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Warm-up and substrate cycling in flight muscles of male bumblebees, Bombus terrestris
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Camp. Bio~h~m. Physiol. Vol. 98A, No. 2,pp. 299-303,1991
C 1991Pergamon Press pie
Printed in Great Britain
WARM-UP AND SUBSTRATE CYCLING IN FLIGHT MUSCLES OF MALE BUMBLEBEES, ~~~~U~ TERRESTRIS BERNHARDSuaHor_r,*t$ HEINRICH GREIVE,* THOMASBAAL* and ANDREAS BERTSCH~ *Zoologisches lnstitut der Westfalischen Wilhelms-Universitlt, Lehrstuhl fur Tierphysiologie, H~ndenburgplatz 55, D-4400 Miinster, F.R.G.; §Fachbereich Biologie der Philipps Universitgt, Karl-von-Frisch-Strasse, D-3550 Marburg, F.R.G. (Received 21 June 1990) Abstract-l I Rates of substrate cycling between fructose &phosphate and fructose bis-phosphate were measured in the flight muscles of male bumblebees (Boamhurterrestris) in viva during four defined activity phases. 2. Shortly after the injection of double-labelled glucose the~~oupies were fixed to the thorax to record surface temperature continuously and to determine the phase of activity every individual bee was in, when finally the cycling rate was measured. 3. Based upon the ‘H/‘4C-ratios, measured in the sugars and hexose-phosphates involved, the following relative cycling rates were determined: torpor: 0.24 k 0.11; warm-up: 9.14 + 3.51; “activated” rest: 1.94 + 0.59, and flight: 0.22 + 0. I 1. Using the data of the glycolytic fluxes, determined in the flight muscles of these bumblebees in previous studies, absolute cycling rates were evaluated as follows: torpor: 0. I I k 0.04; warm-up: 249.9 1 k 96.05; “activated” rest: 17.42 + 5.29, and flight: 5.94 + 2.95 ~moi/min x g muscle. 4. Conversion of these rates of substrate cycling (=rates of ATP-hydrolysis) into rates of heat production demonstrates that in the flight muscles of Bombus terresfris during phases of warming-up without any detectable movements of wings or thorax substrate cycling between fructose 6-phosphate and fructose-bis-phosphate very probably is the predominant mechanism of non-shivering heat production. 5. Possible mechanisms for the regulation of the intensity of this fructose &phosphate/fructose bis-phosphate substrate cycle are discussed.
INTRODUCTION Since unique high activities of fructose l,Qphosphatase have been found in flight muscles of several
bumblebee species (Newsholme et af., 1972), it has remained a matter of debate whether or not this enzyme in l&o, together with Gphospho-fructokinase, forms a “non-futile” substrate cycle (Katz and Rognstad, 1978; Newsholme, 1980: Newsholme et al., 1983; Newsholme et al., 1984; Challis et al., 1984; Moore, 1985). Theoretically. in addition to improving sensitivity in metabolic control (Crabtree and Newsholme, 1985). such a substrate cycle could be a mechanism of heat generation by hydrolysing ATP. Indeed, Clark er al. (1973) were able to demonstrate that such a cycle exists in flight muscles of Bombus uffis, but the cycling rates (i.e. the rates of ATP-hydrolysis) they found were by far too low to generate such amounts of heat to enable a bumblebee to warm-up its flight muscles from 6 to 36°C within 15 min (Heinrich, 1974, 1975). In addition, Kammer and Heinrich (1972) found that warming-up is always coupled with muscle potentials and Heinrich (1979) therefore, describes the essential mechanism of heat tPresent address: Department of Experimental Zoology, University of Utrecht, 8 Padualaan, 3508 TB Utrecht, The Netherlands. SAuthor to whom correspondence should be addressed.
production as muscle shivering, although no movements of the thorax or wings were visually detectable. Recent studies, applying a highly sensitive vibration monitoring system (Surhoit et al., 1990), however, made it extremely improbabie that shivering is the physiological mechanism providing heat production in bumblebee flight muscles. Both, intensive warming-up and muscle potentials (in male Bombus terrestris up to 25 Hz), were recorded, but no vibrations of the thorax. Therefore, in the present study, the method of recording thorax surface temperature, as used in the above study, and the method of measuring the rates of substrate cycling between fructose 6-phosphate and fructose 1,6-bis-phosphate by injection of double-labelled radioactive glucose, as introduced by Bloxham et al. (1973), were combined. This allowed to determine the rates of substrate cycling in the bumblebee flight muscles in z&o during four clearly defined phases of activity of these bees. MATERIALS AND METHODS Animals Bo~bus terrestris were bred under controlled conditions in a greenhouse as described previously (Bertsch, 1984). About one week after they had emerged, male bees were separated from the breeding colonies and labelled individually. Before an experiment, body weight, food uptake and activity pattern were controlled and recorded daily over 7 days as described by Surholt ef al. (1988).
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Chemicals D-[5-‘HI-glucose (21.1 Ci/mmol) and o-[6-‘JC]-glucose (55.0 mCi/mmol) were purchased from Amersham Buchler, Braunschweig. Prior to use the radiochemicals were freezedried to remove traces of radio-labelled water. All biochemicals (enzymes, substrates and co-substrates) were from Boehringer, Mannheim, except fructose 1,6-bis-phosphatase. This, and also Dowex-50-Wx8 and Dowex-2-chlorid, were from Sigma, Taufkirchen. Precoated thin layer-plates came from Machery & Nagel, Diiren; high efficiency emulsifier cocktail ‘Biofluor’ from Du Pont de Nemours, Bad Homburg and Beta-Spark gas (for the linear analyser) from Messer-GrieBheim, Frankfurt a. M., all other chemicals were from Merck, Darmstadt. Experimenlal procedure Basis for measuring cycling rates between fructose 6phosphate and fructose bis-phosphate was the method using D-[5-3H]-glucose and D-[6-‘4C]-glucose introduced by Bloxham et al. (1973). Experiments, however, had to be performed on individual bees, because preliminary experiments had shown that only in this case reproducible results can be achieved. A bumblebee was cooled down to 4°C in a dark refrigerator for 1 hr. Then a differential copper-constantan thermocouple was quickly fixed to the thorax surface as described previously (Surholt er al., 1990). Thereafter the bee was kept in the refrigerator for a further IO min. By transferring it into a deep-freezer (-25’C) for 3 min it then was anaesthetized more intensively and immediately afterwards 3 ~1 double-labelled glucose solution (consisting of 3 PCi o-[S-‘HI-glucose and 3 PCi D-[6-“‘Clglucose) was injected into the abdomen using a 10~1 Hamilton syringe. Then the bee was brought directly into a climate chamber (17°C) and kept in darkness for a further 3 min (for temperature adaptation). During this time the thermocouple was connected to the measuring system (see Surholt et al., 1990). Then the lights were switched on and from then the thorax surface temperature was recorded continuously. Bees keeping their thorax temperature at about the ambient temperature (17°C) for 15 min after the onset of temperature recording were defined as “torpid” bees. Those starting to warm-up 24 min after the onset of the measurement were allowed to continue warm-up for a further 68 min. However, bumblebees starting warming-up even sooner and reaching a plateau-value of thorax temperature (above 30°C) within the first 10 min were left in this activity phase for a further 5 min. Finally. bees starting to fly after warm-up were freed from the thermocouple and were allowed to fly for 5 min. To end an experiment, every bee then was instantly freeze-clamped individually. By this procedure, depending on the course of thorax temperature recorded (or on the flight activity observed) the bees were filed into the four activity groups: “torpor”, “warm-up”, “activated rest” and “flight”.
by adding phospho-fructokinase. Fructose-1.6-bis-phosphate on the other hand, was converted into fructose-6phosphate using fructose bis-phosphatase. It was ensured that reaction conditions allowed complete conversion in all reactions. Thereafter, separations of fructose-mono-phosphate and fructose-bis-phosphate were repeated on freshly prepared Dowex-2-formiate columns. It was shown in separate experiments. that doublelabelled glucose injected into the hemolymph of the bumblebees is converted into trehalose within minutes. “H,‘“C-ratio of trehalose, however, remains unchanged. The trehalose then is taken up by the flight muscles as a fuel for energy production. Therefore, instead of glucose, out of the fraction obtained from the Dowex-2-column containing unloaded components, trehalose was separated as described by Van der Horst et al. (1983). By means of a thinlayer-chromatogram linear analyser (LB 2832. Berthold, Bad Wildbad) labelled trehalose was localized on the plates, eluted
and transferred to a scintillation vial. ‘H/‘4C-ratios of the separated hexose-phosphates and trehalose then were measured by a special scintillation counting program for 3H- and “C-double-labelling using ‘Biofluor’ (NEN) as scintillator in a Packard Tri-Carb 4530 scintillation counter (for details see Surholt, 1981). Relative and absolute cycling rates were calculated from the )H/‘4C-ratios measured by the formula developed by Katz and Rognstad (1976) and Newsholme and Crabtree (I 976). RESULTS
As stated in the Materials and Methods section the rates of substrate cycling between fructose 6-phosphate and fructose 1,6-bis-phosphate were measured in the flight muscles of individual bees. Such an individual bumblebee can stay in different “activity phases”, which can only be determined definitely by fixing thermocouples to the thorax, as done in this study, or by using an infrared-camera. Doing this, the following phases were found: when ambient temperature is low (in these experiments 17-Q bees can be found having a thorax surface temperature only l-2°C higher than the surrounding air. This status can be defined as “torpor” (Fig. 1, line 1). Without any trigger visually detectable outside of the bee, one or another then may start to heat up its thorax temperature (up to inner temperatures of 3640°C) thermogenesis within minutes by “non-shivering” (“C) 16
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I l_“\__,_.z. ,--\,*.c--
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;
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Preparation qf extrac~.~ and isolation qf‘ metabolites After freeze-clamping. perchloric acid extracts from bumblebee flight muscles were prepared and afterwards tritiated water was removed from the extracts as described previously (Surholt and Newsholme, 1983). Separation of hexose-phosphates in principle followed the method of Hammerstedt (1980). To ensure, however, that only ‘H/“C-ratios of fructose-6-P/glucose-6-P and fructose 1,6-bis-phosphate, respectively, were measured, after separating these compounds by anion-exchange column chromatography on Dowex-2-formiate, the separated hexose-phosphates were additionally treated as follows: glucose-6-P was transferred into fructose-6-P using phospho-glucoisomerase and the product fructose-6-P then, together with fructose-6-P already present in this hexose-mono-phosphate fraction, were enzymatically converted into fructose 1,6-phosphate
Time (mid
Fig. 1. Original records of thorax surface temperatures of Bombus terrestris males measured by differential thermocouples representing the four “activity phases”: 1. torpor: 2. warm-up; 3. “activated” rest: 4. flight. Arrows symbolize freeze clamping.
Warm-up in bumble~es (Fig. 1, line 2). After such a phase of warm-up a bumblebee then can remain in an “activated” restphase showing no detectable movements, but keeping its thorax temperature constantly high on that level reached, at least for several minutes (Fig. 1, line 3). From this phase of “activated rest” or immediately from warming-up a bumblebee then directly can go over to flight (Fig. 1, line 4). Considering this, after the injection of the double-labelled glucose into the hemolymph, the surface temperature of the thorax of every single bumblebee was registered during the whole experimental time of 10-1.5 min to determine the activity-phase the bees belonged to. After immediate freeze-clamping, the cycling rate of every individual bee was then estimated. The rates of cycling between fructose bis-phosphate and fructose 6-phosphate found in the flight muscles are summarized in Table 1. As can be seen from this table, there are enormous differences in the intensity of cycling between the four activity phases defined. Not only the absolute cycling rates but already the relative cycling rates differ enormously. During “torpor” and during flight these relative rates are very low, in “torpor” between 0.08 and 0.45 (average: 0.24) and in flight between 0.10 and 0.60 (average: 0.22). They are clearly higher than I in the phase of “activated” rest, values between 1.39 and 3.64 (average: 1.94) were measured. Highest cycling rates, however, were found in those bumblebees steeply warming-up their thorax; relative rates between 5.0 and 24.0 (mean value: 9.14) were determined. Although such very different methods, as measurements of oxygen consumption or fuel utilization, were applied, previous studies on bumblebees resulted in nicely corresponding data for the net-flux through glycolysis in the flight muscles during the four different “activity phases” (Bertsch, 1984; Silvola, 1984 and Surholt et al., 1988). The following net-fluxes were measured in these studies: torpor 0.12, warm-up 7.6, activated rest 2.5 and flight 7.6 pmoles hexose/min x g flight muscles. Using these data and the formula developed by Katz and Rognstad (1976) and Newsholme and Crabtree (1976), it now is possible to evaluate the absolute rates of the fructose 6-phosphate/fructose bis-phosphate cycle in the bumblebee flight muscles. The absolute cycling rates obtained for the four different activity phases of the bees are also given in Table I. Even more obvious than the relative cycling rates, the absolute rates demonstrate that especially during the phase of warming-up, cycling takes place really intensively. While going from torpor to warm-up, within minutes the intensity of substrate cycling increases more than 2200-fold in average and during “activated” rest rates are still more than ISO-fold
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higher than in torpor. During flight they are only approximately 50 times higher. Since absolute cycling rates also represent the rates of ATP-hydrolysis, these data additionally allow calculation of the rates of heat production in the flight muscles and comparison of these to warm-up rates measured in this and in earlier studies (Surholt et al., 1990). The average ATP-hydrolysis measured during warm-up was 249 pmole/min x g muscles or 12.5 pmol/min x bumblebee (body weight of the bee 185 mg, muscle weight 60 mg). Since hydrolysis and immediate resynthesis of 1 mol ATP sets free ap proximately 74 kJ (Karlson, 1988) the average value corresponds to a heat production of 0.0155 J/set x insect. The maximum rate of 655 pmol/min x g muscle found during warm-up even releases 0.04 J/set x bee. DISCUSSION
It is well known that at low ambient temperatures several bumblebee species of cold temperate regions are able to warm-up their thorax temperature. Krogh and Zeuthen (1941) and Heinrich (1972a,b,c; 1975) showed, that those bumblebees can also regulate their inner thorax temperature in a range between 36 and 45°C. Heat production takes place in the flight muscles and is always coupled with the appearance of muscle action potentials, although very often no vibrations of the thorax or wings are visible. The rate of heat production is proportional to the frequency of these potentials (Kammer and Heinrich, 1972; Heinrich and Kammer, 1973). In addition, in workers of ~u~bus ~~~~~~e~~~~~, Kammer and Heinrich (1974) measured that per one potential an average of 2.3 to 2.6p102/g thorax was consumed. Contrary to the interpretation drawn from these findings by Heinrich (1979), recent studies (Surholt et al., 1990) have demonstrated that, during such “warm-ups” without any detectable vibration, heat production must be caused by a non-shivering thermogenesis. The results of the present study suggest that substrate cycling between fructose 6-phosphate and fructose 1,6-bis-phosphate might be the predominant mechanism of this non-shivering heat generation. Clark et al. (1973) already have proved the existence of this cycle in flight muscles of ~~~b~s a&zis. The cycling rates, however, they measured were by far too low to produce enough heat energy to warm-up the thorax with the intensity found in several bumblebee species and casts (Stone and Willmer, 1989). Newsholme and Crabtree (1976) have calculated that the highest cycling rates measured in the flight muscles of ~#~b~~ a~~~~ (10.4 ~moIes/min x g) are not able to generate more than 10% of the heat required.
Tabie I. Relative and absolute cycling rates of fructose fSphosphate/fructose b&phosphate substrate cycle in flight muscles of male Bombus rerresrris during four defined activity phases Activity
Rel. cycl, rates
phase
Torpor (n = 1I) Warm-up (n = II) “Activated” rest (n = Flight (n = 13)
II)
0.24+0.11 9.14 f 3.51 I .94 f 0.59 0.22 * 0.1 I
[~mol:min 0.03 69.42 4.84 1.65
Abs. cycl. rates g insect] [j~mol/min g muscle] i 0.0 I + 26.68 f I .41 + 0.82
0.I 1* 0.04 249.91 + 96.05 17.42 & 5.29 5.94 + 2.95
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BERNHARD SURHOLTel al.
Examining rates of cycling during the phase of “warm-up” directly, as done now in individual males of Bombus terrestris, however, revealed average cycling rates of 250 pmoles/min x g muscle and highest rates of up to 655 pmoles/min. The following calculations of the energy demanded for heat production during this phase confirm that cycling indeed plays that important role in heat generation, as suggested. Above all, the thorax temperature of those bees is influenced by two processes: the active production of heat by the muscles and the passive loss of heat to the colder ambient air. That means, that not only heat production for direct warming-up is required, but that, with continuously increasing temperature difference between thorax and ambient, in addition more and more heat for compensation of the passive loss of body heat must be produced. According to Pyke (1979) and Bertsch (1984) the passive loss of heat of an “average bumblebee thorax” of 60 mg is 0.00167 Jjsec and 1°C difference in temperature. On the other hand, the amount of energy needed to warm-up such a thorax (heat capacity) is 0.2 J/“C (Krogh and Zeuthen, 1941; Heinrich, 1975). Thus the energy demanded for warm-up equals: 0.2 x “,idt + 0.1 x T. (dT/d,= ascent of temperature curve and T = difference in temperature between thorax and ambient air, both at time of measurement.) Applying this formula to the rates of cycling determined, an average cycling rate of 250 pmolesj min x g, which as shown equals to 0.0155 Jjsec and bumblebee for example allows a warm-up of 2.5”C at a given temperature difference of 4.3”C, or of l.S”C/min at 6.3”C difference. The highest cycling rate (655 ~moles/m~n) measured (releasing 0.04 J/set and bee) even allows warm-up rates of 3.5 “Cjmin at 17°C or 2.5”C/min at 19°C difference in thorax-toambient temperature. Finally, a few ideas about mechanisms regulating the intensity of fructose 6-phosphate/fructose bisphosphate substrate cycling in bumblebee flight muscles will be presented. That temperature-depending changes in the concentration of fructose 2,6-his-phosphate may regulate the intensity of this substrate cycle in bumblebee flight muscles by modulating &phosphofructokinase activity has recently been suggested by Leite et al. (1988). Measurements of the concentration of this component in the flight muscles of Bombus terrestris (Bathe and Surholt, unpublished), however, could not confirm this theory. During the four activity phases, as determined by thermocouples, no significant changes were found; an average of 1.34-1.38 f 0.40 nmolesjg muscle fresh weight was measured (n = 10 individual bees in each group). Whatever the biochemically measured data obtained from Bomhus stratus may suggest, they are difficult to interpret because no data upon warming-up rates and cycling rates in V&J seem to be available. Above all, it should be considered that Bombus stratus, as member of the few bumblebee species of the otherwise numerically huge genus Bnmbus living in the (neo-)tropicai region (Roubik, I989), may not need or may not have that enormous capacity to generate heat as those species living in cold temperate or even alpine or arctic regions.
As already has been shown for several of those cold-adapted bumblebee species by Heir&h and co-workers (see Heinrich, 1979) and recently also for Bombus terrestris (Surholt et al., 1990) “warm-ups” are always coupled to muscle action potentials. Although very often following those burst of muscle potentials no vibrations of the thorax were measurable, meaning that the asynchronous flight muscles cannot be mechanically active, those low frequency potentials (recorded up to 20 Hz) should cause an immediate release of calcium ions from the sarcoplasmic reticulum into the cytoplasm (Ashley and Ridgeway, 1970; Rfiegg, 1988). As shown by Greive and Surholt (1990) fructose 1,6-bisphosphatase (FBPase) from Bombus terrestris flight muscles in vitro is markedly activated when free calcium is increased from pCa 9 to 7. Consequently, supposing that this also holds in vivo, the rate of fructose 6-phosphate/fructose bis-phosphate substrate cycle is immediately pushed up, when an action potential is coming in. Since the frequency of those potentials, however, is relatively low and sarcoplasmic reticulum in asynchronous flight muscles is reduced compared to synchronous flight muscles, the concentration of free calcium then probably is reduced gradually before the next potential appears in the fibre. Thus, no highly efficient summing-up of calcium concentration in cytoplasm takes place and cycling rates seem to follow the up and down of free calcium corresponding to the potentials. This probably leads to the relative high standard deviation seen in the cycling rates of the warm-up phase. When the bee starts to fly, however, frequency of muscle potentials recorded is more than doubled (40-60 Hz). Now the concentration of free calcium should increase steeply, because transport back into the sarcoplasmic reticulum cannot be really efficient compared to the efflux caused by these high-frequency potentials, and free Cal+ very quickly should exceed pCa 6. This then causes strong inhibition of FBPase activity concomitant with an immediate slowing down of substrate cycling. Summing-up this hypothesis, concentration of free calcium ions in the cytoplasm of the bumblebee flight muscles probably plays the predominant role. At first, at increasing but relatively low concentrations (pea 7-6), it is the signal for quick acceleration of substrate cycling intensity while later on (i.e. at onset of flight) with concentrations exceeding 10 -6 M, it induces rapid decrease of cycling rates. Secondly, however, these high concentrations then also allow these asynchronous flight muscles to be mechanically active as soon as stretch activation occurs. A~knoM’ledgenlenrs-Wegratefully acknowledge support by the Deutsche Forschungsgemeinschaft (Grant no. SU 82/24 and SFB 305). REFERENCES
Ashley C. C. and Ridgway E. B. (1970) On the relationship between membrane potential, calcium transient and tension in single barnacle muscle fibres. J. P~.Ys~o~. {~~~d~~f 209, 105-130. Bertsch A. (1984) Foraging in male bumble bees (Bombus lucorum L.): maximizing energy or minimizing water load? Oecologiu (Berlin) 62, 325-336.
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Bloxham D. P., Clark M. G., Holland P. C. and Lardy H. A. (1973) A model study of the fructose diphosphatase-phosphofructokinase substrate cycle. Biochem. J. 134, 581-587. Challis R. A. J., Arch J. R. S. and Newsholme E. A. (1984) The rate of substrate cycling between fructose 6-phosphate and fructose 1,6-bisphosphate in skeletal muscle. Biochem. J. 221, 153-161. Clark M. G., Bloxham D. P., Holland P. C. and Lardy H. A. (1973) Estimation of the fructose diphosphatasephosphofructokinase substrate cycle in the highi muscle of Bombus afinis. Biochem. J. 134. 589-597. Crabtree B. and Newsholme E. A. (1985) A quantitative ;yp;;ach to metabolic control. Curr. Top. Cell. Regul. 25, Greive H. and Surholt B. (1990) Dependence offructose-bisphosphatase from flight muscles of the bumblebee (Bombus terrestris L.) on calcium ions. Comp. Biochem. Physiol. WB, 197-200. Hammerstedt R. H. (1980) A rapid method for isolating metabolites involved in substrate cycling. Anal. Biochem. 10!9,443448. Heinrich B (1972a) Energetics of temperature regulation and foraging in a bumblebee, Bombus ferricola Kirby. J. camp. Physiol. 11, 49-64. Heinrich B. (1972b) Patterns of endothermy in bumblebee queens, drones and workers. J. camp. Physiol. 77, 65-79. Heinrich B. (1972~) Temperature regulation in the bumblebee Bombus vagans: a field study. Science 175, 185-187. Heinrich B. (1974) Thermoregulation in bumblebees 1. Brood incubation by Bombus vosnesenskii queens. J. camp. Physiol. 88, 129S140. Heinrich B. (1975) Thermoregulation in bumblebees II. Energetics of warm-up and free flight. J. camp. Physiol. %, 155-166. Heinrich B. (1979) Bumblebee Economics. Harvard University Press, Cambridge, MA. Heinrich B. and Kammer A. E. (1973) Activation of the fibrillar muscles in the bumblebee during warm-up, stabilization of thorax temperature and flight. J. exp. Biol. 58, 6777688. Kammer A. E. and Heinrich B. (1972) Neural control of bumblebee fibrillar muscles during shivering. J. camp. Physiol. 78, 337-345. Kammer A. E. and Heinrich B. (1974) Metabolic rates related to muscle activity in bumblebees. J. exp. Biol. 61, 212-227. Karlson P. (1988) Biochemiefiir Mediziner und Narurwissenschqftler. Georg Thieme, Stuttgart. Katz J. and Rognstad R. (1976) Futile cycles in the metabolism of glucose. Curr. Too. Cell. Repul. 10. 237-289. Katz J. and Rognstad R. (1978) Futile-cycling in glucose metabolism. Trends Biochem. Sci. 3, l7l- 174. Krogh A. and Zeuthen E. (1941) The mechanism of flight preparation in some insects. J. e.xp. Biol. 18, I-10.
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Leite A., Neto J. A., Eyton J. F., Crivellaro 0. and El-Dorry H. A. (1988) Phosphofructokinase from bumblebee flight muscle. J. biol. Chem. 263, 17527-17533. Moore S. A. (1985) Substrate cycles. Trends Biochem. Sri. 10, 386387. Newsholme E. A. (1980) A possible metabolic basis for the control of body weight. N. Engl. J. Med. 302, 400405. Newsholme E. A. and Crabtree B. (1976) Substrate cycles in metabolic regulation and in heat generation. Biochem. Sot. Symp. 41, 61-109. Newsholme E. A., Crabtree B., Higgins S. J., Thornton S. D. and Start C. (1972) The activities of fructose diphosphatase in flight muscles from bumble-bee and the role of this enzyme in heat generation. Biochem. J. 128, 89-97. Newsholme E. A., Arch J. R. S., Brooks B. and Surholt B. (1983) The role of substrate cycles in the metabolic regulation. Biochem. Sot. Trans. 11, 52-56. Newsholme E. A., Challis R. A. J. and Crabtree B. (1984) Substrate cycles: their role in improving sensitivity in metabolic control. Tren& Biochem. Sci. 9, 277-280. Pyke G. H. (1979) Optimal foraging in bumblebees: rule of movement between flowers within influrescences. Anim. Behav. 27, 1167-1181. Roubik D. W. (1989) Ecology and Nafural History of Tropical Bees. Cambridge University Press, Cambridge, NY. Riiegg J. C. (1988) Calcium in Muscle Actitation. Springer, Berlin. Silvola J. (1984) Respiration and energetics of the bumblebee Bombus rerrestris queen. Holarctic Ecology 7, 177-181. Stone G. N. and Willmer P. G. (1989) Warm-up rates and body temperatures in bees: the importance of body size, thermal regime and phylogeny. J. exp. Biol. 147, 303-328. Surholt B. (1981) Substrat-Zyklen in Insektenflugmuskeln. Untersuchungen an den Flugmuskeln des Totenkopfschwarmers Acherontia atropos. Habilitationsschrift, Universitlt Miinster. Surholt B. and Newsholme E. A. (1983) The rate of substrate cycling between glucose and glucose 6-phosphate in muscle and fat-body of the hawk moth (Acheronfia arropos) at rest and during flight. Biochem. J. 210, 49-54. Surholt B., Greive H., Hommel C. and Bertsch A. (1988) Fuel uptake, storage and use in male bumble bees Bombus terrestris L. J. camp. Physiol. 158, 263.-269. Surholt B., Greive H., Baa1 T. and Bertsch A. (1990) Non-shivering thermogenesis in asynchronous flight muscles of bumblebees? Comparative studies on males of Bombus rerrestris, Xylocopa sulcatipes and Achrrontia arropos. Comp. Biochem. Physiol. 97A, 493-499. Van der Horst D. J., Abbink J. H. M., Van Dorn J. M., Van Marrewijk W. J. A. and Beenakkers A. M. Th. (1983) Glycerol dynamics and metabolism during flight of the locust, Locusta migratoria. Inseci Biochem. 13, 45-55.
Camp. Bio~h~m. Physiol. Vol. 98A, No. 2,pp. 299-303,1991
C 1991Pergamon Press pie
Printed in Great Britain
WARM-UP AND SUBSTRATE CYCLING IN FLIGHT MUSCLES OF MALE BUMBLEBEES, ~~~~U~ TERRESTRIS BERNHARDSuaHor_r,*t$ HEINRICH GREIVE,* THOMASBAAL* and ANDREAS BERTSCH~ *Zoologisches lnstitut der Westfalischen Wilhelms-Universitlt, Lehrstuhl fur Tierphysiologie, H~ndenburgplatz 55, D-4400 Miinster, F.R.G.; §Fachbereich Biologie der Philipps Universitgt, Karl-von-Frisch-Strasse, D-3550 Marburg, F.R.G. (Received 21 June 1990) Abstract-l I Rates of substrate cycling between fructose &phosphate and fructose bis-phosphate were measured in the flight muscles of male bumblebees (Boamhurterrestris) in viva during four defined activity phases. 2. Shortly after the injection of double-labelled glucose the~~oupies were fixed to the thorax to record surface temperature continuously and to determine the phase of activity every individual bee was in, when finally the cycling rate was measured. 3. Based upon the ‘H/‘4C-ratios, measured in the sugars and hexose-phosphates involved, the following relative cycling rates were determined: torpor: 0.24 k 0.11; warm-up: 9.14 + 3.51; “activated” rest: 1.94 + 0.59, and flight: 0.22 + 0. I 1. Using the data of the glycolytic fluxes, determined in the flight muscles of these bumblebees in previous studies, absolute cycling rates were evaluated as follows: torpor: 0. I I k 0.04; warm-up: 249.9 1 k 96.05; “activated” rest: 17.42 + 5.29, and flight: 5.94 + 2.95 ~moi/min x g muscle. 4. Conversion of these rates of substrate cycling (=rates of ATP-hydrolysis) into rates of heat production demonstrates that in the flight muscles of Bombus terresfris during phases of warming-up without any detectable movements of wings or thorax substrate cycling between fructose 6-phosphate and fructose-bis-phosphate very probably is the predominant mechanism of non-shivering heat production. 5. Possible mechanisms for the regulation of the intensity of this fructose &phosphate/fructose bis-phosphate substrate cycle are discussed.
INTRODUCTION Since unique high activities of fructose l,Qphosphatase have been found in flight muscles of several
bumblebee species (Newsholme et af., 1972), it has remained a matter of debate whether or not this enzyme in l&o, together with Gphospho-fructokinase, forms a “non-futile” substrate cycle (Katz and Rognstad, 1978; Newsholme, 1980: Newsholme et al., 1983; Newsholme et al., 1984; Challis et al., 1984; Moore, 1985). Theoretically. in addition to improving sensitivity in metabolic control (Crabtree and Newsholme, 1985). such a substrate cycle could be a mechanism of heat generation by hydrolysing ATP. Indeed, Clark er al. (1973) were able to demonstrate that such a cycle exists in flight muscles of Bombus uffis, but the cycling rates (i.e. the rates of ATP-hydrolysis) they found were by far too low to generate such amounts of heat to enable a bumblebee to warm-up its flight muscles from 6 to 36°C within 15 min (Heinrich, 1974, 1975). In addition, Kammer and Heinrich (1972) found that warming-up is always coupled with muscle potentials and Heinrich (1979) therefore, describes the essential mechanism of heat tPresent address: Department of Experimental Zoology, University of Utrecht, 8 Padualaan, 3508 TB Utrecht, The Netherlands. SAuthor to whom correspondence should be addressed.
production as muscle shivering, although no movements of the thorax or wings were visually detectable. Recent studies, applying a highly sensitive vibration monitoring system (Surhoit et al., 1990), however, made it extremely improbabie that shivering is the physiological mechanism providing heat production in bumblebee flight muscles. Both, intensive warming-up and muscle potentials (in male Bombus terrestris up to 25 Hz), were recorded, but no vibrations of the thorax. Therefore, in the present study, the method of recording thorax surface temperature, as used in the above study, and the method of measuring the rates of substrate cycling between fructose 6-phosphate and fructose 1,6-bis-phosphate by injection of double-labelled radioactive glucose, as introduced by Bloxham et al. (1973), were combined. This allowed to determine the rates of substrate cycling in the bumblebee flight muscles in z&o during four clearly defined phases of activity of these bees. MATERIALS AND METHODS Animals Bo~bus terrestris were bred under controlled conditions in a greenhouse as described previously (Bertsch, 1984). About one week after they had emerged, male bees were separated from the breeding colonies and labelled individually. Before an experiment, body weight, food uptake and activity pattern were controlled and recorded daily over 7 days as described by Surholt ef al. (1988).
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Chemicals D-[5-‘HI-glucose (21.1 Ci/mmol) and o-[6-‘JC]-glucose (55.0 mCi/mmol) were purchased from Amersham Buchler, Braunschweig. Prior to use the radiochemicals were freezedried to remove traces of radio-labelled water. All biochemicals (enzymes, substrates and co-substrates) were from Boehringer, Mannheim, except fructose 1,6-bis-phosphatase. This, and also Dowex-50-Wx8 and Dowex-2-chlorid, were from Sigma, Taufkirchen. Precoated thin layer-plates came from Machery & Nagel, Diiren; high efficiency emulsifier cocktail ‘Biofluor’ from Du Pont de Nemours, Bad Homburg and Beta-Spark gas (for the linear analyser) from Messer-GrieBheim, Frankfurt a. M., all other chemicals were from Merck, Darmstadt. Experimenlal procedure Basis for measuring cycling rates between fructose 6phosphate and fructose bis-phosphate was the method using D-[5-3H]-glucose and D-[6-‘4C]-glucose introduced by Bloxham et al. (1973). Experiments, however, had to be performed on individual bees, because preliminary experiments had shown that only in this case reproducible results can be achieved. A bumblebee was cooled down to 4°C in a dark refrigerator for 1 hr. Then a differential copper-constantan thermocouple was quickly fixed to the thorax surface as described previously (Surholt er al., 1990). Thereafter the bee was kept in the refrigerator for a further IO min. By transferring it into a deep-freezer (-25’C) for 3 min it then was anaesthetized more intensively and immediately afterwards 3 ~1 double-labelled glucose solution (consisting of 3 PCi o-[S-‘HI-glucose and 3 PCi D-[6-“‘Clglucose) was injected into the abdomen using a 10~1 Hamilton syringe. Then the bee was brought directly into a climate chamber (17°C) and kept in darkness for a further 3 min (for temperature adaptation). During this time the thermocouple was connected to the measuring system (see Surholt et al., 1990). Then the lights were switched on and from then the thorax surface temperature was recorded continuously. Bees keeping their thorax temperature at about the ambient temperature (17°C) for 15 min after the onset of temperature recording were defined as “torpid” bees. Those starting to warm-up 24 min after the onset of the measurement were allowed to continue warm-up for a further 68 min. However, bumblebees starting warming-up even sooner and reaching a plateau-value of thorax temperature (above 30°C) within the first 10 min were left in this activity phase for a further 5 min. Finally. bees starting to fly after warm-up were freed from the thermocouple and were allowed to fly for 5 min. To end an experiment, every bee then was instantly freeze-clamped individually. By this procedure, depending on the course of thorax temperature recorded (or on the flight activity observed) the bees were filed into the four activity groups: “torpor”, “warm-up”, “activated rest” and “flight”.
by adding phospho-fructokinase. Fructose-1.6-bis-phosphate on the other hand, was converted into fructose-6phosphate using fructose bis-phosphatase. It was ensured that reaction conditions allowed complete conversion in all reactions. Thereafter, separations of fructose-mono-phosphate and fructose-bis-phosphate were repeated on freshly prepared Dowex-2-formiate columns. It was shown in separate experiments. that doublelabelled glucose injected into the hemolymph of the bumblebees is converted into trehalose within minutes. “H,‘“C-ratio of trehalose, however, remains unchanged. The trehalose then is taken up by the flight muscles as a fuel for energy production. Therefore, instead of glucose, out of the fraction obtained from the Dowex-2-column containing unloaded components, trehalose was separated as described by Van der Horst et al. (1983). By means of a thinlayer-chromatogram linear analyser (LB 2832. Berthold, Bad Wildbad) labelled trehalose was localized on the plates, eluted
and transferred to a scintillation vial. ‘H/‘4C-ratios of the separated hexose-phosphates and trehalose then were measured by a special scintillation counting program for 3H- and “C-double-labelling using ‘Biofluor’ (NEN) as scintillator in a Packard Tri-Carb 4530 scintillation counter (for details see Surholt, 1981). Relative and absolute cycling rates were calculated from the )H/‘4C-ratios measured by the formula developed by Katz and Rognstad (1976) and Newsholme and Crabtree (I 976). RESULTS
As stated in the Materials and Methods section the rates of substrate cycling between fructose 6-phosphate and fructose 1,6-bis-phosphate were measured in the flight muscles of individual bees. Such an individual bumblebee can stay in different “activity phases”, which can only be determined definitely by fixing thermocouples to the thorax, as done in this study, or by using an infrared-camera. Doing this, the following phases were found: when ambient temperature is low (in these experiments 17-Q bees can be found having a thorax surface temperature only l-2°C higher than the surrounding air. This status can be defined as “torpor” (Fig. 1, line 1). Without any trigger visually detectable outside of the bee, one or another then may start to heat up its thorax temperature (up to inner temperatures of 3640°C) thermogenesis within minutes by “non-shivering” (“C) 16
-q--.
I l_“\__,_.z. ,--\,*.c--
14 1 12 -1
,,‘a
;
,i
Preparation qf extrac~.~ and isolation qf‘ metabolites After freeze-clamping. perchloric acid extracts from bumblebee flight muscles were prepared and afterwards tritiated water was removed from the extracts as described previously (Surholt and Newsholme, 1983). Separation of hexose-phosphates in principle followed the method of Hammerstedt (1980). To ensure, however, that only ‘H/“C-ratios of fructose-6-P/glucose-6-P and fructose 1,6-bis-phosphate, respectively, were measured, after separating these compounds by anion-exchange column chromatography on Dowex-2-formiate, the separated hexose-phosphates were additionally treated as follows: glucose-6-P was transferred into fructose-6-P using phospho-glucoisomerase and the product fructose-6-P then, together with fructose-6-P already present in this hexose-mono-phosphate fraction, were enzymatically converted into fructose 1,6-phosphate
Time (mid
Fig. 1. Original records of thorax surface temperatures of Bombus terrestris males measured by differential thermocouples representing the four “activity phases”: 1. torpor: 2. warm-up; 3. “activated” rest: 4. flight. Arrows symbolize freeze clamping.
Warm-up in bumble~es (Fig. 1, line 2). After such a phase of warm-up a bumblebee then can remain in an “activated” restphase showing no detectable movements, but keeping its thorax temperature constantly high on that level reached, at least for several minutes (Fig. 1, line 3). From this phase of “activated rest” or immediately from warming-up a bumblebee then directly can go over to flight (Fig. 1, line 4). Considering this, after the injection of the double-labelled glucose into the hemolymph, the surface temperature of the thorax of every single bumblebee was registered during the whole experimental time of 10-1.5 min to determine the activity-phase the bees belonged to. After immediate freeze-clamping, the cycling rate of every individual bee was then estimated. The rates of cycling between fructose bis-phosphate and fructose 6-phosphate found in the flight muscles are summarized in Table 1. As can be seen from this table, there are enormous differences in the intensity of cycling between the four activity phases defined. Not only the absolute cycling rates but already the relative cycling rates differ enormously. During “torpor” and during flight these relative rates are very low, in “torpor” between 0.08 and 0.45 (average: 0.24) and in flight between 0.10 and 0.60 (average: 0.22). They are clearly higher than I in the phase of “activated” rest, values between 1.39 and 3.64 (average: 1.94) were measured. Highest cycling rates, however, were found in those bumblebees steeply warming-up their thorax; relative rates between 5.0 and 24.0 (mean value: 9.14) were determined. Although such very different methods, as measurements of oxygen consumption or fuel utilization, were applied, previous studies on bumblebees resulted in nicely corresponding data for the net-flux through glycolysis in the flight muscles during the four different “activity phases” (Bertsch, 1984; Silvola, 1984 and Surholt et al., 1988). The following net-fluxes were measured in these studies: torpor 0.12, warm-up 7.6, activated rest 2.5 and flight 7.6 pmoles hexose/min x g flight muscles. Using these data and the formula developed by Katz and Rognstad (1976) and Newsholme and Crabtree (1976), it now is possible to evaluate the absolute rates of the fructose 6-phosphate/fructose bis-phosphate cycle in the bumblebee flight muscles. The absolute cycling rates obtained for the four different activity phases of the bees are also given in Table I. Even more obvious than the relative cycling rates, the absolute rates demonstrate that especially during the phase of warming-up, cycling takes place really intensively. While going from torpor to warm-up, within minutes the intensity of substrate cycling increases more than 2200-fold in average and during “activated” rest rates are still more than ISO-fold
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higher than in torpor. During flight they are only approximately 50 times higher. Since absolute cycling rates also represent the rates of ATP-hydrolysis, these data additionally allow calculation of the rates of heat production in the flight muscles and comparison of these to warm-up rates measured in this and in earlier studies (Surholt et al., 1990). The average ATP-hydrolysis measured during warm-up was 249 pmole/min x g muscles or 12.5 pmol/min x bumblebee (body weight of the bee 185 mg, muscle weight 60 mg). Since hydrolysis and immediate resynthesis of 1 mol ATP sets free ap proximately 74 kJ (Karlson, 1988) the average value corresponds to a heat production of 0.0155 J/set x insect. The maximum rate of 655 pmol/min x g muscle found during warm-up even releases 0.04 J/set x bee. DISCUSSION
It is well known that at low ambient temperatures several bumblebee species of cold temperate regions are able to warm-up their thorax temperature. Krogh and Zeuthen (1941) and Heinrich (1972a,b,c; 1975) showed, that those bumblebees can also regulate their inner thorax temperature in a range between 36 and 45°C. Heat production takes place in the flight muscles and is always coupled with the appearance of muscle action potentials, although very often no vibrations of the thorax or wings are visible. The rate of heat production is proportional to the frequency of these potentials (Kammer and Heinrich, 1972; Heinrich and Kammer, 1973). In addition, in workers of ~u~bus ~~~~~~e~~~~~, Kammer and Heinrich (1974) measured that per one potential an average of 2.3 to 2.6p102/g thorax was consumed. Contrary to the interpretation drawn from these findings by Heinrich (1979), recent studies (Surholt et al., 1990) have demonstrated that, during such “warm-ups” without any detectable vibration, heat production must be caused by a non-shivering thermogenesis. The results of the present study suggest that substrate cycling between fructose 6-phosphate and fructose 1,6-bis-phosphate might be the predominant mechanism of this non-shivering heat generation. Clark et al. (1973) already have proved the existence of this cycle in flight muscles of ~~~b~s a&zis. The cycling rates, however, they measured were by far too low to produce enough heat energy to warm-up the thorax with the intensity found in several bumblebee species and casts (Stone and Willmer, 1989). Newsholme and Crabtree (1976) have calculated that the highest cycling rates measured in the flight muscles of ~#~b~~ a~~~~ (10.4 ~moIes/min x g) are not able to generate more than 10% of the heat required.
Tabie I. Relative and absolute cycling rates of fructose fSphosphate/fructose b&phosphate substrate cycle in flight muscles of male Bombus rerresrris during four defined activity phases Activity
Rel. cycl, rates
phase
Torpor (n = 1I) Warm-up (n = II) “Activated” rest (n = Flight (n = 13)
II)
0.24+0.11 9.14 f 3.51 I .94 f 0.59 0.22 * 0.1 I
[~mol:min 0.03 69.42 4.84 1.65
Abs. cycl. rates g insect] [j~mol/min g muscle] i 0.0 I + 26.68 f I .41 + 0.82
0.I 1* 0.04 249.91 + 96.05 17.42 & 5.29 5.94 + 2.95
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BERNHARD SURHOLTel al.
Examining rates of cycling during the phase of “warm-up” directly, as done now in individual males of Bombus terrestris, however, revealed average cycling rates of 250 pmoles/min x g muscle and highest rates of up to 655 pmoles/min. The following calculations of the energy demanded for heat production during this phase confirm that cycling indeed plays that important role in heat generation, as suggested. Above all, the thorax temperature of those bees is influenced by two processes: the active production of heat by the muscles and the passive loss of heat to the colder ambient air. That means, that not only heat production for direct warming-up is required, but that, with continuously increasing temperature difference between thorax and ambient, in addition more and more heat for compensation of the passive loss of body heat must be produced. According to Pyke (1979) and Bertsch (1984) the passive loss of heat of an “average bumblebee thorax” of 60 mg is 0.00167 Jjsec and 1°C difference in temperature. On the other hand, the amount of energy needed to warm-up such a thorax (heat capacity) is 0.2 J/“C (Krogh and Zeuthen, 1941; Heinrich, 1975). Thus the energy demanded for warm-up equals: 0.2 x “,idt + 0.1 x T. (dT/d,= ascent of temperature curve and T = difference in temperature between thorax and ambient air, both at time of measurement.) Applying this formula to the rates of cycling determined, an average cycling rate of 250 pmolesj min x g, which as shown equals to 0.0155 Jjsec and bumblebee for example allows a warm-up of 2.5”C at a given temperature difference of 4.3”C, or of l.S”C/min at 6.3”C difference. The highest cycling rate (655 ~moles/m~n) measured (releasing 0.04 J/set and bee) even allows warm-up rates of 3.5 “Cjmin at 17°C or 2.5”C/min at 19°C difference in thorax-toambient temperature. Finally, a few ideas about mechanisms regulating the intensity of fructose 6-phosphate/fructose bisphosphate substrate cycling in bumblebee flight muscles will be presented. That temperature-depending changes in the concentration of fructose 2,6-his-phosphate may regulate the intensity of this substrate cycle in bumblebee flight muscles by modulating &phosphofructokinase activity has recently been suggested by Leite et al. (1988). Measurements of the concentration of this component in the flight muscles of Bombus terrestris (Bathe and Surholt, unpublished), however, could not confirm this theory. During the four activity phases, as determined by thermocouples, no significant changes were found; an average of 1.34-1.38 f 0.40 nmolesjg muscle fresh weight was measured (n = 10 individual bees in each group). Whatever the biochemically measured data obtained from Bomhus stratus may suggest, they are difficult to interpret because no data upon warming-up rates and cycling rates in V&J seem to be available. Above all, it should be considered that Bombus stratus, as member of the few bumblebee species of the otherwise numerically huge genus Bnmbus living in the (neo-)tropicai region (Roubik, I989), may not need or may not have that enormous capacity to generate heat as those species living in cold temperate or even alpine or arctic regions.
As already has been shown for several of those cold-adapted bumblebee species by Heir&h and co-workers (see Heinrich, 1979) and recently also for Bombus terrestris (Surholt et al., 1990) “warm-ups” are always coupled to muscle action potentials. Although very often following those burst of muscle potentials no vibrations of the thorax were measurable, meaning that the asynchronous flight muscles cannot be mechanically active, those low frequency potentials (recorded up to 20 Hz) should cause an immediate release of calcium ions from the sarcoplasmic reticulum into the cytoplasm (Ashley and Ridgeway, 1970; Rfiegg, 1988). As shown by Greive and Surholt (1990) fructose 1,6-bisphosphatase (FBPase) from Bombus terrestris flight muscles in vitro is markedly activated when free calcium is increased from pCa 9 to 7. Consequently, supposing that this also holds in vivo, the rate of fructose 6-phosphate/fructose bis-phosphate substrate cycle is immediately pushed up, when an action potential is coming in. Since the frequency of those potentials, however, is relatively low and sarcoplasmic reticulum in asynchronous flight muscles is reduced compared to synchronous flight muscles, the concentration of free calcium then probably is reduced gradually before the next potential appears in the fibre. Thus, no highly efficient summing-up of calcium concentration in cytoplasm takes place and cycling rates seem to follow the up and down of free calcium corresponding to the potentials. This probably leads to the relative high standard deviation seen in the cycling rates of the warm-up phase. When the bee starts to fly, however, frequency of muscle potentials recorded is more than doubled (40-60 Hz). Now the concentration of free calcium should increase steeply, because transport back into the sarcoplasmic reticulum cannot be really efficient compared to the efflux caused by these high-frequency potentials, and free Cal+ very quickly should exceed pCa 6. This then causes strong inhibition of FBPase activity concomitant with an immediate slowing down of substrate cycling. Summing-up this hypothesis, concentration of free calcium ions in the cytoplasm of the bumblebee flight muscles probably plays the predominant role. At first, at increasing but relatively low concentrations (pea 7-6), it is the signal for quick acceleration of substrate cycling intensity while later on (i.e. at onset of flight) with concentrations exceeding 10 -6 M, it induces rapid decrease of cycling rates. Secondly, however, these high concentrations then also allow these asynchronous flight muscles to be mechanically active as soon as stretch activation occurs. A~knoM’ledgenlenrs-Wegratefully acknowledge support by the Deutsche Forschungsgemeinschaft (Grant no. SU 82/24 and SFB 305). REFERENCES
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