`Futile cycle' enzymes in the flight muscles of North American bumblebees
Department of Biology, University of Western Ontario, London, Ontario, Canada, N6A 5B7
* Author for correspondence (e-mail: jfstaple{at}uwo.ca)
Accepted 8 December 2003
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Summary |
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Key words: thermogenesis, flight muscle, fructose-1,6-bisphosphatase, phosphofructokinase, futile cycle, enzyme, bumblebee, Bombus
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Introduction |
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The mechanisms of heat production in non-flying bees have been debated
since the 1970s. One theory holds that heat is generated by an enzymatic
`futile cycle' within the flight muscles. Newsholme et al.
(1972) found high activities
of both phosphofructokinase (PFK) and fructose-1,6-bisphosphatase (FbPase) in
the flight muscles of several European Bombus species. It was
hypothesized that the unusually high (for muscle) activities of FbPase would
rapidly hydrolyse fructose-1,6-bisphosphate produced by PFK, providing more
substrate for PFK and resulting in a cycle between the two enzymes
(Fig. 1). Each turn of this
cycle would result in the net hydrolysis of 1 ATP. If it were to function at
high rates, this cycle would produce significant amounts of heat that could
warm the thorax in the absence of muscular contraction. This mechanism is
thought to produce heat in malignant hyperthermia in pigs
(Clark et al., 1973b
). In
Bombus affinis, the rate of cycling in resting bees increases as
Ta decreases and completely stops when flight is initiated
(Clark et al., 1973a
). The
cycle can be deactivated during flight, as bumblebee FbPase is inhibited by
Ca2+ (Grieve and Surholt,
1990
; Storey,
1978
) that is released into the flight muscle cytoplasm when
activated by stretching or motor nerve action potentials. Further support for
this theory comes from observations that the activities of FbPase among
European Bombus species correlate negatively with body mass
(Newsholme et al., 1972
) and
positively with foraging activity on massed flowers
(Prys-Jones, 1985
). From this,
it was concluded that bees that foraged primarily by walking on massed flowers
could not rely on heat generated by flight muscle contractions and thus there
would be selective pressures favouring alternative mechanisms of thermogenesis
(Prys-Jones, 1985
).
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Although elegant, the futile cycle theory has been challenged. Some
investigators estimate that the rates of heat production, either at observed
in vivo cycling rates (Clark et
al., 1973a) or at cycling rates defined by maximal PFK and FbPase
activities, are insufficient to account for the observed rate of thoracic
warming (Kammer and Heinrich,
1978
; Newsholme and Crabtree,
1976
). Beyond this, it is clear that, during pre-flight warm-up,
action potentials are delivered to the flight muscles of Bombus
terrestris, although there is no apparent movement of the wings
(Surholt et al., 1990
) and
only minute deformations of the thorax
(Esch et al., 1991
). This has
been interpreted as the simultaneous tetanic contraction of the dorsoventral
and dorsal longitudinal flight muscles
(Esch and Goller, 1991
). In
this way, metabolic rate and heat production are stimulated through
actinomyosin hydrolysis of ATP, but no `shivering' per se is
obvious.
If the PFK/FbPase futile cycle is a common thermogenic mechanism, one would
predict that activities of both enzymes, especially FbPase, would be high
among bumblebees. Although PFK and FbPase activities have been determined in
seven Bombus species found in western Europe
(Newsholme et al., 1972), only
one North American species has been examined to our knowledge
(Clark et al., 1973a
;
Newsholme et al., 1972
). The
purpose of the present study is to measure these enzymes in several bumblebee
species from central North America and to relate them to animal mass.
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Materials and methods |
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Enzyme assays
All assays were performed at 37°C using a Varian DMS80 dual-beam
spectrophotometer. The PFK assay was based on the methods of Suarez et al.
(1996). Thoraxes were
homogenized in nine volumes of buffer containing 25 mmol l-1
Tris-KH2PO4 (pH 7.8 at 4°C), 2 mmol l-1
EDTA, 0.5% (v/v) Triton X-100, 1 mmol l-1 fructose-6-phosphate and
0.1% (v/v) ß-mercaptoethanol. Glucose-6-phosphate (G6P) was omitted from
the homogenization medium to allow the assay of both PFK and FbPase in the
same homogenate. Preliminary experiments showed that the omission of G6P did
not affect the activity of PFK. Thoraxes were minced with scissors and
homogenized on ice with three 15 s passes (30 s between passes) of a small
homogenizer (Tissue Tearor). The homogenate was then sonicated on ice using
three 15 s pulses (30 s between pulses). The homogenate was then centrifuged
at 10 000 g for 5 min at 4°C and the supernatant was used
for enzyme assays.
The PFK assay medium contained 50 mmol l-1 Tris (pH 8.0 at 37°C), 10 mmol l-1 MgCl2, 100 mmol l-1 KCl, 0.1% (v/v) ß-mercaptoethanol, 20 mmol l-1 fructose-6-phosphate (omitted for determination of control rates), 2 mmol l-1 ATP, 0.01 mmol l-1 fructose-2,6-bisphosphate, 0.15 mmol l-1 NADH, 0.3 units ml-1 aldolase (Sigma, St Louis, MO, USA), 3.6 units ml-1 triosephosphate isomerase (Sigma) and 0.5 units ml-1 glycerol-3-phosphate dehydrogenase (Sigma).
The FbPase assay was based on the method of Storey
(1978). FbPase was assayed in
the same homogenate used for PFK assays. Preliminary experiments showed no
significant difference in activity when compared with homogenates prepared
according to Storey (1978
).
Measuring both PFK and FbPase activities in the same homogenate allowed us to
examine the ratio of the activities of the two enzymes among individual
animals. The FbPase assay medium contained 50 mmol l-1 Tris (pH 7.4
at 37°C), 6 mmol l-1 MgCl2, 0.2 mmol l-1
fructose-1,6-bisphosphate (omitted for determination of control rates), 0.2
mmol l-1 NADP, 10 units ml-1 phosphoglucose isomerase
(Roche, Laval, QC, Canada) and 2 units ml-1 glucose-6-phosphate
dehydrogenase (Roche). Coupling enzymes from another supplier (Sigma) appeared
to be contaminated with FbPase, giving significant changes in optical density
in the absence of muscle homogenate. In preliminary experiments, supernatants
from the crude homogenates were centrifuged through a desalting column to
remove small-molecular-mass metabolites
(Helmerhorst and Stokes,
1980
), but this had no significant effect on FbPase activity.
Data analysis
Enzyme activities were calculated relative to thorax mass. Activities of
PFK and FbPase and ratios of FbPase/PFK were compared among species by one-way
analysis of variance (ANOVA; =0.05). Thorax mass-specific enzyme
activities and whole-animal masses were log10 transformed, and the
relationships analysed by least-squares regression. All statistical
calculations and transformations were performed by SigmaStat (version 2.03,
SPSS Inc.).
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Results |
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FbPase activities in North American bees are generally low, only
0.02-0.3-fold those of B. terrestris
(Fig. 2). The one exception is
B. rufocinctus, where FbPase activities are not significantly
different from those of B. terrestris. FbPase activity could not be
detected in P. citrinus thoraxes. In B. terrestris, our
FbPase data correspond to 54.9±4.2 units g-1 muscle
(N=10), close to the 65 units g-1 muscle reported by
Newsholme et al. (1972). As
reported by other investigators (Newsholme
et al., 1972
; Storey,
1978
), we found B. terrestris FbPase to be sensitive to
Ca2+ (33% inhibition at 0.125 mmol l-1) and
fructose-2,6-bisphosphate (41% inhibition at 0.05 mmol l-1; data
not shown). No detailed enzyme kinetic studies were performed in this
experiment.
The ratio of FbPase to PFK calculated for individual animals is depicted in Fig. 3. Relative to PFK, there is significantly more FbPase in B. terrestris than in any of the North American species analysed. The ratio in B. rufocinctus is significantly higher than in the other North American bees.
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There is an approximately 10-fold range in body mass of bees used in this study, allowing for allometric analysis of enzyme activities among individuals. From Figs 4, 5, it is evident that there is a significant negative allometric relationship between body mass and thorax-mass-specific activity of PFK (P=0.011) and FbPase (P=0.002). For PFK, the allometric exponent is -0.18 (r2=0.13), while for FbPase it is -1.33 (r2=0.21).
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Discussion |
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Bumblebee flight muscle contains very low activities of enzymes from
gluconeogenic and pentose phosphate pathways
(Clark et al., 1973a;
Newsholme et al., 1972
),
limiting the possible roles of FbPase in this tissue. Given the role of PFK in
powering flight metabolism, any capacity for thermogenesis by the PFK/FbPase
cycle is probably best reflected by FbPase activity, especially when expressed
relative to PFK activity. In a broad sampling of European bumblebee species,
the average ratio of FbPase/PFK activities was reported to be 1.10, with a
range of FbPase activity of 14-114 units g-1 muscle
(Newsholme et al., 1972
).
Because of the higher PFK activities in the present study (probably due to an
improved homogenization technique; see Results), the FbPase/PFK for B.
terrestris (0.6) is lower than the 1.05 reported by Newsholme et al.
(1972
). Despite this, the
ratio is significantly higher in B. terrestris than in any North
American species measured here (Fig.
3). In the North American B. rufocinctus, the FbPase/PFK
ratio is significantly lower than B. terrestris but is still higher
than the other North American species sampled here, and the FbPase activity
(43.1 units g-1 thorax) is not significantly different from B.
terrestris. In the present study, we found very low FbPase activity in
B. affinis (3.4 units g-1 thorax; N=2; range
2.2-4.5 units g-1 thorax). This contrasts with earlier studies that
show high (45.3 units g-1 muscle) FbPase activities
(Clark et al., 1973a
). The
reasons for this discrepancy are not immediately obvious but may relate to
geographic intraspecific differences (Wisconsin vs Ontario), the
small sample size available for this study, and potential contamination of the
coupling enzymes used by Clark et al.
(1973a
) in the FbPase assay
(see Materials and methods).
To ascertain whether this substrate cycle could functionally contribute to
thermogenesis in a whole bumblebee requires, in part, a calculation of how
much heat could be produced by the cycle. It is estimated that heat production
at a rate of 92.1 J g-1 muscle min-1 (22 cal
g-1 muscle min-1) is required to maintain a thoracic
temperature 27°C higher than Ta
(Heinrich, 1972;
Newsholme and Crabtree, 1976
).
The PFK/FbPase cycle can produce some heat directly from the hydrolysis of
ATP, yielding 30.6 kJ mol-1 (7.3 kcal mol-1) ATP. The
maximal possible cycling rate is determined by the maximal catalytic
capacities of the two enzymes. In the present study, the species with the
highest FbPase activity (B. rufocinctus) could theoretically support
a cycling rate of 57.5 µmol g-1 muscle min-1. Heat
production from ATP hydrolysis due to cycling (7.3 kcal mol-1)
would produce only 1.7 J g-1 muscle min-1 (0.4 cal
g-1 muscle min-1). Even during flight, the tissue
content of ATP in bumblebee flight muscle decreases only slightly
(Newsholme et al., 1972
). It
is, therefore, reasonable to assume that most of the ADP produced in the cycle
would be rephosphorylated through the complete oxidation of glucose. This
would produce a further 4.6 J g-1 muscle min-1 (1.1 cal
g-1 muscle min-1; assuming 686 kcal and 36 mol ATP
mol-1 glucose oxidized). In total, we estimate that, at maximal
cycling rates, the PFK/FbPase cycle could produce less than 7% of the heat
required to maintain thoracic temperature on a cold day. Estimates using
14C- and 3H-labelled glucose suggest that maximum in
vivo cycling rates are only 10.4 µmol g-1 muscle
min-1 (Clark et al.,
1973a
). This rate of cycling would produce less than 2% of the
heat required to keep a bumblebee thorax warm on a cold day. It is worth
noting that another study on B. terrestris males has estimated that
during the pre-flight `warm-up' phase cycling occurred at a rate of 249
µmol g-1 muscle min-1, and this could contribute
significantly to thermogenesis (Surholt et
al., 1991
). We suspect, however, that this result is an artefact
of the isotopic method employed, as the reported cycling rate is
4-fold
greater than PFK and FbPase activities found in B. terrestris workers
(Newsholme et al., 1972
). This
is unlikely because in other Bombus species FbPase activities in
males and workers are comparable
(Newsholme et al., 1972
).
Our data do not support a significant thermogenic role for substrate
cycling between PFK and FbPase in bumblebees. This leaves open the question of
why B. rufocinctus and several European Bombus species have
relatively high levels of FbPase activity in a tissue with no significant
capacities for gluconeogenesis or pentose phosphate metabolism. Operation of
this cycle at low levels probably allows for greater sensitivity of PFK and/or
FbPase to allosteric regulators and therefore greater amplification of net
glycolytic flux in the transition from rest to flight
(Newsholme and Crabtree,
1973). Such amplification may be possible with the relatively low
FbPase activities found in most North American bees in this study
(Newsholme and Crabtree,
1973
), although the conditions necessary for significant
amplification are limited (Fell,
1997
). An amplification role for this cycle does not explain,
however, why other regionally endothermic flying hymenopterans, such as
Psithyrus spp. and Apis mellifera, have virtually no FbPase
(Newsholme et al., 1972
) or
why European bumblebees generally have higher FbPase activities than most
North American congeners.
Our data do not show any apparent pattern of cycling capacity among Bombus subgenera. High FbPase activities were found in members of Cullumanobombus (B. rufocinctus) and Bombus (sensu strictu) (B. terrestris), while another member of the latter subgenus has low FbPase activities (B. affinis; present study). None of the members of the subgenera Pyrobombus (B. bimaculatus, B. perplexus, B. impatiens, B. vagans) or Separatobombus (B. griseocollis) have FbPase activities comparable with those of B. terrestris or B. rufocinctus. Future studies evaluating the capacity for PFK/FbPase cycling in relation to phylogeny would be informative.
The negative allometric relationship (exponent -0.18) between body mass and
mass-specific PFK activity (Fig.
4) reflects the primary role of PFK in powering flight.
Whole-animal oxygen consumption in euglossine bees scales with a greater
allometric exponent of -0.42 (Casey and
Ellington, 1990). If the scaling of bumblebee oxygen consumption
follows a similar pattern, it would suggest that glycolytic flux in smaller
bees is closer to maximal PFK capacity and would result in higher enzyme
fractional velocities. Support for this hypothesis will require a rigorous
examination of glycolytic flux and enzyme capacities within individuals of
Bombus species spanning a large mass range. We are currently
performing such experiments. PFK activity in European Bombus species
was found to scale with an allometric exponent of -0.6
(Newsholme et al., 1972
), much
higher than the exponent reported in the present study. This difference
probably reflects the improved homogenization technique used in the present
study, which better preserved PFK activity. It is also worth noting that our
analysis was confined to workers, while Newsholme et al.
(1972
) considered all
castes.
FbPase activity scales with an allometric exponent of -1.33
(Fig. 5), comparing favourably
with the exponent of -1.4 found in European bumblebees
(Newsholme et al., 1972). This
more intense scaling relative to PFK is consistent with a thermogenic role of
the PFK/FbPase cycle - smaller bees cool more quickly, and one may predict
that the capacity for thermogenesis by cycling would increase more quickly
than the maximal capacity for glycolytic flux (reflected by mass-specific PFK
activity). Our data suggest, however, that in B. rufocinctus this
cycle could contribute, at most, 7% of the required heat to maintain
Tth on a cold day and that other mechanisms of
thermogenesis would be more important. Cycling may contribute more
significantly when differences between Ta and
Tth are less than 27°C. The differential scaling of
PFK and FbPase may also indicate that smaller bees have a greater need for
amplification of glycolytic flux when moving from rest to flight.
In summary, we found generally low levels of FbPase activity in the flight muscles of North American bumblebees, with the exception of B. rufocinctus. This results in low capacities for substrate cycling compared with European Bombus species. Combined with our calculation of maximal rates of heat production, these data do not support a significant thermogenic role of a PFK/FbPase cycle. This cycle may serve to amplify glycolytic flux in rest-to-flight transitions. Both PFK and FbPase activities show negative allometric scaling with body mass, but the scaling of FbPase activity is much more intense. These results are consistent with the negative allometric scaling of whole-animal oxygen consumption and a greater role for glycolytic amplification by PFK/FbPase cycling in smaller bees.
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Acknowledgments |
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Footnotes |
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