BASAL METABOLIC RATE AND LIPID AND LIVER GLYCOGEN IN MICE INFECTED BY THE NEMATODE ~E~~T~~P~~~~~E~ ~~~r~~~ L. E. A. SYMONS and
Division of Animal Health, C.S.I.R.O., Private
McMaster N.S.W., 2037,
1, P.O. Glebe,
Abstract---SYMoNs L. E. A. and JONESW. 0. 1974. Basal metabolic rate and lipid and liver glycogen in mice infected by the nematode Nematospiroides duhius. Internntional JoutwalfLw Purasitofogy 4: 3Ol305. The basal metabolic rate (BMR) of mice losing weight about two weeks after infection by Nematospiroides dubiru was lower than that of uninfected mice gaining weight when fed ari libitum or losing weight on quantjtatively reduced rations. There was no difference in BMR between the latter two groups. Following the injection of ‘%Z-glucose, the high specific activity of expired CO, from infected and reduced ration mice was considered to be due to the utilization of energy reserves. The levels of lipid and liver glycogen were low in these two groups of mice and their specific activities, particularly in the severely affected animals. were high. It was concluded that the depressed BMR of infected mice is unrelated to anorexia, which did, however, explain the low levels of lipid and liver glycogen. INDEX KEY WORDS: NematrtspiroicJes rJm%rtsI carbon dioxide; lipid: anorexia; “C-glucose.
with intestinal nematode infections had shown that skeletal muscle protein synthesis was depressed, while liver protein synthesis was elevated in mice and guinea pigs infected with Nrmatospivoi&.s citrbilr.~ and T&/tostro~l~y~~is ~~~~t~b~J~o~/~~~,~, respectively. The rate of whole-body protein catabolism was increased. It was concluded that anorexia was an important cause of these changes of protein metabolism. The effect on other aspects of metabolism of these infections had not been investigated. It was not known whether the basal metabolic rate (BMR) was changed and it was expected from post-mortem exalninat~on and as a normal response to anorexia that energy reserves such as glycogen and lipid would be depleted. The possible relationship between energy reserves and changes in BMR was the
also unknown. For these reasons
rate; liver: gtycogen:
quantitatively reduced rations so that they lost weight at rates comparable with infected animals.
the expiration of CO, as indicative of BMR, and liver glycogen and carcase lipid were measured in separate experiments in mice infected with N. rhbilts. In both experiments CO,?, glycogen and lipid were labelled by prior injections of “C-glucose so that their specific radioactivities could be measured. The importance of anorexia was assessed in each instance in uninfected mice fed
Growing male white mice were used throughout. In all experiments uninfected mice that were gaining weight (controls) were compared with mice that were losing weight after about two weeks infection by stomach tube with 300 to 350 larvae of N. drrbios. Infected mice selected for measurement of expired CO, had lost weight for at least 24 h before the injection of “C-glucose and continued to do so over the next 24 h. On occasions described below, groups of uninfected mice were fed quantitatively reduced rations so that they lost weight at about the same rate as did infected mice. Apart from these, all mice were fed stock laboratory pellets and water UC/fib. RMR The BMR was determined by a measurement of expired CO,. Twenty-four hours before collection of CO,, the animals were given an intraperitoneal injection of 0.5 vCij20 g of body wt of liniformly labelled “C-glucose after which they were fasted overnight for ahout I? h: hut given water ad lib. Two pairs of mice (one infected or one reduced ration mouse and one control per pair) were placed in the apparatus illustrated in Fig. I. The CO, was collected for 6 h after a IO-15 min equilibration period, during which the mice settled down. At the end
L. E. A. SYMONS
and W. 0. JONES
One pair of Perspex
25cm Expired CO, trap
1. Diagramatic illustration of the experimental apparatus for collection of Ethanolamjne-n~ethyl cellosolve according !o Jeffay & Alvarez (1961).
of this period the volume of the ethanolamine-methyl cellosolve (ethylene gtycol monomethyl ether) mixture was re-made to 15 ml and 1 ml duplicate samples taken for radioactive counting. Ten ml of the mixture was back titrated to the initial pH with I.94 N NaOH with an automatic titrator (Radiometer Type TTTIC). The samples for counting were added to I5 ml of the scintillant of Jeffay & Alvarez (1961 f. The total pmoles CO, expired in 6 h was calculated from the volume of NaOH used in the back titration. The specific activity of the CO, was calculated from the total CO, radioactivity expired. The BMR was expressed as pmoles CO, expired/hr/g body wt. The results were statistically analyzed by paired “t” tests as the mice were used in pairs as described.
Mice were given an intrapcritoneal injection of gici uniformly lahelled 11C-glucose/21g body wtiday for three days at about 9 a.m. On the morning of the fourth day they were killed with a blow on the head, the liver removed rapidly and weighed for glycogen analysis. The skin, head, lower limbs, tail and all viscera except the kidneys were also removed. The remainder, consisting of skeletal muscle, skeleton and kidneys and henceforth referred to as the carcase, was weighed and the lipid extracted. Liver glycogen was estimated by the method of Carroll rl al. (1956). The specific activity was determined on the trichloracetic acid extract. to IO ml of which was added 50 ml ethanol. After standing overnight. the precipitate was centrifuged and the tube inverted for IO min. The glycogen was dissolved in NCS solublizer fAmersham/
and IO ml of scinGllant (Symons &
Jones, 1971) was added. The carcase lipid was Folch or al. (1957)and the weighing the dried extract after adding the scintillant. The uniformly labelled was purchased from the
estimated by the method of specific activity determined by and counting the radioactivity llC-glucose (335 mCi/mmole) Radiochemical Centre. Amer-
sham, England. Radioactive counting was carried out in a Packard (Model 3375) scintillation spectrometer with external standards to correct for quenching. RESULTS
The severity of infection was assessed subjectively from the clinical appearance of the animal, and objectively by the amount of weight lost. Severely affected animals losing weight were obviously depressed and tended to remain stationary unless disturbed. When deliberately disturbed, they quickly reverted to their original attitude. In contrast, uninfected mice losing weight when fed a reduced ration rapidly became hyperactive when disturbed, and continued in this state for some time afterwards. They were often seen to move about the cage as if searching for food. The mean loss of weight of infected and reduced ration mice in the experiments in which liver glycogen and carcase lipid as well as BMR were measured are shown in Table I. In the former experiment, the severely affected lost twice as much weight as did the moderately affected animals, whereas the mean weight lost by those on reduced rations was intermediate between those two groups. In the BMR experiment the weight.; lost by the infected and reduced ration mice were equivalent to the ranges of both the moderately and severely affected animals in the giycogen and lipid experiment. Although the reduced ration mice were not pair-fed with infected animals the comparable weight losses indicated a comparable effect. BMR In spite of the differences in appearances and behaviour between the infected and the uninfected
LOSSES OF MICE INFECTED WITH
of mice infected
with N. dubi~s
BMR (pmoles Controls (gaining
QUANTITATIVELY OF CONTROLS
Specific activity (dis/min per mmoles CO*)
I, p <.-o.o::99\
Infected Moderately affected (5) Severely affected (7) Reduced ration (4)
GlycoEen and lipid
3.3 6.6 4-5
3.0-3.5 5.5-9.5 4.0-5-O
Mean Maximim Weight of all mice ~~ 25 g; range 19.5-33 g. Numbers in parenthesis ~~ number of mice.
! 7.4 7
Ins. Reduced ration (losing weight)
mice that have been described above, no observable differences occurred in their behaviour in the apparatus during the 6 h collecting period. Apart from occasional short bursts of activity, they settled down quickly into one position after a short exploratory phase during the equilibration period. The experimental results are set out in Table 2. The mean BMR expressed as pmoles COY expired/h/g body wt of 12 infected mice was lower than that of their uninfected controls gaining weight when fed ad lib. This difference was statistically highly significant. On the other hand there was no significant difference between the BMR of six reduced ration mice losing weight and their controls. The specific activites of the CO, expired by the infected mice and of the reduced ration mice were significantly higher than that of their respective controls.
* Standard deviation. Statistical analysis: n.s. ~ Statistically Liver glycogen
I 2938 J
Paired “t” test (see text). not significantly different.
and cat-case lipid
From Table 3 it is clear that the severe infections drastically reduced the levels of liver glycogen, but that moderate infections had little effect upon it. Quantitatively reducing the ration of uninfected animals also appreciably reduced the glycogen levels. Infection, whether severe or moderate, reduced the lipid content of the carcase. The mean lipid level of the severely affected animals was lower than that of the latter group, but the difference was not statistically significant (0.05
Glycogen, 2. The specific activities of liver glycogen affected A severely affected, A, uninfected,
p <- 0.01
B.M.R. Infected (12) Reduced ration (6)
‘: 0 -
in mice after the injection fed ad lib. H. uninfected,
of ‘V-glucose. Moderately fed reduced rations 0.
L. E. A. SYMONS and W. 0. JONES
TABLE ~-LIVER INFECTED
FED A QUANTITATIVELY
* Standard Numbers
Glycogen (mg/g fresh liver) Controls (12) Infected Moderately affected (5) Severely affected (7) Reduced ration (4)
dubius OR LOSING WEIGHT
9.2 4~ 2.8
22.1 -t 7.2 0.93 $ 0.69 2.5 + 3.3
deviation. in parenthesis
4.8 + 2.4 2.7 I I.6 2.6 I I.6
The specific activities of liver glycogen in the three groups of mice are illustrated in Fig. 2. They rose steeply at levels of glycogen lower than about 2 mg/g fresh liver. At glycogen levels higher than 10 mg/g the specific activities were approximately equal. Although there were only four mice that lost weight on a reduced ration and hence had low levels of liver glycogen, they too had higher specific activities than the control mice with greater amounts of glycogen. The specific activities of carcase lipid in these groups of mice are shown in Fig. 3. In both the infected and uninfected control mice there was a marked inverse relationship between specific activity
Lipid, n-q x10+ FIG. 3. The specific activities of carcase lipid in mice after the injection of ‘T-glucose. Moderately affected A, severely affected A, uninfected fed ad lib. n , uninfected, fed reduced rations r-1.
and mg of lipid, but it was also clear that the two populations differed from one another in that the specific activity/mg of lipid was appreciably higher in the latter group. It is not possible to make a precise statement about the uninfected reduced ration group, except that they too illustrated the inverse relationship between specific activity and weight of lipid. Because no constant relationship could be detected between body weight loss as a measure of the severity of the infection and the specific activity of expired CO, in the BMR experiment, this is not illustrated in this report. DISCUSSION BMR
The differences of the rate of expiration of CO, between infected mice and their controls could not be explained by differences in physical activity during the collection period of 6 h. This length of time was chosen to allow for any short periods of higher respiratory activity due to any intermittent spells of exploratory movement, which occurred most commonly and were completed during the equilibration period before the collection of CO, began. The differences between the magnitudes of the parameters measured for the uninfected mice gaining weight in the two parts of the experiment cannot be readily explained. The experimental conditions of the two parts were identical, except that there may have been unrecorded differences in room temperatures as they were carried out some months apart. This variation between the halves of the experiment eliminated any possibility of a meaningful comparison of BMR between the infected and reduced ration mice, but did not invalidate the comparison between uninfected mice gaining weight when fed ad lib. or losing weight on a reduced ration. The lower BMR of infected mice losing weight is consistent with their depression and relative inactivity. This contrasted with that of the reduced ration mice whose BMR did not differ significantly from their uninfected controls despite their apparent hyperexcitability and lower food consumption. Steel (1972), found that the irreversible loss of CO, from the blood of sheep infected with T. colubr(/hwis and from uninfected sheep pair-fed with them was clearly lower than that from uninfected sheep fed ad lib. If the irreversible loss of CO, can be taken as an index of energy expenditure (Corbett et al.. 1971) then, in common with the mice, the energy expenditure of infected sheep was lower than adequately fed uninfected sheep. Unlike the sheep, there was no difference between reduced ration mice and their uninfected control group fed ad lib. This difference between the two experiments does not necessarily imply a difference between the responses of uninfected sheep and mice to reduced rations, as the mice unlike the sheep. were fasted for 17 h in addi-
I.J.P. VOL. 4. 1974
tion to their reduced collections were made. Liver
of mice infected with N. rluhius
Because of the anorexia that had already been reported for infected mice (Symons & Jones, 1971) it was not unexpected that the liver glycogen and carcase lipid levels were reduced, although glycogen fell below control levels only in severely affected animals. Nor was it unexpected that there were falls comparable to the severely affected mice in the reduced ration group. The high specific activities of glycogen and lipid in severely affected mice do not necessarily indicate faster rates of synthesis. The probable explanation of the high specific activities is the incorporation of 14C-glucose into the low energy reserves of these infected animals. If the specific activity of glycogen of the moderately affected animals is indicative of synthesis then there appeared to be no change in the infected animals as these did not differ from their uninfected controls (Fig. 2). The apparent difference between the populations of the infected and uninfected mice illustrated in Fig. 3, in which the latter group is to the right, suggested a lower rate of incorporation of ‘YY into lipid in the infected group. At comparable levels of carcase lipid the specific activity would be higher in the uninfected animals. Without knowledge of the specific activities of the labelled pools it is impossible to determine the relative rates of synthesis of these substances in infected and uninfected animals. Because of these uncertainties, it cannot be stated whether infection has a similar effect upon glycogen and lipid synthesis as it does upon skeletal muscle protein synthesis in infected mice and guinea pigs. The effect of the reduced ration of uninfected mice upon the specific activities of glycogen and lipid was similar to that of infection. It is necessary to consider the interrelationships between the results of the two parts of the work described in this paper. The high specific activities of the CO1, expired by the infected and reduced ration mice, can be explained readily as the utilization of their energy reserves of glycogen and lipid which have been labelled with “C. This is supported by the low levels of these substances in these animals, and their high specific activities, particularly in the severely affected animals. Symons & Jones (I971 and 1972) showed that the changes in muscle protein synthesis and in wholebody protein catabolism in infected mice and guinea pigs were largely, if not solely related to anorexia. The two parts of this work present further evidence on this question. The low levels and higher specific activities of liver glycogen and carcase lipid in both
reduced ration and infected mice, particularly the more severely affected animals, support the conclusion that anorexia is important in the pathology of the disease. On the other hand, it was clear that the anorexia per se was not responsible for the lower BMR in infected mice as reduced food consumption in uninfected mice did not affect the BMR. From this and the earlier work referred to, it is possible to construct a description of the response to intestinal nematode infection. The utilization of liver glycogen, carcase lipid and whole-body protein, particularly skeletal muscle protein, is a predictable response to anorexia. The relationship between this utilization of energy reserves and depression of muscle protein synthesis remains uncertain although some aspects of the mechanism of the latter have been revealed (Symons & Jones, I97 I ). An explanation of anorexia and the causal relationships between it, general depression and a lower BMR in mammalian nematode infections, are questions that remain to be answered. Acknowledgements-We lsberg for her valuable
wish to thank technical assistance.
REFERENCES CARROLL N. V., LONGLEY R. W. & ROE J. H. 1956. The determination of glycogen in liver and muscle by use of anthrone reagent. Journal of Biological Chemistry 220: 583-593. CORBET~ J. L., FARRELL D. J., LENG R. A., MCCLYMON~ G. L. & YOUNG B. A. 1971. Determination of the energy expenditure of penned and grazing sheep from estimates of carbon dioxide entry rate. Brifish Journal of Nutrition 26: 277-291. FOLCH J., LEES M. & STANLEY G. H. S. 1957. A simple method for the isolation and purification of total lipid from animal tissues. Journal of Bic~lo~~ical Chemistry 226: 497-509. JEFFAY H. & ALVAREZ J. 1961. Liquid scintillation counting of carbon-14. Use of ethanolamine-ethylene glycol monomethyl-ether-toluene. Analytical Chemistry 33: 612-615. STEEL J. W. 1972. Effects of the intestinal nematode Trichostrongylus colubr(formis on ruminal acetate metabolism in young sheep. Proceedings Australian Society fbr Animal Production 9: 402407. SYMONS L. E. A.&JONES W. 0. 1971. Protein metabolism -1. Incorporation of *lC-L-leucine into skeletal muscle and liver protein of mice and guinea pigs infected with Nematospiroides dubius and Trichostrongylus colubriformis. E.uperimental Parasitology 29: 230-241. SYMONS L. E. A. &JONES W. 0. 1972. Protein metabolism -11. Protein turnover, synthesis and muscle growth in suckling, young and adult mammals infected with Nematospiroides dubius or Trichostrongylus colubrifbrmis. Experimental Parasitology 32: 335-342.