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The effect of the α2-adrenoceptor agonist, guanfacin, on energy expenditure, intake and deposition in rats
Camp. Biochem. Physiol. Vol. 112C.No. 1, pp. 29-34, 1995
Pergamon
Copyright 0 1995Elsevier Science Inc. Printed in Great Britain. All rights reserved 0742-8413195 $9.50+ .OO
0742-8413(95)00072-O
The effect of the q-adrenoceptor agonist, guanfacin, on energy expenditure, intake and deposition in rats Carlo Gazzola Queensland Department of Primary Industries, Tropical Beef Centre, PO Box 5545, Rockhampton Mail Centre, Qld 4702, Australia The effect of guanfacin on the energy expenditure, food intake and body composition of female Wistar rats at maintenance was studied. Rats on a restricted food intake (about 75% of ad lihitum), treated for 6 days with twice-daily injections of guanfacin.HCI (0.5 mg/kg) showed large reductions in energy expenditure (30%, p < 0.001) compared to control animals hut no significant differences in liveweight gain, dry mass gain, or gains in total body energy content, protein, fat and ash. The treated group had a lower intake of metabolixable energy (9%, p = 0.01) partially due to a lower intake of food. However, this lower intake of energy did not fully account for the lowered energy expenditure in the treated animals. The unaccounted lowered energy expenditure was not due to changes in the diurnal pattern of energy expenditure since continuous indirect calorimetry with rats fed ad libitum, over 3 days, showed that twice-daily injections of guanfacin (0.5 mglkg per injection) achieved a sustained decrease in energy expenditure. The lack of an increased growth rate of rats treated with guanfacin was attributed to behavioural changes; to a decreased food intake and to other, unknown, factors related to energy expenditure. Key words: Guanfacin;
Energy expenditure;
Energy deposition;
Growth; Rat.
Comp. Biochem. Physiol. 112C, 29-34, 1995.
Introduction The deposition of fat or protein in humans or commercial animals is generally manipulated by changes in dietary energy intake. Where dietary energy intake is fixed, especially at submaintenance levels, deposition can be manipulated by altering thermogenesis or energy expenditure (EE). In humans, exercise is the method of choice for increasing EE, while in commercial animals where the opposite effect is desirable, antithermogenic compounds would be useful in lowering EE. A link between drug-induced lowering of EE and increased liveweight gain has been convincingly demonstrated in pigs (Yen and Neinaber,
1992). In cattle, it can be predicted, on theoretical grounds, that moderate decreases (10%) in the maintenance energy requirements of beef cattle grazing on low-quality native Australian pastures can result in the economically and environmentally desirable outcome of a significant reduction in the time required to achieve a saleable liveweight (Hunter et al., 1992). As a tool for lowering EE in cattle, the cw,-adrenoceptor agonist, guanfacin, may be useful since it decreases resting energy expenditure (REE) by up to 20%, in rats (Gazzola, 1993), in mice (Sillence et al., 1990) and in cattle (Hunter, 1992). Hunter (1992) reported that under-ted cattle infused with guanfacin lost less liveweight than control animals. In many additional studies at our centre with mice and with rats, both under ad libitum and restricted food intakes, guanfacin treatment always lowered metabolic rate, but treated animals rarely showed enhanced liveweight gain over controls. The
Correspondence
10: Carlo Gazzola, Queensland Department of Primary Industries, Troical Beef Centre, P.O. Box 5545, Rockhampton Mail Centre, Qld 4702, Australia. Received 13 November 1994; revised 26 April 1995; accepted 27 April 1995.
29
30
C. Gazzola
All animal experiments were approved by the Animal Experimentation Ethics Committee of the Tropical Cattle Research Centre, Rockhampton, Australia. Female Wistar rats, liveweights noted below, were housed and cared for as previously described (Gazzola, 1993). Guanfacin.HCl was obtained from Sandoz (Base& Switzerland) and administered in isotonic saline, subcutaneously (SC), as a 0.5 mg/mL solution. Injections were of a volume in FL equal to the animal’s liveweight in grams giving a dose rate of 0.5 mg/kg. Control animals received saline injections exactly analogous to the regimen for the treated animals. All injections were given between 0930 and 1000 for once-daily treatments and a second injection was given at 2130 for twice-daily treatments.
Over a period of 6 days, all the rats were gradually restricted from ad libitum intake of food to an intake of 900 kJld/kg0~7S(about 75% of ad libitum). Food was given at 1600 daily and for the remainder of the experiment the rats were offered this same amount of food. For the next 6 days, pretreatment period, all the rats were injected twice-daily with saline. At the end of this 6-day period the rats in group Z were slaughtered by carbon dioxide anaesthesia and cervical dislocation, and frozen whole. The other two groups were treated during the next 6 days, treatment period, twice-daily, with saline (group C, control group) or guanfacin (group T, treated group). Resting oxygen consumption was measured on the third day of the period. After 6 days of treatment these rats were also slaughtered and frozen. All the rats’ whole bodies were autoclaved, homogenized and lyophilized to a constant dry mass. The energy content of the dried bodies, food samples and cardboard bases were determined using an adiabatic bomb calorimeter (Parr Instrument Co., U.S.A.). Protein was determined using Kjeldahl digestion and auto analyser methods (Industrial method 218-72A, Technicon, Tarrytown, NY, U.S.A.) with a nitrogen to protein conversion factor of 6.25. Fat was determined by the Folch procedure (Christie, 1973) and ash content was measured by ashing at 550°C.
Growth trial
Continuous
Routine resting oxygen consumption measurements and training of female Wistar rats were as previously described (Gazzola, 1993). Briefly, oxygen consumption measurements were taken by removing 5 rats at a time from their cages to individual, flow-through respiration chambers for 30 min. After a settling down period during which the rats were observed to be resting, measurements of air flow, oxygen consumption and air temperature were automatically measured for each of the six chambers in sequence. Measurements were taken over approx. 1 min for each chamber and repeated four times within the 30-min measurement period. Twenty-seven rats, mean liveweight (SEM) of 241(7)g, were randomly allocated to three treatment groups (2, C and T). During this experiment rats were housed in plastic cages with raised wire floors on trays. On the trays were placed tared pieces of cardboard which absorbed the rat’s urine and any spilled water. The cardboard, faeces, spilled food and other debris (hair, skin) were collected, air dried, weighed, pulverized and the total energy content measured by bomb calorimetry.
Female Wistar rats (liveweights 194-263 g) were continuously monitored for their individual oxygen consumption rates during 3-day experimental periods. Indirect calorimetry procedures previously described (Gazzola, 1993) were modified as follows. Large perspex calorimetry chambers (SL) were constructed allowing rats freedom of movement and access to water and food ad libitum. The chambers had wire floors over adsorbent sawdust for animal comfort. They were ventilated at about 1.5 L/min, flow rate being accurately measured as previously described (Gazzola, 1993). The oxygen consumption rate for each chamber was measured at 10.5 min intervals. Experiment 1. Five rats were placed, singly, into metabolism chambers for 3 days. They were given once-daily injections of saline for 3 days while their oxygen consumption rates were continuously monitored. Experiment 2. Five rats were given oncedaily injections of saline (day l), guanfacin (day 2) and saline (day 3). Experiment 3. Four rats were given oncedaily injections of saline (day l), guanfacin (day 2) and guanfacin (day 3).
only consistent observation of liveweight change has been that guanfacin, given orally to mice and rats, even at relatively low doses, always resulted in a lessened liveweight gain (Sillence et al., 1990; Spiers et al., 1990; Gazzola, unpublished). Since large decreases in REE produced by treatment with guanfacin have rarely resulted in enhanced growth, the aim of the present work was to account for all energy fluxes in control and guanfacin-treated animals.
Methods
energy expenditure
experiments
31
The effect of guanfacin on rats
protein, fat and ash of 261(8)g, 86(3)g, 2.O(O.l)MJ, 51(3)g, 18.0(0.4)g and 9.8(0.4)g, respectively. Within. this range of liveweights, the relationship between dry mass (g) and Calcukztions and data analysis liveweight (g) could be described by a straight Results are presented here as the mean line of slope 0.61 and intercept of -65 (p < (standard error of the mean). Metabolizable 0.01). Lipid (g) was related to dry mass (g) by energy (ME) intake was calculated as the en- a linear relationship with a slope of 0.60 and ergy of the food offered minus the energy of intercept of -32 (p < 0.01). Total body enfood left, food spilled, faeces, urine and other ergy (MJ) was related to dry mass (g) by a debris as collected on the cardboard bases. slope of 0.033 and intercept - 0.83 (p < 0.05). No account was taken of any gaseous or vola- Protein and ash were found to be constant tile excretions. Oxygen consumption rates proportions of dry mass (61(4)% and were converted to watts per unit of metabolic 11.5(0.3)%, respectively). With the assumpmass (W/kg0.75)using a value for the energy tion that all three groups (Z, C and T) conequivalence factor (Jequiver and Felber, 1987) sisted of equivalent animals at this point in the of 20.565 kJ/L. This factor was estimated on experiment, these relationships were used to the assumption that animals at maintenance estimate values for individual unslaughtered meet their energy requirements fully from the animals in groups C and T. These estimated diet. Here the diet was composed of 76.5% starting values are reported in Table 1. Values for liveweight, dry mass, total body carbohydrate, 20% protein and 3.5% fat by weight (manufacturer’s analysis; Norco, Lis- energy, protein, fat and ash after treatment are presented in Table 1. No significant differmore, Australia). The body composition of animals in groups ences were found between the control and C and T at the start of treatment was esti- treated groups and no significant gains (final mated from the composition of animals in value minus measured or estimated starting group Z by relating dry mass to liveweight and value) were found for any body component (p total body energy, protein, fat and ash to dry > 0.05, not shown in the table). There were mass. Where a statistically significant linear significant differences in the ME intake (9%, relationship (F-statistic) was found this was p = 0.012) and REE (30%, p < 0.001). Two used, otherwise a constant ratio was used. of the treated animals left a small amount of Sets of data were compared using the two- food and all spilled some food onto the base so that the differences in ME intake were at tailed, unpaired t-test. least partially due to a lower intake of food. Results Continuous energy expenditure
Experiment 4. Four rats were given twicedaily injections of saline (day I), guanfacin (day 2) and guanfacin (day 3).
Growth experiment
Animals in group Z (n = 9), slaughtered at the start of treatment had values for liveweight, dry mass, total body energy content,
The rates of energy expenditure (EE) for five rats injected with saline at 1000 daily for 3 days are shown in Fig. 1A. The pattern was as expected (Mount and Willmott, 1967) with
Table 1. Changes in body composition and energy intake and expenditure in rats on treatment with guanfacin.HCI (0.5 mg/kg twice daily for 6 days)
Starting liveweight (g) Final liveweight (g) Starting dry mass (g)S Final dry mass (g) Starting protein (g)S Final protein (g) Starting fat (g)S Final fat (g) Starting ash (g)$ Final ash (g) Starting body energy (MJ)S Final body energy (MJ) Metabolize energy intake (MJ) Resting energy expenditure (W/kg0.75)
Control*
Treated*
245(7) 249(7) 83(4) 86(3) 51(3) 4g(3) 180) 20(l) 9.6(0.5) 9.5(0.4) 2.qo.l) 1.9(0.1)
245(7) 250(7) 83(4) 85(3) 51(3) 49(3) 18(3) 20(2) 9.6(0.6) 10.9(0.9) 2.0(0.1) 1.9(0.1)
Significancet
NS NS NS NS NS NS NS NS NS NS NS NS 1.13(0.02) 1.03(0.03) p = 0.012 6.1(0.3) 4.2(0.2) p
C. Gazzola
32 1.5
1.0
0.5 1.5
5
1.0
.!! 0 $ 0.5 '; 1.5
E : ‘Z $ 1.0 nZ
0.5 1.5
1.0
0.5
1
I
Day
1
Day
2
1
Day
3
Fig. 1. Continuous rate of energy expenditure over 3 days for rats treated with 0.5 mgikg guanfacin.HCI either once or twice daily. Rates are presented as relative to the mean rate of energy expenditure on day I. Days are the 24 hr starting at 1000. Measurements taken every 10.5 min. Morning injections were given at 0930 and evening injections at 2130. A: Control. Results are the mean relative metabolic rates of 5 rats injected twice daily with saline on each of the 3 days. B: Results are the means for 5 rats injected with saline on the mornings of day 1 and day 3 and guanfacin on the morning of day 2. C: Means for four rats injected with saline on the morning of day 1 and guanfacin on the mornings of days 2 and 3. D: Means for 4 rats injected with saline on the morning and evening of day 1and guanfacin on the mornings and evenings of days 2 and 3.
(TDEE) to REE, TDEE: REE, for the 3 days of this experiment, were 1.20(0.03), l.lg(O.02) and 1.15(0.03), respectively. It can be seen that in spite of a tendency for the EE rates to increase over the 3 days (Fig. I), possibly due to the stress of being in the metabolism chambers, the ratios, and therefore the diurnal patterns of energy expenditure, were not significantly different over the 3 days. When 5 animals were injected with guanfacin on the second day (Experiment 2), the EE during the first 12 hr after injections on day 2 were depressed relative to normal but returned to normal during the subsequent 12 hr period (Fig. 1B). The changed diurnal pattern on day 2 was reflected in the ratio of TDEE : REE (I .34(0.06) which was significantly different (p < 0.01) from the ratio on day 1 (1.09(0.04) of the same experiment. On the third day, following a saline injection, both the diurnal pattern and the ratio of TDEE: REE (1.05(0.03)) returned to normal. In Experiment 3, 4 animals were given guanfacin on days 2 and 3 (Fig. 1C). The diurnal pattern of EE and the ratio for each treated day were similar (1.26(0.03) and (1.33(0.01) but they were different from the ratio on day 1 (1.22(0.03); day l/day 2, NS, day I/day 3, p < 0.01). In Experiment 4, 4 animals which were injected twice-daily (saline day 1, guanfacin days 2 and 3) showed a constant lowering of EE over both days of treatment (Fig. ID). In spite of the lowering of EE, the diurnal pattern of EE remained normal on the treated days so that the ratios of TDEE : REE on the treated days were not different from each other (1.14(0.01) and 1.13(0.03), respectively), nor from the ratio on the untreated day (1.12(0.04). It can be concluded, therefore, that relative changes in REE (measurements of EE taken between 1000 and 1600) accurately reflect relative changes in total daily energy expenditure when animals were treated with guanfacin twice-daily.
Discussion animals resting during the day and being most active during the night. During the resting periods, animals slept, occasionally stirring and rarely eating. During the active periods, the animals were observed eating, grooming and moving about. The measurements of EE taken during resting period corresponded to the routine measurement of REE as used in the growth experiment so that the rate of EE during the period from 1000 to 1600 was taken as the REE for these experiments. The ratios relating total daily energy expenditure
The primary aim of this work was to explain the absence of an increased liveweight gain in guanfacin-treated animals in spite of a lowering in REE. The results from the growth experiment suggest that the lack of growth rate effect (enhanced gain in liveweight or dry mass) was partially due to a decreased intake of metabolizable energy. Some of this decreased ME intake was due to a decreased intake of food. The decrease in ME intake on treatment with guanfacin in this experiment was O.lMJ or about 9% (p = 0.012). Neither
The effect of guanfacin on rats
the control nor the treated rats showed any significant deposition of energy, protein or fat, so the animals were genuinely at maintenance. There was no evidence of repartitioning as for example fat being mobilized for energy while protein was deposited. Therefore the reduced intake of energy should have been evident as a reduction in energy expenditure. However the measured difference in resting energy expenditure on treatment with guanfacin was much larger (30%, p < 0.001) than the difference in intake of metabolizable energy. One must allow the possibility that the REE as measured (Gazzola, 1993) did not accurately reflect the TDEE so that TDEE in the treated animals was in fact normal or even increased. The antihypertensive effects of guanfacin are long-lived (half-life about 21 hr (Schotysik et al., 1980)) but its plasma levels have a half-life of only 4 hr (Kamimura et al., 1980). The results here clearly showed that the effect of a single guanfacin injection on EE was short-lived, lasting at most 12 hr, so that changes in REE did not directly reflect changes in TDEE. A sustained lowering of EE was achieved by repeated, multiple daily injections so that the proportional lowering of TDEE was the same as the proportional lowering of REE. Even though the animals in these experiments were fed ad libitum, as opposed to the restricted diet of the animals in the growth experiment, the proportional lowering of EE was maintained both after the morning injection which was followed by 12 hr during which the rats were resting and eating very little and the evening injection which preceded 12 hr during which the animals ate and were active. It is reasonable to expect then that the differences in REE between the treated animals and the control animals in the growth experiment, where twice-daily injections were used, accurately reflected differences in total energy expenditure between the two groups. For both control animals and twice-daily treated animals, the ratio of TDEE : REE can confidently be placed in the range 1.10 to 1.25. Using the REE values measured in the growth experiment, it can be estimated that the total EE during the 6 days of treatment was 1.2- 1.4 MJ for the control animals and 0.8-I .O MJ for the treated animals. A conservative estimate of the EE difference between the control and treated groups (0.3-0.4MJ) is three times the difference in ME intake and is equivalent to 26 g less food eaten or 7.3 g more fat deposited or 7.4 g more protein deposited (Lindsay et al., 1993). Differences of this magnitude would have been detected in these experiments. However, only a small amount of food
33
was not eaten by the treated animals and no extra deposition of fat or protein occurred. Since the EE of these animals during the morning period (10.00-16.00) is known, only extra EE during the other periods could explain the unaccounted ME intake in the treated animals. One possibility is an increment in the thermic effect of feeding, but this is unlikely since the continuous energy expenditure experiment suggested otherwise. It is possible that the treated animals were more active than the control animals but this was never observed to be the case, although no systematic measurements of activity were taken. It seems unlikely that the rats could have selectively had a higher body temperature during the evening periods, with increased heat output to the environment, since the effect of guanfacin is to lower body temperature under the ambient conditions of these experiments (Ben-Zvi and Leibson, 1980). Although guanfacin lowered resting energy expenditure sufficiently to have produced large increases in energy deposition, no effect on growth was observed. This result was only partially explained by a decreased intake of metabolizable energy. One must conclude, therefore, that treatment with guanfacin resulted in behavioural changes, one of which was decreased food intake. Other, as yet unidentified, behaviours which selectively increased energy expenditure during the nighttime active period must also be invoked. Unless these activities can be identified and eliminated, guanfacin treatment can not result in a net saving of energy which can be used by the animal for increased growth. Acknowledgements-i gratefully acknowledge the financial support of the Meat Research Corporation (Australia). I thank MS V. Grant for her capable technical assistance and diligence.
References Ben-Zvi Z. and Leibson V. (1989) Thermoregulation and clI-adrenergic agonists: effects on environmental temperature and sex. In Thermoregulation: Research and Clinical Applications. 7th International Symp. Pharmacology of Thermoregulation, Odense, Denmark. (Edited by Lomax and Schonbaum), pp. 187-189. Karger, Basel. Christie W. M. (1973) Lipid Analysis, p. 39. Pergamon, Oxford, UK. Gazzola C. (1993) Adrenoceptor-mediated effects on resting energy expenditure. Inr. .I. Obesity 17, 637441. Hunter R. A. (1992) The effect of the (Yeadrenergic agonist, guanfacin, on the energy metabolism of steers fed low-quality roughage diets. Brit. J. Nurr. 67, 337-343. Hunter R. A., Sillence M. N., Gazzola C. and Spiers W. G. (1992) Increasing annual growth rates of cattle by reducing maintenance energy. Aust. J. Ag. Res. 44, 579-595.
C. Gazzola
34 Jequiver E. and Felber J. (1987) Indirect calorimetry. Bailliere’s
Clinical
Endocrinology
In
and Metabolism.
(Edited by Alberti K. G. M. M., Home P. D. and Taylor R., pp. 911-935. Bailliere-Tindall, London. Kamimura S.. Okuda M.. Kitihara N.. Totori T.. Tanaka Y., Kanno S. and Hay&hi R. (1980) Metabolic studies on N-aminidino-2(2,6-dich1orophenyl)acetamide hydrochloride (guanfacine), a new antihypertensive agent (1) absorption, distribution and excretion in rats after single and repeated oral administration. Pharmacometrics zo, 741-744. Lindsay D. B., Hunter R. A., Gazzola C., Spiers W. G. and Sillence M. N. (1993) Enerav and growth. Aust. J. Agricultural
Research
44, 875-k.
-
Mount L. E. and Willmott J. V. (1967) The relation between spontaneous activity, metabolic rate and the 24
hour cycle in mice at different environmental temperatures. J. Physiol. 190, 371-380. Schotysik G., Jirie P. and Picard C. W. (1980) Guanfacine. In Pharmacology of Antihypertensive Drugs. (Edited by Scriabine A.), pp. 79-98. Raven Press, NY. Sillence M. N., Matthews M. L., Spiers W. G. and Lindsay D. B. (1990) Effects of an crz-agonist on growth and metabolic rate in mice. Proc. Nutr. Sot. Australia 15, 170.
Spiers W. G., Sillence M. N. and Lindsay D. B. (1990) cY,-Agonist induced effects on the growth of rats. Proc. Nutr. Sot. Aust. 15, 172. Yen J. T. and Neinaber J. A. (1992) Influence of carbadox on fasting oxygen consumption by portal vein drained organs and by the whole animal in growing pigs. J. Anim. Sci. 70, 478-483.
Pergamon
Copyright 0 1995Elsevier Science Inc. Printed in Great Britain. All rights reserved 0742-8413195 $9.50+ .OO
0742-8413(95)00072-O
The effect of the q-adrenoceptor agonist, guanfacin, on energy expenditure, intake and deposition in rats Carlo Gazzola Queensland Department of Primary Industries, Tropical Beef Centre, PO Box 5545, Rockhampton Mail Centre, Qld 4702, Australia The effect of guanfacin on the energy expenditure, food intake and body composition of female Wistar rats at maintenance was studied. Rats on a restricted food intake (about 75% of ad lihitum), treated for 6 days with twice-daily injections of guanfacin.HCI (0.5 mg/kg) showed large reductions in energy expenditure (30%, p < 0.001) compared to control animals hut no significant differences in liveweight gain, dry mass gain, or gains in total body energy content, protein, fat and ash. The treated group had a lower intake of metabolixable energy (9%, p = 0.01) partially due to a lower intake of food. However, this lower intake of energy did not fully account for the lowered energy expenditure in the treated animals. The unaccounted lowered energy expenditure was not due to changes in the diurnal pattern of energy expenditure since continuous indirect calorimetry with rats fed ad libitum, over 3 days, showed that twice-daily injections of guanfacin (0.5 mglkg per injection) achieved a sustained decrease in energy expenditure. The lack of an increased growth rate of rats treated with guanfacin was attributed to behavioural changes; to a decreased food intake and to other, unknown, factors related to energy expenditure. Key words: Guanfacin;
Energy expenditure;
Energy deposition;
Growth; Rat.
Comp. Biochem. Physiol. 112C, 29-34, 1995.
Introduction The deposition of fat or protein in humans or commercial animals is generally manipulated by changes in dietary energy intake. Where dietary energy intake is fixed, especially at submaintenance levels, deposition can be manipulated by altering thermogenesis or energy expenditure (EE). In humans, exercise is the method of choice for increasing EE, while in commercial animals where the opposite effect is desirable, antithermogenic compounds would be useful in lowering EE. A link between drug-induced lowering of EE and increased liveweight gain has been convincingly demonstrated in pigs (Yen and Neinaber,
1992). In cattle, it can be predicted, on theoretical grounds, that moderate decreases (10%) in the maintenance energy requirements of beef cattle grazing on low-quality native Australian pastures can result in the economically and environmentally desirable outcome of a significant reduction in the time required to achieve a saleable liveweight (Hunter et al., 1992). As a tool for lowering EE in cattle, the cw,-adrenoceptor agonist, guanfacin, may be useful since it decreases resting energy expenditure (REE) by up to 20%, in rats (Gazzola, 1993), in mice (Sillence et al., 1990) and in cattle (Hunter, 1992). Hunter (1992) reported that under-ted cattle infused with guanfacin lost less liveweight than control animals. In many additional studies at our centre with mice and with rats, both under ad libitum and restricted food intakes, guanfacin treatment always lowered metabolic rate, but treated animals rarely showed enhanced liveweight gain over controls. The
Correspondence
10: Carlo Gazzola, Queensland Department of Primary Industries, Troical Beef Centre, P.O. Box 5545, Rockhampton Mail Centre, Qld 4702, Australia. Received 13 November 1994; revised 26 April 1995; accepted 27 April 1995.
29
30
C. Gazzola
All animal experiments were approved by the Animal Experimentation Ethics Committee of the Tropical Cattle Research Centre, Rockhampton, Australia. Female Wistar rats, liveweights noted below, were housed and cared for as previously described (Gazzola, 1993). Guanfacin.HCl was obtained from Sandoz (Base& Switzerland) and administered in isotonic saline, subcutaneously (SC), as a 0.5 mg/mL solution. Injections were of a volume in FL equal to the animal’s liveweight in grams giving a dose rate of 0.5 mg/kg. Control animals received saline injections exactly analogous to the regimen for the treated animals. All injections were given between 0930 and 1000 for once-daily treatments and a second injection was given at 2130 for twice-daily treatments.
Over a period of 6 days, all the rats were gradually restricted from ad libitum intake of food to an intake of 900 kJld/kg0~7S(about 75% of ad libitum). Food was given at 1600 daily and for the remainder of the experiment the rats were offered this same amount of food. For the next 6 days, pretreatment period, all the rats were injected twice-daily with saline. At the end of this 6-day period the rats in group Z were slaughtered by carbon dioxide anaesthesia and cervical dislocation, and frozen whole. The other two groups were treated during the next 6 days, treatment period, twice-daily, with saline (group C, control group) or guanfacin (group T, treated group). Resting oxygen consumption was measured on the third day of the period. After 6 days of treatment these rats were also slaughtered and frozen. All the rats’ whole bodies were autoclaved, homogenized and lyophilized to a constant dry mass. The energy content of the dried bodies, food samples and cardboard bases were determined using an adiabatic bomb calorimeter (Parr Instrument Co., U.S.A.). Protein was determined using Kjeldahl digestion and auto analyser methods (Industrial method 218-72A, Technicon, Tarrytown, NY, U.S.A.) with a nitrogen to protein conversion factor of 6.25. Fat was determined by the Folch procedure (Christie, 1973) and ash content was measured by ashing at 550°C.
Growth trial
Continuous
Routine resting oxygen consumption measurements and training of female Wistar rats were as previously described (Gazzola, 1993). Briefly, oxygen consumption measurements were taken by removing 5 rats at a time from their cages to individual, flow-through respiration chambers for 30 min. After a settling down period during which the rats were observed to be resting, measurements of air flow, oxygen consumption and air temperature were automatically measured for each of the six chambers in sequence. Measurements were taken over approx. 1 min for each chamber and repeated four times within the 30-min measurement period. Twenty-seven rats, mean liveweight (SEM) of 241(7)g, were randomly allocated to three treatment groups (2, C and T). During this experiment rats were housed in plastic cages with raised wire floors on trays. On the trays were placed tared pieces of cardboard which absorbed the rat’s urine and any spilled water. The cardboard, faeces, spilled food and other debris (hair, skin) were collected, air dried, weighed, pulverized and the total energy content measured by bomb calorimetry.
Female Wistar rats (liveweights 194-263 g) were continuously monitored for their individual oxygen consumption rates during 3-day experimental periods. Indirect calorimetry procedures previously described (Gazzola, 1993) were modified as follows. Large perspex calorimetry chambers (SL) were constructed allowing rats freedom of movement and access to water and food ad libitum. The chambers had wire floors over adsorbent sawdust for animal comfort. They were ventilated at about 1.5 L/min, flow rate being accurately measured as previously described (Gazzola, 1993). The oxygen consumption rate for each chamber was measured at 10.5 min intervals. Experiment 1. Five rats were placed, singly, into metabolism chambers for 3 days. They were given once-daily injections of saline for 3 days while their oxygen consumption rates were continuously monitored. Experiment 2. Five rats were given oncedaily injections of saline (day l), guanfacin (day 2) and saline (day 3). Experiment 3. Four rats were given oncedaily injections of saline (day l), guanfacin (day 2) and guanfacin (day 3).
only consistent observation of liveweight change has been that guanfacin, given orally to mice and rats, even at relatively low doses, always resulted in a lessened liveweight gain (Sillence et al., 1990; Spiers et al., 1990; Gazzola, unpublished). Since large decreases in REE produced by treatment with guanfacin have rarely resulted in enhanced growth, the aim of the present work was to account for all energy fluxes in control and guanfacin-treated animals.
Methods
energy expenditure
experiments
31
The effect of guanfacin on rats
protein, fat and ash of 261(8)g, 86(3)g, 2.O(O.l)MJ, 51(3)g, 18.0(0.4)g and 9.8(0.4)g, respectively. Within. this range of liveweights, the relationship between dry mass (g) and Calcukztions and data analysis liveweight (g) could be described by a straight Results are presented here as the mean line of slope 0.61 and intercept of -65 (p < (standard error of the mean). Metabolizable 0.01). Lipid (g) was related to dry mass (g) by energy (ME) intake was calculated as the en- a linear relationship with a slope of 0.60 and ergy of the food offered minus the energy of intercept of -32 (p < 0.01). Total body enfood left, food spilled, faeces, urine and other ergy (MJ) was related to dry mass (g) by a debris as collected on the cardboard bases. slope of 0.033 and intercept - 0.83 (p < 0.05). No account was taken of any gaseous or vola- Protein and ash were found to be constant tile excretions. Oxygen consumption rates proportions of dry mass (61(4)% and were converted to watts per unit of metabolic 11.5(0.3)%, respectively). With the assumpmass (W/kg0.75)using a value for the energy tion that all three groups (Z, C and T) conequivalence factor (Jequiver and Felber, 1987) sisted of equivalent animals at this point in the of 20.565 kJ/L. This factor was estimated on experiment, these relationships were used to the assumption that animals at maintenance estimate values for individual unslaughtered meet their energy requirements fully from the animals in groups C and T. These estimated diet. Here the diet was composed of 76.5% starting values are reported in Table 1. Values for liveweight, dry mass, total body carbohydrate, 20% protein and 3.5% fat by weight (manufacturer’s analysis; Norco, Lis- energy, protein, fat and ash after treatment are presented in Table 1. No significant differmore, Australia). The body composition of animals in groups ences were found between the control and C and T at the start of treatment was esti- treated groups and no significant gains (final mated from the composition of animals in value minus measured or estimated starting group Z by relating dry mass to liveweight and value) were found for any body component (p total body energy, protein, fat and ash to dry > 0.05, not shown in the table). There were mass. Where a statistically significant linear significant differences in the ME intake (9%, relationship (F-statistic) was found this was p = 0.012) and REE (30%, p < 0.001). Two used, otherwise a constant ratio was used. of the treated animals left a small amount of Sets of data were compared using the two- food and all spilled some food onto the base so that the differences in ME intake were at tailed, unpaired t-test. least partially due to a lower intake of food. Results Continuous energy expenditure
Experiment 4. Four rats were given twicedaily injections of saline (day I), guanfacin (day 2) and guanfacin (day 3).
Growth experiment
Animals in group Z (n = 9), slaughtered at the start of treatment had values for liveweight, dry mass, total body energy content,
The rates of energy expenditure (EE) for five rats injected with saline at 1000 daily for 3 days are shown in Fig. 1A. The pattern was as expected (Mount and Willmott, 1967) with
Table 1. Changes in body composition and energy intake and expenditure in rats on treatment with guanfacin.HCI (0.5 mg/kg twice daily for 6 days)
Starting liveweight (g) Final liveweight (g) Starting dry mass (g)S Final dry mass (g) Starting protein (g)S Final protein (g) Starting fat (g)S Final fat (g) Starting ash (g)$ Final ash (g) Starting body energy (MJ)S Final body energy (MJ) Metabolize energy intake (MJ) Resting energy expenditure (W/kg0.75)
Control*
Treated*
245(7) 249(7) 83(4) 86(3) 51(3) 4g(3) 180) 20(l) 9.6(0.5) 9.5(0.4) 2.qo.l) 1.9(0.1)
245(7) 250(7) 83(4) 85(3) 51(3) 49(3) 18(3) 20(2) 9.6(0.6) 10.9(0.9) 2.0(0.1) 1.9(0.1)
Significancet
NS NS NS NS NS NS NS NS NS NS NS NS 1.13(0.02) 1.03(0.03) p = 0.012 6.1(0.3) 4.2(0.2) p
C. Gazzola
32 1.5
1.0
0.5 1.5
5
1.0
.!! 0 $ 0.5 '; 1.5
E : ‘Z $ 1.0 nZ
0.5 1.5
1.0
0.5
1
I
Day
1
Day
2
1
Day
3
Fig. 1. Continuous rate of energy expenditure over 3 days for rats treated with 0.5 mgikg guanfacin.HCI either once or twice daily. Rates are presented as relative to the mean rate of energy expenditure on day I. Days are the 24 hr starting at 1000. Measurements taken every 10.5 min. Morning injections were given at 0930 and evening injections at 2130. A: Control. Results are the mean relative metabolic rates of 5 rats injected twice daily with saline on each of the 3 days. B: Results are the means for 5 rats injected with saline on the mornings of day 1 and day 3 and guanfacin on the morning of day 2. C: Means for four rats injected with saline on the morning of day 1 and guanfacin on the mornings of days 2 and 3. D: Means for 4 rats injected with saline on the morning and evening of day 1and guanfacin on the mornings and evenings of days 2 and 3.
(TDEE) to REE, TDEE: REE, for the 3 days of this experiment, were 1.20(0.03), l.lg(O.02) and 1.15(0.03), respectively. It can be seen that in spite of a tendency for the EE rates to increase over the 3 days (Fig. I), possibly due to the stress of being in the metabolism chambers, the ratios, and therefore the diurnal patterns of energy expenditure, were not significantly different over the 3 days. When 5 animals were injected with guanfacin on the second day (Experiment 2), the EE during the first 12 hr after injections on day 2 were depressed relative to normal but returned to normal during the subsequent 12 hr period (Fig. 1B). The changed diurnal pattern on day 2 was reflected in the ratio of TDEE : REE (I .34(0.06) which was significantly different (p < 0.01) from the ratio on day 1 (1.09(0.04) of the same experiment. On the third day, following a saline injection, both the diurnal pattern and the ratio of TDEE: REE (1.05(0.03)) returned to normal. In Experiment 3, 4 animals were given guanfacin on days 2 and 3 (Fig. 1C). The diurnal pattern of EE and the ratio for each treated day were similar (1.26(0.03) and (1.33(0.01) but they were different from the ratio on day 1 (1.22(0.03); day l/day 2, NS, day I/day 3, p < 0.01). In Experiment 4, 4 animals which were injected twice-daily (saline day 1, guanfacin days 2 and 3) showed a constant lowering of EE over both days of treatment (Fig. ID). In spite of the lowering of EE, the diurnal pattern of EE remained normal on the treated days so that the ratios of TDEE : REE on the treated days were not different from each other (1.14(0.01) and 1.13(0.03), respectively), nor from the ratio on the untreated day (1.12(0.04). It can be concluded, therefore, that relative changes in REE (measurements of EE taken between 1000 and 1600) accurately reflect relative changes in total daily energy expenditure when animals were treated with guanfacin twice-daily.
Discussion animals resting during the day and being most active during the night. During the resting periods, animals slept, occasionally stirring and rarely eating. During the active periods, the animals were observed eating, grooming and moving about. The measurements of EE taken during resting period corresponded to the routine measurement of REE as used in the growth experiment so that the rate of EE during the period from 1000 to 1600 was taken as the REE for these experiments. The ratios relating total daily energy expenditure
The primary aim of this work was to explain the absence of an increased liveweight gain in guanfacin-treated animals in spite of a lowering in REE. The results from the growth experiment suggest that the lack of growth rate effect (enhanced gain in liveweight or dry mass) was partially due to a decreased intake of metabolizable energy. Some of this decreased ME intake was due to a decreased intake of food. The decrease in ME intake on treatment with guanfacin in this experiment was O.lMJ or about 9% (p = 0.012). Neither
The effect of guanfacin on rats
the control nor the treated rats showed any significant deposition of energy, protein or fat, so the animals were genuinely at maintenance. There was no evidence of repartitioning as for example fat being mobilized for energy while protein was deposited. Therefore the reduced intake of energy should have been evident as a reduction in energy expenditure. However the measured difference in resting energy expenditure on treatment with guanfacin was much larger (30%, p < 0.001) than the difference in intake of metabolizable energy. One must allow the possibility that the REE as measured (Gazzola, 1993) did not accurately reflect the TDEE so that TDEE in the treated animals was in fact normal or even increased. The antihypertensive effects of guanfacin are long-lived (half-life about 21 hr (Schotysik et al., 1980)) but its plasma levels have a half-life of only 4 hr (Kamimura et al., 1980). The results here clearly showed that the effect of a single guanfacin injection on EE was short-lived, lasting at most 12 hr, so that changes in REE did not directly reflect changes in TDEE. A sustained lowering of EE was achieved by repeated, multiple daily injections so that the proportional lowering of TDEE was the same as the proportional lowering of REE. Even though the animals in these experiments were fed ad libitum, as opposed to the restricted diet of the animals in the growth experiment, the proportional lowering of EE was maintained both after the morning injection which was followed by 12 hr during which the rats were resting and eating very little and the evening injection which preceded 12 hr during which the animals ate and were active. It is reasonable to expect then that the differences in REE between the treated animals and the control animals in the growth experiment, where twice-daily injections were used, accurately reflected differences in total energy expenditure between the two groups. For both control animals and twice-daily treated animals, the ratio of TDEE : REE can confidently be placed in the range 1.10 to 1.25. Using the REE values measured in the growth experiment, it can be estimated that the total EE during the 6 days of treatment was 1.2- 1.4 MJ for the control animals and 0.8-I .O MJ for the treated animals. A conservative estimate of the EE difference between the control and treated groups (0.3-0.4MJ) is three times the difference in ME intake and is equivalent to 26 g less food eaten or 7.3 g more fat deposited or 7.4 g more protein deposited (Lindsay et al., 1993). Differences of this magnitude would have been detected in these experiments. However, only a small amount of food
33
was not eaten by the treated animals and no extra deposition of fat or protein occurred. Since the EE of these animals during the morning period (10.00-16.00) is known, only extra EE during the other periods could explain the unaccounted ME intake in the treated animals. One possibility is an increment in the thermic effect of feeding, but this is unlikely since the continuous energy expenditure experiment suggested otherwise. It is possible that the treated animals were more active than the control animals but this was never observed to be the case, although no systematic measurements of activity were taken. It seems unlikely that the rats could have selectively had a higher body temperature during the evening periods, with increased heat output to the environment, since the effect of guanfacin is to lower body temperature under the ambient conditions of these experiments (Ben-Zvi and Leibson, 1980). Although guanfacin lowered resting energy expenditure sufficiently to have produced large increases in energy deposition, no effect on growth was observed. This result was only partially explained by a decreased intake of metabolizable energy. One must conclude, therefore, that treatment with guanfacin resulted in behavioural changes, one of which was decreased food intake. Other, as yet unidentified, behaviours which selectively increased energy expenditure during the nighttime active period must also be invoked. Unless these activities can be identified and eliminated, guanfacin treatment can not result in a net saving of energy which can be used by the animal for increased growth. Acknowledgements-i gratefully acknowledge the financial support of the Meat Research Corporation (Australia). I thank MS V. Grant for her capable technical assistance and diligence.
References Ben-Zvi Z. and Leibson V. (1989) Thermoregulation and clI-adrenergic agonists: effects on environmental temperature and sex. In Thermoregulation: Research and Clinical Applications. 7th International Symp. Pharmacology of Thermoregulation, Odense, Denmark. (Edited by Lomax and Schonbaum), pp. 187-189. Karger, Basel. Christie W. M. (1973) Lipid Analysis, p. 39. Pergamon, Oxford, UK. Gazzola C. (1993) Adrenoceptor-mediated effects on resting energy expenditure. Inr. .I. Obesity 17, 637441. Hunter R. A. (1992) The effect of the (Yeadrenergic agonist, guanfacin, on the energy metabolism of steers fed low-quality roughage diets. Brit. J. Nurr. 67, 337-343. Hunter R. A., Sillence M. N., Gazzola C. and Spiers W. G. (1992) Increasing annual growth rates of cattle by reducing maintenance energy. Aust. J. Ag. Res. 44, 579-595.
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34 Jequiver E. and Felber J. (1987) Indirect calorimetry. Bailliere’s
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Mount L. E. and Willmott J. V. (1967) The relation between spontaneous activity, metabolic rate and the 24
hour cycle in mice at different environmental temperatures. J. Physiol. 190, 371-380. Schotysik G., Jirie P. and Picard C. W. (1980) Guanfacine. In Pharmacology of Antihypertensive Drugs. (Edited by Scriabine A.), pp. 79-98. Raven Press, NY. Sillence M. N., Matthews M. L., Spiers W. G. and Lindsay D. B. (1990) Effects of an crz-agonist on growth and metabolic rate in mice. Proc. Nutr. Sot. Australia 15, 170.
Spiers W. G., Sillence M. N. and Lindsay D. B. (1990) cY,-Agonist induced effects on the growth of rats. Proc. Nutr. Sot. Aust. 15, 172. Yen J. T. and Neinaber J. A. (1992) Influence of carbadox on fasting oxygen consumption by portal vein drained organs and by the whole animal in growing pigs. J. Anim. Sci. 70, 478-483.