetic
acid (IAA) in quantities which inhibited aerobic
glycolysis did not remove the inhibitory effect of
glucose on oxygen consumption (19, 4~, 45, 48,
57, 80, 84, 10~). On the other hand, it has been
reported that IAA (90), oL-glyceraldehyde (90), or
bromoacetate (81) stimulated respiration in the
presence of glucose, but not endogenously, thus
partially releasing the Crabtree effect. Seelich and
Letnansky (86) claim that IAA releases the Crabtree effect only in the presence of phosphate. However, El'tsina and Seitz (81) obtained a release of
inhibition in bicarbonate buffer, whereas other
workers (48, 80) who obtain no effect of IAA on
respiration employed phosphate buffers. Theoretically, these differences in findings could be attributed to the fact that the glycolytic inhibitors
prevent the accumulation of H +, which also inhibits respiration. However, the data of the workers who find a release of the Crabtree effect by an
inhibitor of the triose dehydrogenase show no evidence of a large pH change caused by lactate accumulation. In fact the kinetic data of El'tsina
and Seitz (31) suggest that the pH was well controlled. Possibly the results vary because of the use
of a different strain of Ehrlich ascites tumor cells.
The endogenous respiration appears to be more
susceptible to hydrogen ion (~6, 50, 85) and
fluoride ion (47) than is the respiration in the
presence of glucose. Therefore, these agents tend
to release the Crabtree effect. Cells which have
grown in the animal for a shorter time have a
smaller rate of endogenous respiration than do
cells withdrawn from the animal at a later period
after inoculation; yet the respiration in the presence of glucose remains constant (8).
I t has been reported that, by increasing the
phosphate concentration in the medium, the per
cent of inhibition of oxidation caused by glucose
can be decreased (15~, 51, 10~). More recently, it
has been demonstrated that the effect of phosphate is caused by three different factors and that,
when Pi in the media is greater than 10 raM, the
direct effect of phosphate on respiration is probably insignificant (50). When the supplementary
Vol. ~1, A u g u s t 1961
buffer capacity is low and the glucose concentration high, Pi decreases the effect of glucose on
respiration by maintaining the pH more constant
(50, 83). When the Pi in the media is less than
5 mM there may be a direct effect of this ion on
oxidation (50). However, the majority of the nonbuffer effect of Pi is due to the influence of this
ion on glycolysis (50). Decreased glucose utilization was reflected in the oxygen consumption
values due to prolongation of the Crabtree effect.
Another example of the lack of a direct effect of
phosphate on respiration is shown in the data of
Seelich et al. (88), who find comparable oxygen
consumption values in phosphate and in bicarbonate buffers for both the endogenous respiration
and respiration in the presence of glucose. Concentrations of Pi higher than 80 m• have been reported to be inhibitory to respiration (71).
The addition of glucose to ascites cells also upsets the endogenous equilibrimn by sparing the
oxidation of lipide (73, 74, 90) and amino acids
(78, 81). In addition to providing substrate for
oxidation, glucose also is utilized as a building
block for many intermediates and apparently
transforms the cell from a catabolic to an anabolic
condition (e9, 80).
Although there is an initial re-equilibration
period after gh, cose addition, during which the
adenosine triphosphate (ATP) level is greatly reduced and the adenosine diphosphate (ADP) level
is correspondingly raised, these values quickly return to levels approaching those found before
glucose addition (44, 48, 10o,). When ~-deoxyglucose is added, all the adenine nucleotides other
than adenylic acid (AMP) appear to be quickly
utilized and remain at low levels for an extended
period 1 (48). The same is true when glucose is incubated in the presence of IAA (10~). Interestingly enough, there is no initial upward surge in the
A D P level when ~-deoxyglucose is phosphorylated
(48). This is possibly owing to the much lower rate
of phosphorylation of 52-deoxyglucose relative to
glucose and the use of the A D P so produced by
the mitoehondrial oxidations (48).
Some workers (8, s
57) find a slightly increased A T P level in cells incubated in the presence of glucose relative to those depending upon
endogenous substrate, whereas others find that
the A T P content remains constant (44, 48, 49,
105~). The incorporation of pa2 into A T P is not
affected by glucose (5~5, ~9). This is true despite
the observation (5~9) of an increased amount of
ATP being formed in the presence of glucose and
a decrease in the A T P level endogenously. The
decrease of A T P is felt to be due to utilization of
this compound into maeromoleeules, whereas the
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IBSEN~Crabtree Effect: Review
increase found in the presence of glucose was due
to the de novo synthesis of A T P from ribose (39).
The endogenous decrease might be related to
nucleoprotein synthesis which may be greater in
bicarbonate buffer, in which a decrease of A T P
was noted (3, 39), than in phosphate buffer in
which no decrease of A T P was noted (48).
Creaser et al. (35) have shown that de novo synthesis is increased by ascites fluid. Alternatively,
the endogenous decrease may occur during the extensive washing employed to remove p3~ after incubation and before analysis. Ibsen et al. (48) did
not find an increased A T P content but did note
an increased level of pentosides and of total adenine nucleotides. The latter was transitory. I t
appears that, whether or not glucose can be shown
to increase the A T P content, one must conclude,
in agreement with Quastel and Bickis (79), that
the energy derived through glycolysis is equal to
the energy lost from the decreased rate of oxygen
consumption.
Sorbose, maltose, glucosamine, lactose, sucrose,
galactose, trehalose, raffinose, xylose, ribose,
glycine, hydroxyproline, DL-lactate, L-aspartate,
glycolate, pyruvate, succinate, L-glutamate, oxalacetate, DL-alanine, citrate, a-ketoglutarate, and
malate do not either affect or stimulate respiration from the endogenous level (56, 71, 80, 85).
The longer fatty acids have been reported to
depress (38, 56, 85) or to have no effect on the
oxT~gen consumption (73). Of the carbohydrates
listed above the only one which has been shown
to be glycolyzed is glucosamine 1 (85). This compound is metabolized at one-quarter the rate of
glucose. 1 Some investigators report that the glucose-inhibited respiration can be slightly stimulated by succinate (93), lactate (10), and pyruvate
(10), whereas others have indicated that citrate
(I3), succinate (I3, 17), or a-ketoglutarate (13)
had no effect on respiration. The Crabtree effect
has also been reported to the almost overcome by
the addition of malate to the glucose-inhibited cell
(5). K v a m m e (59) has found that, when cells are
partially blocked with malonate, fumarate overcame the Crabtree effect, but citrate or a-ketoglutarate did not.
There is less information concerning the pattern
of inhibition when it is caused by fructose, mannose, or 3-deoxyglucose. The degree of inhibition
is the same for equal concentrations of glucose,
fructose, or mannose when measured manometrically for 1 hour (13, 80). Equal quantities of
glucose or fructose appear to hold the cell in inhibition equally as long, whereas mannose maintains the inhibition longer (13). This difference
may indicate t h a t mannose is glycolyzed more
831
slowly. The depth of inhibition caused by 3deoxyglucose is also the same as that caused by
glucose (48). However, the inhibition lasts longer
in the presence of 3-deoxyglucose probably because this compound is phosphorylated at a
slower rate than glucose (48). The fructose and
mannose inhibition is released by dinitrocresol in
a manner similar but not identical to glucose (31),
whereas D N P releases the 3-deoxyglucose inhibition, but only briefly (47). I t has been shown also
that mannose (76) and 3-deoxyglucose (47, 76)
stimulate respiration more than glucose does during the early pre-inhibitory period. The effects of
fructose addition seem to be delayed, since Packer
and Golder could show no effect with this sugar
during the few minutes' duration of their studies
(76). This delayed effect may be due to the relatively low affinity of fructose toward the hexokinase of this cell (56).
T H E P R E S E N C E OF A C R A B T R E E
EFFECT IN TISSUE OTHER
THAN E H R L I C H ASCITES
Table 1 summarizes some of the essential factors concerning the characteristics of the Crabtree
effect in a variety of tissues. The tissues listed in
the table are divided into three groups. Group A
includes those normal tissues which have shown
an effect under the conditions studied and are not
known to need special treatment, before or after
removal from the body, in order to make their
respiratory system sensitive to glycolysis. Group
B includes those non-neoplastic tissues which show
a Crabtree effect only when they are pretreated
or incubated under special conditions or when the
animal of their origin needs to be stressed in a particular manner. Group C includes all the neoplastic tissues which show a Crabtree effect regardless of the treatment of the cell. The leukocytes are treated separately in Table 3 because of
lack of agreement among the various investigators.
I t is impossible to evaluate these heterogeneous
data statistically, and therefore a critical view is
highly desirable. Particularly questionable are the
data which suggest t h a t rat liver, diaphragm, or
yolk sac show a glucose-induced inhibition. In
none of these cases did the authors themselves
indicate that the data represented a real effect,
but they are reviewed for the sake of completeness.
Furthermore, glucose may be observed to have
no inhibitory effect on respiration in a tissue which
potentially could show a Crabtree effect. The effect could be missed for many reasons. Some of
these, such as the hormonal balance of the animal
or choice of the buffer system, are essentially a
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TABLE 1
TISSUES OTHER THAN LEUKOCYTES WHICH SHOW A CRABTREE EFFECT
Approx. per cent
depression of
.Ioxygen consumption
Tissue
Reference
Re marks
A. Non-neoplastic (under no specified necessary condition)
Rat liver
Rat yolk sac
Rat diaphragm
H u m a n reticuloeytes
Human thrombocytes
Bull spermatozoa
Bovine articulate cartilage
2-10
4
8
14
19
42
63
(24, 27, 28, 99)
Questionable significance
(27)
(99)
(81)
Not released by D N P but released by bromoacetate
(pH effect?)
(67)
Succinate and pyruvate stimulate respiration in
presence of glucose. M a n n o ~ but not fructose
also inhibits respiration
(83)
(60)
B. Non-neoplastic tissues which show an effect, but only under specific conditions*
Yea st
t I u m a n synovial membrane
Monkey heart
Monkey kidney
Developing retina
Adult retina
Rat thymus
Rat messenteric lymph
nodes
30
Preincubation with a substrate
3O
30
35
Possibly from rheumatoid subjects only
From tissue culture only. Not released by IAA
From tissue culture only. Partially released by ]AA.
Released by methylene blue
Only if incubateo in bicarbonate-free buffer
Only if incubated in bicarbonate-free buffer
Only from adrenalectomized animals
32-4(I
O, 10-30
30
Jensen rat sarcoula
1-15
Gardner lymphosarcoma
0-~7
Philadelphia #1
Crocker sarcoma
5-10
10
Chicken tumor #13 of
Stubbs and Furth
Mouse tar carcinoma 2146
Mouse sarcoma S-37
14
14-19
20
HeLa (cultured)
20
I~.Ep. #2 (cultured)
Walker rat sarcoma 519
(cultured)
Flexner-Jobling rat
sarcoma
Ehrlich ascites carcinoma
Hepatoma
Krebs-2 ascites
24
(4)
(41)
(42, 7.5)
(46, 75)
(s2)
(s2)
Effect appears to be more marked in phosphate than
in hicarbonate buffer. Fructose stimulates respiration
The Crabtree effect appears to be dependent on the
phosphate level of the buffer. In the absence of
phosphate there is no effect ~
Value relative to the oxygen consumption in the
presence of xylose
I
(24, 27)
(~, 2s)
(2,)
(13)
(24)
Octanoate releases, due to greater inhibitory effect
on endogenous respiration
D N P & IAA inhibit respiration in presence or absence of glucose
Partially released by IAA (5%)
(ss)
(loo)
(4)
(9s)
23
27
25-50
35
35
Also inhibited by 2-deoxyglucose
~5-5"2
Partially released by phosphate:
(99)
See Section
(29)
Also inhibited by 2.deoxyglucose, fructose, and
mannose
Yoshida ascites rat
sarcoma
(77)
30
C. Neoplastic tissue
Chicken tumor #11 of
Furth
Rous sarcoma
Rhabdomyo~reoma
aseites cells
S-~A aseites m a m m a r y
ca rcino ll]a
(s)
(98, 103)
(29, 54, 86)
50
(is)
((~1)
35-50
(34)
49
5O
DN~P releases
(32)
* The necessary conditions are listed under the column "Remarks."
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IBsEN~Crabtree Effect: Review
matter of chance. The effect of phosphate appears
variable, apparently increasing the inhibition in
the Jensen sarcoma (~7) and Gardner lymphosarcoma, 2 while decreasing the inhibition in the
Yoshida ascites ~ (86). Retina shows a glucose effect only in the absence of bicarbonate (46). In
other instances the effect may be missed because
of utilizing a too high cell-to-glucose ratio, in
which case the glucose might be fully utilized before the effect was evaluated. An effect may also be
833
glucose stimulated, rather than inhibited, respiration. A final way in which the Crabtree effect may
be missed is by allowing the cell to utilize its
endogenous substrate before beginning the experiment. Such a case was illustrated in sperm by
Lardy and Phillips (60). Actually, glucose, if
metabolized, probably would stimulate respiration in any starved cell; therefore, by pre-feeding
yeast cells Belitzer (8) was able to prime them so
that glucose might then cause a respiratory in-
TABLE 2
SUMMARY OF THE REPORTED VALUES FOR THE CRABTREE EFFECT FROM A
VARIETY OF LEUKOCYTES
Approx. per cent
Crabtree effect
Incubation
medium
Source
Reference
A. Leukocytes from normal donors
0
14
30
59
?
Phosphate
Bicarbonate
Serum
Phosphate
] Huma.n
(87)
(87)
(66)
"
"
Guinea pig
(69, 7~)
(35)
B. Lymphocytes
No effect
0
0
Phosphate
Serum
Bicarbonate
8
29
]9
Phosphate
Bicarbonate
Phosphate
'30
Phosphate
Rat
Human
Human lymphatic
letLkemia
tt
~
Mouse leukemic lymph
nodes
Lymph nodes of
adrcnalectomizcd
rats only
(~3)
(66)
(63)
(87)
(87)
(94)
(8~.)
C. Leukocytcs from patients with myeloid leukemia
0
0
26
s
Bicarbonate
Phosphate
Human
~erllrn
t~
Bicarbonate
(63)
(87)
(66)
(87)
D. Chicken myeloblasis
I
3-17
Gey's saline
missed because of the decreased rate of endogenous respiration with time. The data of Hopkinson and Kerly (46) show how the Crabtree effect
in the retina would have been missed, as it had
been previously (55), had they reported the results
on the basis of only 1-hour values. In 40 minutes
~280 ~zn~oles 02 were utilized endogenously, whereas in 60 minutes only 307 #moles were utilized.
However, in the presence of 15 mM glucose 245
umolcs 02 were used in 40 minutes and 864 pmoles
in 60 minutes. Therefore, if in this case only 1hour values were reported, it would appear as if
2 W. E. Hull, private communication.
I Short-time culture
I
(7)
hibition. I t is possible that a more correct impression of the inhibitory effect of glucose could be
ascertained by adding glucose to a cell already
stimulated by another oxidizable substrate.
In cells which produce lactate aerobically the
glucose-induced respiratory inhibition may be
caused by the increased hydrogen ion concentration in the media. Such a pII effect is not the
metabolically significant Crabtree effect which is
the primary cause of the respiratory inhibition in
the Ehrlich ascites cell. Therefore, an inhibition
caused by acid can be termined a "quasi" effect.
T h a t the inhibition caused by decreased p H of the
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Cancer Research
medium is not the primary cause of the respiratory
inhibition in Ehrlich ascites cells can be demonstrated by the following facts: (a) glucose inhibition does not increase progressively with incubation and is immediately removed upon utilization
of the glucose (48) ; (b) titration of the formed acid
with alkali does not relieve the inhibition (71) ; (c)
glucose in concentrations too low to affect pH
appreciably still causes an inhibition of respiration
(19, 50); (d) glucose plus monoiodoacetic acid and
2-deoxyglucose can inhibit oxygen consumption
while not changing the pH (48, 80); and (e) D N P
releases the inhibition but does not decrease the
pH change found in the presence of glucose alone
(48). To say that the respiratory inhibition is not
caused by the pH change of the media does not
necessarily mean the metabolically significant effect is not caused by the production of acid at a
specific site within the cell. This possibility is discussed in the next section.
Despite the fact that the "quasi" effect will always be added to the true metabolically significant Crabtree effect whenever there is a pH
drop and respiration is pH-sensitive, the degree of
the two effects can be estimated. If the "quasi"
effect is minimal, the rate of oxygen consumption
after release from inhibition will be equal to the
pre-inhibitory rate (48). On the other hand,
kinetic data will also demonstrate a ease which is
truly due to the accumulation of H + in the media;
in this case the onset of inhibition will be delayed;
the effect will be aeeumulatory and will not be
released when all the glucose is utilized.
I t follows, therefore, that, if in any tissue a
kinetic study was done and the glucose effect is immediate and not aeeumulatory, if acid production
can be stopped without releasing the inhibition, or
if respiration can be stimulated without decreasing
the pH change, then the Crabtree effect is probably due to an effect other than pH production in
the media and can be called a metabolically
significant effect.
Although Crabtree originally thought that the
effect of glucose on respiration was a characteristic of tumors only (24), it is obvious from Table 1
that this effect is not limited to neoplastic tissues;
yet there is a pronounced tendency toward such a
relationship. Of all the studies found, in which the
respiration of neoplastic tissue was compared in
the presence and absence of glucose, only the
Walker 256 (28) and the DBA mouse aseites
thymoma (62) were reported to have no Crabtree
effect. However, many non-neoplastic tissues ineluding brain (27, 28, 55), testis (27, ~8), kidney
(24, ~7, 28, 41), spleen (27), adipose tissue (53),
and rat, rabbit, and chicken embryos (~7) have
Vol. 21, A u g u s t 1961
been reported to show no Crabtree effect. Furthermore, still other tissues, such as liver and diaphragm, show an effect which is of doubtful
statistical meaning, or which may be due to a pH
effect such as in the reticulocyte or thrombocyte.
Finally, the majority of non-neoplastic tissues
which show an effect do so only under rather
specific conditions as demonstrated in Table lB.
One of these conditions, tissue culturing, is known
to cause several other neoplastic-like alterations
(of. 41). I t has also been found that the induction
of a neoplastic growth in a tissue which previously
showed no Crabtree effect can induce the effect
in this tissue (61). It has been suggested (66) that
there is a positive relation between the amount of
aerobic glycolysis and the Crabtree effect. Although this relationship does appear to be true in
general, it may not explain all the facts. For instance, testieular cells which show no Crabtree
effect have a high rate of aerobic glyeolysis (~7).
Also, adrenaleetomy which induces a Crabtree effect in several tissues does not stimulate glyeolysis
in these cells (8~).
I t has been suggested (2, 48) that the existence
of a Crabtree effect in Ehrlieh ascites cells is related to the location of the hexokinase in the mitochondria. In this regard, it is of interest to note
that culturing creates a tendency for the hexokinase of cells to become more active and to relocate into the mitoehondria (36, 41).
M E C H A N I S M OF T H E C R A B T R E E
EFFECT
Ehrlich ascites cells.--Several mechanisms have
been proposed to explain the Crabtree effect in the
Ehrlieh ascites cells. Of these the suggestion that
a pH effect caused the inhibition has a minimum
of experimental support. Tiedemann (9~) first
noted that the respiration was pH-sensitive and
that the endogenous metabolism could be reduced
to that of the respiration in the presence of glucose
by It +. He suggested that the observed respiratory
inhibition induced by glucose was caused by the
concomitant production of H + through glyeolysis.
As previously discussed, it is very unlikely, if not
impossible, that the inhibition is due to the general
pH decrease of the medium. Bloeh-Frankenthal
and Weinhouse (11), however, refined the pH concept and suggested that the production of H + at
specific sites within the cell may cause the inhibition. Such a concept has gained some experimental
support, since it has been found that the pH
change may be greater within the cell than in the
media (26). Nonetheless, to explain the inhibitory
effect of deoxyglueose or of glucose in the presence
of IAA, it becomes necessary to assume that in
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]BSEN~Crabtree Effect: Review
these cases a different mechanism operates or that
the critical p H change is associated with the early
phosphorylations. Furthermore, it becomes necessary to assume that D N P somehow protects these
sites, since D N P does not inhibit the production of
H + by glucose. Emmelot and Bos (3~) present
further evidence and a lucid discussion on the unlikelihood t h a t H + induces the Crabtree effect in
S3A ascites cells.
As early as 1936 Belitzer (8) suggested that the
Crabtree effect was due to a competition between
glycolysis and respiration for a common intermediate. Since D N P has been shown to uncouple phosphorylation from oxidation (65) and
does release the Crabtrec effect, it was suggested
that this competition might be for inorganic phosphate or adenine nucleotide. The competition
would then be removed, since the rate-limiting
mitochondrial phosphorylations would become uncoupled from oxidation by D N P and the substrates of phosphorylation could no longer limit
the respiration, which would then proceed at a
greater rate.
Besides the D N P effect, the evidence which suggested a Pi involvement is: (a) the Pi level is
lowered during glycolysis, (3, 43, 48, 80, 101); (b)
raising the Pi level of the medium reduces the
Crabtree effect (1r 10~); and (c) limitations of Pi
can cause a respiratory inhibition in reconstituted
systems (40).
On the other hand, there is evidence which suggests that Pi is at least not the only factor involved
m the Crabtree effect. First, it has recently been
established that much of the stimulatory effect
which Pi has been reported to cause on the respiration in the presence of glucose was due to the effect of this ion on glycolysis, but not to a direct
effect on respiration (50). K v a m m e (57) and
Bloch-Frankenthal and R a m (10) have found that
increasing the :Pi level of the medium may even increase rather than reduce the Crabtree effect.
Secondly, although all the investigators found an
initial drop in the Pi level, Hess and Chance (43)
find that the level returns to the endogenous level
within 3 minutes, and Ibsen et al. (48) found that
in Pi-rich media the intracellular Pi level found
in the presence of glucose approached the endogenous value before respiration was released from
the inhibition. Furthermore, increased Pi in the
media increases the cellular Pi level (~t5, 48). I t
has been suggested that, when the Pi level of the
medium is above 10 raM, Pi has no effect on
respiration and the Crabtree effect, but when the
extracellular level is below 5 mM the Pi probably
does have a direct effect on respiration (50). Similarly, Creaser et al. (~5) found that increasing the
8S5
Pi of the incubation medium did not influence the
amount of p3~ incorporated into A T P or ADP,
provided the Pi level was over 8 raM.
K v a m m e (58) had previously advanced a similar concept of a critical level of Pi in the medium.
He felt that, in a buffer with less than 20 mM Pi,
the Crabtree effect could be explained by a competition for Pi between glycolysis and phosphorylation(s) at the substrate level in the a-ketoglutarate-succinate reaction. However, this concept
was based largely on the observation that D N P
did not release the Crabtree effect when the Pi
level of the incubation medium was lower than ~0
mu. As previously mentioned, this observation
had not been made by others (50, 97) and may
have been due to the greater change of p H which
would be expected as the total buffer capacity was
reduced. Such a p H effect is suggested by Kvamtoe's kinetic data (58, Fig. ~), which show that the
inhibition does not begin until almost ~0 minutes
after glucose addition. This is characteristic of the
"quasi" Crabtree effect as discussed previously.
Since in these experiments the final pII was about
5.1-5.3, since glycolysis itself is pH-dependent in
these cells (~6, 50, 85), and since Tris, which has
no buffering capacity below p H 7, was used to
replace phosphate, it is meaningless to try to
evaluate the p H effect by comparing the final p H
in different samples. I t would be the rate of change
of p H which is most important.
There is a considerable body of evidence, apart
from the effect of uncouplers, which suggests that
A D P limitation m a y be of importance in the
respiratory inhibition. This evidence includes the
following: (a) A D P is present in low concentration
endogenously and returns to an equally low or
lower level shortly after glucose addition (44, 48);
(b) in isolated mitochondria A D P controls respiration more effectively than Pi (16); (c) the initial
stimulatory phase after glucose addition is very
reminiscent of the effect of A D P addition to nfitochondria in which respiration was limited by
A D P (15, 16, 18, ~0); (d) an equal quantity of
phosphorylation occurs in the presence or absence
of glucose under aerobic conditions (s ~9); (e)
Gatt and Racker (40) have shown that limiting
A D P can inhibit respiration in reconstructed
systems.
Of the evidence given above there is some controversy over point (c). Ibsen el al. (48) were unable to find the initial stimulation but suggested
this was because of an inadequate measuring device. In subsequent work (47) it was possible to
show the stimulation, although never to the same
degree as reported by Chance and Hess (15, 16,
18, co).
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There appears to be, therefore, good reason to
suspect A D P as a factor limiting respiration.
However, again a competitive mechanism could
not explain a Crabtree effect induced by ~-deoxyglucose or glucose and IAA. If the decreased rate
of oxygen consumption was indeed due to the decreased availability of A D P at the mitochondrial
sites of phosphorylation, it would be necessary to
suppose either that the mechanism causing the inhibition was entirely different when it was induced by
2-deoxyglucose or by glucose in the presence of
IAA or that the A D P limitation was not due to a
direct competition with glycolysis (48). A second
reason for suggesting that if A D P became limiting
it must do so indirectly is that it appeared as if the
oxygen inhibition occurred at a time when the
ADP level was much higher than it was endogenously (48). I t must be recognized, however,
that this observation may be invalid, because the
cell concentration was lower when the change of
oxygen consumption was measured than when the
nucleotides were measured (48).
Two different theories involving the concept of
intracellular barriers and a resulting indirect
competition for ADP were published. The first of
these suggested that A T P became trapped
within the mitochondria because of the initial increased rate of oxidation and an associated change
in mitochondrial permeability (15, 80). This
theory has been supported by Packer and Golder
(76), who have demonstrated structural changes
in the mitochondria seemingly associated with
the early phase of stimulated oxidation and high
cytoplasmic ADP, and by Hess and Chance (44),
who find more A T P in mitochondria of cells which
have been exposed to glucose. This hypothesis can
also be used to explain the Pasteur effect along
the lines proposed by Lynch and Koenigsberger
(68). The second theory of compartmentalization
of nucleotides was based upon Siekevitz and
Potter's theory (89) of nucleotide transport
through the mitochondrial membrane. I t was supposed that, upon initiation of glycolysis, the
hexokinase, which is located in the mitochondria
(s 101), utilized a portion of the normal mitochondrial membrane ATP, making it unavailable
to aid in the transport of cytoplasmic A D P
through the membrane to the sites of oxidative
phosphorylation within the mitochondria (48).
This theory as formulated was no stronger than
the Siekevitz-Potter hypothesis of transport upon
which it is based. However, the hypothesis of
Siekevitz and Potter has been criticized (14) on
the grounds that A M P did not stimulate oxygen
consumption as quickly as did ADP. Mitochondria
in which the respiratory rate was controlled by
Vol. ~1, A u g u s t 1961
limiting amounts of phosphate acceptor were used
in both cases. However, in the author's opinion,
there is no suggestion, in the Siekevitz-Potter hypothesis, of a primary role of A M P as the phosphate acceptor in oxidative phosphorylation and
therefore no reason to believe that A M P would
stimulate respiration as fast as ADP, particularly
in the absence of substrate quantities of ATP. In
fact, the dual adenylatekinase conversions suggested by Siekevitz and Potter might be regarded
as a funnel directing A D P from the cytoplasm to
sites of oxidative phosphorylation in the mitochondria. The Siekevitz-Potter hypothesis, therefore, could be considered as a means by which the
mitochondria could enhance their capacity to
compete with the cytoplasmic reactions for ADP.
The theories of Chance's and McKee's groups
(15, 48), pronmlgated as an attempt to explain the
Crabtree effect, are similar in many respects.
Both groups feel that A D P becomes limiting at the
mitochondrial sites of phosphorylation, that this
limitation is not due to a direct competition with
glycolysis but rather an indirect one involving
compartmentalization of the nucleotides, that the
mitochondrial deficiency of A D P is in some manner related to the phosphorylations of the carbohydrate, and that this compartmentalization effect
may act only during the first few minutes of the
total time of depression. By accentuating the
effect of phosphorylation, these theories would
explain how even ~-deoxyglucose or glucose plus
IAA can initiate the Crabtree effect. Nevertheless, it must not be forgotten that it is possible
that the Crabtree effect is caused by different
mechanisms when induced by different agents.
Although there is a fair amount of evidence suggesting an A D P involvement in the mechanism of
respiratory inhibition with additional Pi effects
when this ion is present in limiting quantities, an
argument can also be offered for the possible imbalance in the oxidative reductive state of the
tissue as a cause of the Crabtree effect when it is
induced by glucose.
Such an effect of glucose could be proposed to
explain the following: the capacity of methylene
blue to release the inhibition (41), the reported low
specific activity of lactate from C 14 glucose (5),
the ability of oxamic acid--a lactate dehydrogenase inhibitor--to release the inhibition (78),
and the observation that sulfhydryl-containing
compounds completely release the inhibition (37).
Bennett et al. (9) have found two types of respiratory inhibition in the same cell. At lower lactate
levels they find a normal glucose effect; but, when
glucose is added to cells respiring in the presence
of 0.16 M lactate, respiration is almost completely
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IBSEN--Crabtree Effect: Review
inhibited. This latter inhibition can be overcome
by succinate but by no other Krebs cycle intermediate nor by DNP. I t is conceivable that,
whereas the usual Crabtree effect is dependent
mainly upon the nucleotide balance, the latter
effect is dependent mainly upon the redox state
within the cell.
The specific stimulatory effect of succinate
suggests that the variant type of Crabtree effect
discovered by Bennett et al. is indeed related to
pyridine nucleotide. Also, since D N P does not
release the inhibition, the mechanism inhibiting
respiration in the presence of excess lactate must
be different from the mechanism in operation
when lactate is present in lesser amounts. Since
glucose can then cause respiratory inhibition to
occur by two distinct mechanisms in the same cell
under different conditions, it is conceivable that
the normal Crabtree effect is not due to one
mechanism but to a series of different mechanisms,
the relative important of each being dependent
upon the circumstances. If this concept were true
it may explain why there is some divergence in the
observations reported by different laboratories.
Some evidence can be found which favors such
a concept of multiplicity of mechanisms. Chance
and Hess (16, 19) have indicated that the inhibitory phase of the glucose effect could be
divided into a period of deep inhibition and a
period of the regular steady-state inhibition.
Lonberg-Holm (64) has data which may suggest
even further subdivision of the glucose effect. The
more recent studies (50) on the effect of Pi on the
Crabtree effect could be best explained as an introduction of an additional limiting factor without
suggesting an elimination of the original controlling mechanisms.
In considering the possible effect of glucose on
the redox balance of the cell, it is interesting to
note that ~-deoxyglucose, which is only phospho~,lated, stimulates lactate production from
endogenous substrate (48).
Ascites tumors other than Ehrlich.--Emmelot
and Bos (3~) suggested that the Crabtree effect in
the S3A ascites cells is due to a lack of Pi or phosphate acceptor because: (a) D N P removes the
inhibition of oxidation caused by glucose, (b)
exogenous tricarboxylic acid intermediates decrease glycolysis in a manner which seems to be
correlated to the amount of phosphorylation associated with their oxidation; and (c) the endogenous respiration can be stimulated by DNP,
which indicates that, even in the absence of glucose, Pi or ADP limits oxidation. Furthermore,
they find no stimulatory effect of exogenous Pi on
respiration and present evidence which suggests
837
that neither the intra- or extra-cellular pH change
causes the inhibition (82). Also, as in the Ehrlich
cell, glucose replaces fatty acid as a source of substrate for oxidation (83).
Yushok and Batt (103) have found that glucose, s
fructose, and mannose produce an inhibition of oxygen consumption in the
Krebs-~ ascites carcinoma cell. Furthermore, they
find that glucosone, which has a high affinity for
the hexokinase but is very slowly phosphorylated,
does not cause a Crabtree effect itself and prevents
other carbohydrates from causing the effect. This
would appear to relate the hexokinase reaction to
the Crabtree effect in these cells. Interestingly, as
in the Ehrlich ascites cell, the hexokinase is particulate (70).
On the other hand, Seclich and Letnansky (86),
who conclude that Pi is not a limiting factor in the
Ehrlich cells, think that in Yoshida ascites sarcoma cells the inhibition of oxidation is due to the
competitive removal of Pi. Hull 2 has data which
suggest that in these cells even the endogenous
respiration is grossly stimulated by an increase of
the phosphate in the buffering medium. Kieler (54)
has found that an atmosphere containing 1 per
cent carbon dioxide neutralizes the Crabtrec effect
in Yoshida but not in Ehrlich ascitcs cells. However, other investigators (0.9, 86) found a marked
Crabtree effect in the Yoshida ascites cells while
using a bicarbonate buffer. Kieler attributes the
carbon dioxide effect to a possible ADP-sparing
effect caused by shuttling phosphoenolpyruvate to
oxaloacetic acid via the Utter-Kurahashi reaction.
From the limited data available it may be
tentatively assumed that the Ehrlich ascites, the
Krebs-s ascites (103), and the S3A ascites carcinoma
cells (3~) have their respiration controlled by similar mechanisms, whereas, in the Yoshida ascites
sarcoma cell, respiration is more directly controlled by Pi or 1)y other mechanisms.
Retina.--Retina shows a Crabtree effect only
when incubated in the absence of bicarbonate
(46). Furthermore, only the nonvisual elements of
the retina display a Crabtrec effect (75). This has
been shown by destruction of the visual elements
with IAA (75) and by utilizing immature retinas
which have not yet developed the visual elements
(~, 75).
In addition to removing the glucose effect, bicarbonate greatly stinmlates oxidation (46). This
stimulation has been felt not to be due to the provision of tricarboxylic acid intermediates alone,
but to be due also to the production of oxidized
T P N via the TPN-malic dehydrogenase reaction
(46). If it is true that respiration is limited by the
T P N + concentration in the absence of bicarbonate,
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this limitation might be expected to be made more
severe by glucose due to stimulation of the oxidative shunt and perhaps indirectly through the
formation of D P N H . Assuming glucose inhibited
respiration by decreasing T P N +, one would expect that glucose would stimulate respiration in
the presence of bicarbonate and that IAA might
increase the inhibition. Both these exceptions are
met (46), but it is not obvious why D N P released
the inhibition (75). It is possible that D N P stimulates visual or other cells not affected by glucose.
Leukocytes.--It is obvious from Table ~ that
the leukocyte picture is confuse(]. Seelich et al.
(87) found that, under their experimental conditions, increasing Pi levels resulted in a tendency to
decrease the per cent of inhibition due to the
Crabtree effect in normal leukocytes, although
they did not feel that the effect in the absence of
phosphate was necessarily significant. Yet MeKinney and associates (69, 7~) found a large
Crabtree effect, even though they employed phosphate buffer, and Luganova et al. (66) found a
Crabtree effect in normal cells incubated in serum.
These differences may be due to the method of
preparation, one method perhaps being richer in a
particular cell type.
Seelich et al. (87) found the minimal effect in
normal leukocytcs but obtained the largest effect
in leukocytes obtained from patients with lymphatic leukemia and with chronic myelocytic
leukemia. Yet, Luganova et al. (66) found no effect
with leukocytes obtained from patients with lymphatic leukemia but reported a large effect in
leukocytes from normal individuals and from
patients with chronic myelocytic leukemia. These
inconsistencies suggest that the lack of agreement
is not duc to differences in the experimental
methodology such as total buffer capacity, etc.
The data of Roberts and White (85~) show that the
corticoid hormonal balance of the animal affects
the degree of Crabtrec effect in thymus, ruesenteric lymph nodes, and lymphosarcoma and
suggest that some of the reported differences may
bc duc to variance in the hormonal balance of the
leukocyte donors.
T H E PSYCIIOLOGICAL S I G N I F I C A N C E
OF T H E C R A B T R E E E F F E C T
No in vitro study is complete unless its biological meaning, if any, is assessed. In considering
what role the Pasteur or Crabtrec effects might
play in living cells one cannot help observing that,
if considered in the reverse manner from which
they are studied, they are both excellent clncrgency measures. The releasing of the Pasteur effect
would allow glycolysis to I)rocccd more rapidly
Vol. s
A u g u s t 1961
when the cell's oxygen level dropped to a low
level, thus maintaining a continuous source of
energy. Similarly, the release of the Crabtree
effect would oxidatively provide needed energy
normally supplied by glyeolysis when the cell's
supply of carbohydrate substrate becomes exhausted. However, one can expect the glucose and
oxygen supply to remain relatively constant for
any given mammalian cell other than cancer cells
or perhaps rapidly dividing fetal cells. Therefore,
these emergency mechanisms would be of greater
advantage for primitive cells living under less
organized conditions. It is possible that both effects are chemical remnants of evolution, the
Pasteur effect generally remaining, whereas the
Crabtree effect usually remains only in a latent
form, to be taken advantage of by those cells
which for some reason break away from the controlling mechanisms. Although entirely teleological, this concept could be considered to be supported by the following evidence: (a) the development of a tumor in a tissue has been shown to
induce a Crabtree effect in that tissue (61); (b)
growing the tissue outside the body in tissue culture causes tile induction of a Crabtree effect (4,
41); (c) adrenalectomy can induce a glucose effect
in several tissues which normally have no Crabtree effect (8~).
On the other hand, it is possible that the energy
relations are only a coincidental fact related to the
mechanism inducing the inhibitions and that the
Pasteur and Crabtree effects either have no
physiological meaning or are artificially induced
reflections of normal intracellular control mechanisms. It has been demonstrated that the total
amount of phosl)horylation in the Ehrlich aseites
or hepatoma cell remains constant under aerobic
conditions in the presence or absence of glucose
(~25, ~29). This should mean that all energy-needing
reactions wouht proceed as well in the absence or
presence of glucose. This is true in some instances,
such as in amino acid uptake (79); but on the
other hand, Acs (1) has found that K + accunmlation in the Ehrlich ascites cell depends upon
glyeolysis. This K + accumulation could not be
associated with glyconeogenesis, since these cells
have no phosphorylase (91). Similarly, phagocytosis in leukocytes appears to be completely dependent upon glycolysis even under aerobic conditions (39). These facts suggest that the energy
from glycolysis may be channeled differently from
that energy which is derived by oxidative processes. If there is such a variance of effect from
the energy obtained through the different catabolic routes, then the cell needs to have a mechanism capable of shifting from one catabolic
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IBsEN--Crabtree Effect: Review
process to another. This shift, magnified by artificial c o n d i t i o n s , m a y b e w h a t is b e i n g s t u d i e d a s
the Crabtree or Pasteur effect.
Since glucose has been shown to be necessary
f o r a n a b o l i c m e t a b o l i s m of E h r l i c h a s c i t e s cells in
vitro (~5, ~9, 31) o n e w o u l d e x p e c t t h a t , b y inc r e a s i n g t h e a v a i l a b i l i t y of s u g a r in vivo, t h e t u m o r
cells' growth would be enhanced. However, the
opposite appears to be true, since hyperglycemic
m i c e s u r v i v e d o s e s of E h r l i c h cells s i g n i f i c a n t l y
l o n g e r t h a n d o n o r m a l m i c e (5~).
A CKNOWLED GMENTS
The author wishes to thank Drs. thfll, Gal, Garcia, and
McKee for their critical apl)raisal of this paper. He wishes to
thank Miss Ardella Sweek for aid in checking the references
and Dr. MeKee for making his reference files available.
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Research.
The Crabtree Effect: A Review
Kenneth H. Ibsen
Cancer Res 1961;21:829-841.
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