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Evolution of glucose induced hyperglycemia during the annual cycle of a sahelian lizard (Varanus exanthematicus)
EVOLUTION OF GLUCOSE INDUCED HYPERGLYCEMIA DURING THE ANNUAL CYCLE OF A SAHELIAN LIZARD (VARANUS EXANTHEMATICUS)” Laboratoire de Neurophysiologie,
YVETTEADIOVI FacultC des Sciences et Techniques, Universitt de Nice, 06100 France (Received 13 October 1980)
Abstract--During the ecophysiological cycle of Varanus exanthematicus: 1. Normoglycemia can be defined only by referring to each period of the ecophysiological cycle. 2. Intravenous injection of glucose (0.6 g/kg body weight) induces an immediate and durable hyperglycemia. (a) V. exanthematicus shows a glucose tolerance which can be compared to that reported in other lizards (for a duration of 48 hr). (b) Between the two characteristic periods of the ecophysiological cycle, significant differences have been found at the beginning of the response to induced hyperglycemia: the lizards captured in October (nourishment period) present a glucose cellular assimilation slower and less efficient than that of animals captured in December (beginning of the starvation period) or February (middle period of starvation). (c) The lizards captured in June (transition between starvation and nourishment period) show variables responses. 3. Our results concernine the elvcemic reeulation in V. exanthemaricus are compared to those reported by different authors in othir Ligaids.
INTRODUCTION Numerous studies concerning the blood glucose levels in reptiles exhibited important variations according to different species. The minimal values were observed in turtles, crocodiles and snakes (Dessauer, 1970; Skoczylas & Sidorkiewicz, 1974); the maximal ones were found in lizards (see Dessauer, 1970, and data in Discussion, Table 4). These variations of blood glycemic levels characterize the reptilian species from mammals where glycemia is rather stable from one species to another. In addition, seasonal’ variations of climatic types such as: temperature, sunlight and hygrometry, determine modifications in the biological activity of many reptiles. The biorhythm rest/fasting induces changes in blood chemical constituents and specially variations in blood glucose level. This was observed in alligators (Coulson et al., 1950; Hernandez & Coulson, 1952) turtles: Pseudemys scripta elegans (Hutton, 1960) Emys orbicularis (Vladescu, 1964) and less frequently in snakes: Vipera aspis (Agid et al., 1961), Natrix natris (Skoczylas & Sidorkiewicz, 1974). Among lizards these variations were particularly pointed out in Anolis carolinensis (Dessauer, 1952, 1953) vat-anus greseus (Haggag et al., 1966), Egernia cunninghami (Moore, 1967) and more precisely in Vromastix aegyptia (Khalil & Yanni, 1959) Vromastix hardwicki (Jahangeer et al., 1973) and V. exanthematicus (Cisse & Demaille, 1975) (see data in Discussion Table 4). In all the reptiles, the response to hyperglycemia induced by oral or intraperitoneal administration of glucose indicates a glucose tolerance really lower than that of mammals. This was pointed out in snakes: Bothrops jararaca (Prado, 1946). Xenodon merremii *Supported by a grant research from D.G.R.S.T. of Senegal (No. 2.806 art. 4031.1). 529
(Houssay & Penhos, 1960), turtles: Pseudemys sorbignyi (Lopes et al., 1954), alligators (Coulson & Hernandez, 1953; Penhos er al., 1967) and lizards: Eumeces sp (Miller and Wurmster, 1958) Tupinambis sp (Penhos et al., 1965) V. hardwicki (Kumar & Khanna, 1975). After we have verified that V. exanthematicus exhibits the same characteristics, our study was limited to the short-time response to hyperglycemia in relation to the biorhythm. MATERIALS AND METHODS Lizards were captured in the Sahelian area of Senegal, 8 to 10 days prior to study. During their captivity in the laboratory, they were held under similar hygrothermic conditions to those of their biotope (Cisse, 1980), but they were fasting. The animals were studied at each characteristic period of their ecophysiological cycle: -in October. nourishment period and sexual post-activity -in December, beginning of starvation -in February. period of starvation and rest tdiapause) -in June. end of starvation. Each group was made of 4 to 5 animals of both sexes, weighing from 750 to 1500 g (usually, females were inferior to 1OOOgin weight). The experiments were performed at laboratory temperature, between 25 and 28°C. An influence of anaesthetics on plasmatic glucose level (approximate increase 27%) was noticed. Consequently, the hyperglycemia test was performed on awake animals after a local anaesthesia of abdominal teguments. Blood takings and glucose administration were made in the abdominal part of the posterior vena cava. The experiment took place as follows: (1) Taking of reference blood (2) Fast injection of a hypertonic solution of glucose _ (Aguettant Laboratory): 0.6 gikg body weight. (3) Samolinas of blood, at regular intervals: 10. 20. 30. 50, 70, 90 and-180 min after glucose administration (to). The blood samples were collected in sodium fluoride tubes and centrifuged at 5000 rev/min for 5 min.
530
YVETTE ADJW
Glucose was estimated by glucose oxydase and peroxydase method (kit Sigma) with a net-spectrometer “Labospat” (Jobin & Yvon).
The mean values are given f standard errors and i confidence limits for (N - 1) degrees of freedom. The level of significance was set at 0.05. Statistical comparisons were made using Student’s r-test.
RESULTS
Establishment animuls
of normoyl~cemiu
stcrndrrrd of cuptire
An induced hyperglycemia study cannot be signihcant without relation to the standard glycemia level for a considered population and its seasonal variations. We have called this parameter standard of normoglycemic evolution. The glycemic analysis of a population of lizards similar to ours was carried out by Cisse et d. (1975) immediately after capture. In our study. glycemia was estimated just before experiments (Table I). The standard curve obtained with captive animals is by all account inferior to that of animals in their own environment. This difference is particularly marked for the animals captured during nourishment period (September and October) and at the end of the starvation period (June). The mean values of normoglycemia estimated during October are statistically different from those of December and February (P = 0.05). Analysis
of induced h_vperylycemia
The analysis of response-curves to hyperglycemia was carried out in relation to three parameters. According to Conard (1955) “the most probable law which expresses the speed of disappearance of injected glucose is an exponential curve corresponding to the formula: C = A e- k’, in which C is the glucidic concentration at time t. e the basis of neperian logarithms, A and K, constants”. When sugar concentrations are plotted on a logarithmic ordinate. a straight line of regression is obtained which represents the temporal evolution of the hyperglycemia (Fig. I). In addition to the response-curves obtained, significant physiological values are given: (a) The constant K. angular coefficient of the straight line. which expresses the speed of decrease of the induced hyperglycemia following cellular assimilation of glucose; (b) The yield UhO. expresses the yield of the chemical reaction. basis of the assimilation process during the first hour of the experiment; (c) The residue of hyperglycemia. R. is determined for the time f = 180 min expressed in percentage of the glycemia for I = 20min. It gives information about glucose tolerance of k’. eurrrltltemlrtic,u.s. in our experimental conditions. Comparison of response-curves fdr the unimrds tested in October, December and Fehruur),. The results of
hyperglycemia tests reported in Table 2 and Fig. I. permit us to notice the following. During the first 20 min after the glucose administration, the plasmatic glucose level decreases more or less rapidly. This corresponds to glucose diffusion into extracellular space. We observe that. whatever the normoglycemia of the
531
Evolution of glucose induced hy~rgIy~emia
_
400-
r
z
?!
z‘:
tooA
--
100’
0
iz
n cl:
.R
I
5
Ix
_-___---I
1
10
20
t
30
SO
f
III
__-I-----
___----
I
i
-27
t 90
70
tlmrs
t mn.b
Fig. 3. Theoretical curve of induced hyperglycemia. A = Theoretical concentration, I : period of extracellular
K =
repartition;
1ogA - 1ogc t
,
U6p = t - emksa5 or
II: beginning of glucose assimilation;
A-C Us0 = -. A
III: period of glucose
tolerance.
the blood glucose levels are quite similar at I = 20 min from one group to another (no significant differences). Between t = 20 min and t = 90 min, the speeds of cellular assimilation of’ glucose, K, and Ihe yields, UeO, present significant differences (P = 0.05) between the mean values of October compared to the mean values of December, the mean values
animals,
of October present significant differences when compared to the mean values of February (UbO in February is twice as high as in October), but no significant differences are observed between December and February. If we relate these results to the ecophysio~ogi~al cycle of the animals, we can observe that all the ani-
Fig. 2. Hyperglycemic curves related to ecophysiological cycle of V. exanthernuticus. A-Blood glucose Ievels in normal ordinate. B-Blood glucose levefs in logarithmic ordinate. Mean values F standard errors.
* Standard
errors.
r=180min R (“, Hyperglycemia
GIucose assimilation K cl hO
t contidence
f = 20 min)
t & + + + + +
67.05 (t 106.69) 17.23(&27.41) 23.45 ( i: 37.31) 20.68(&32.91) 14.10( k22.44) 19.61 (k31.20) 58.91 (k93.73)
during
11.13.59)
limits of the mean values for (N) determinations
80.67 f 8.54
0.27 lo-’ i_ 0.02 lo-’ (kO.03 0.152 k 0.011 (+0.018)
398.12 311.50 299.25 287.12 271.50 256.00 239.62
Evolution of induced hyperglycemia in mg/lOO ml t= 10mm 20 30 50 70 90 180
12.1(*)(+7.3)(t)
(5)t
estimated
OCTOBER
hyperglycemia
83.8 f
of induced
r = 0 Normogly~mia in mg/lOO ml
Table 2. Evolution
and P: 0.05.
IO- “)
nourishment (5)
and starvation
66.15 7t 8.59(+13.66)
0.43 lo-> & 0,10~0-~(~0.14 0.228 i: 0.047 ( i: 0.075)
f 13.40(+21.32) f 43.37( &69.00) rf: 40.40 ( k64.27) I: 41.56 (f66.13) + 36.07 ( k 57.40) It 35.05 (k55.77) rf: 39.74 (k63.23)
57.7 c?: 10.7 (,8.2)
DECEMBER
(October)
448.25 317.37 296.87 274.37 255.25 234.25 209.87
period
10 2)
period
k & & + It + t:
71.47(+113.71) 48.39 ( If:77.00) 40.81(*64.94) 27.44 (k43.65) 30.27(*48.17) 30.22 (k48.08) 26.28 (k41.82)
12.9)
(5)
64.93 + 5.11
I k8.14)
0.49 IO- ’ f 0.07~~0.12) 0.320 F 0.042 f kO.067)
338.00 281 SO 253.00 216.25 204.00 198.25 195.75
44.8 f 10.4(*
FEBRUARY
(December-February)
533
Evolution of glucose induced hyperglycemia
mals in starvation exhibit the same reaction to hyperglycemia (K and Uho present no significant differences) while those captured during the nourishment period (October) indicate a lower cellular assimilation of glucose (K and UhO significantly inferior to the values of December and February) [Fig. 21. For t = 180min. R is higher in October than in December or February (P = 0.05). The results for the 3 hr following the administration of glucose show that the disappearance of blood glucose is slower during the activity and nourishment period than during starvation. Response-curre.s of the unimtrls in June. During June. period of transition between starvation and the food-intake period. the variability of the responses do not permit us a statistical treatment of the data. At the beginning of the experiment (t = 20min), the rapid drop in glucose level induces values of glycemia similar to those reported for the animals of December and February. Yet, this glycemia is high and significantly different from the glycemia of the animals of October (P = 0.05). Between t = 20 min and t = 90 min. the individual differences are markedly important. specially for K and Lie,, (see Table 3). For t = I80 min. R is not very different from one animal to another. The variability of these responses is probably due to modifications in climatic conditions of the biotope which determine physiological modifications in the lizards and prepare them to enter into the nourishment period (CissC, 1980). DISCUSSION
Tables 4 and 5 show values of normoglycemia expressed in mg/lOOml in different species of lizards. Within these results a great disparity can be noticed which may come from different origins such as: methods of blood sampling or analysis, use or nonuse of anaesthesia, sex, ecophysiological period of the animal when captured or tested.
When ecophysiological period has been mentioned the highest values concern the period of nourishment and metabolic activity; the lowest values are in relation to the period without food-intake (fasting and starvation). This can also be found in V. exanthematicus, even in captivity (see Table 1). When normoglycemia has been estimated all over the year (Table 5) we can notice again that, firstly, some lizards keep all over the year a low normoglycemia compared to the others, and in this case, this could be in relation to the place of blood-sampling (dorsal aorta for Uromastix aegyptia, inferior vena cava for U. hardwicki) or to the assay methods (Gawadi method or ortho-toluidine photometric method). Secondly, the values of glycemia are markedly different between January-February-March and JulyAugust, or, according to the biorhythms, between rest period and activity period. For the three cases reported in this table the increase of normoglycemia during March-June could be partly due to the elevation of hygrometric and thermal conditions of the biotope (Moore, 1967; Cisse, 1980). In the experiments of induced-hyperglycemia which were carried out in various species of lizards (Di Maggio & Dessauer, 1963; Vladescu, 1965; Kumar & Khanna, 1975), the glucose administration was done by oral or intraperitoneal route. In our study, oral administration of glucose provides a great diversity in the hyperglycemia test results. This could come from individual differences in intestinal absorption. The amounts of glucose administrated in the different experiments reported herewith vary between 1 and 5 g/kg body weight. The dose we used (0.6 g/kg) allowed us to go beyond the maximal normoglycemia and to remain close to physiological limits. From the point of view of glucose tolerance in lizards, in A. carolinensis the glucose level stays high during less than 36 hr in a period lasting from March to July; on the other hand, during Autumn and Winter, this level remains for more than 48 hr higher
Table 3. Induced hyperglycemia estimated in June, phase of transition between starvation and nourishment period I
2
3
92.5
89.5
84.0
Evolution of induced hyperglycemia (mg/tOO ml) f = 10min 20 30 50 70 90 180
369.0 352.5 365.0 357.0 343.5 362.0 274.5
387.0 353.5 319.0 295.0 282.0 284.0 261.0
427.0 359.0 346.5 336.5 328.5 305.0 258.0
Glucose assimilation K UhO
0.053 lo-’ 0.020
0.312 lo-’ 0.248
0.177 lo-’ 0.107
77.87
73.83
71.86
r = 0 normoglycemia in mg/lOO ml
r=180min R (“d Hyperglycemia t = 20)
11 30 7 14.46
265 f 236 & 11O_t 136.5 $:
Vuranus monitor
A-Metabolic activity and nourishment period. B---Fasting state. C-Starvation period. *-Mean values + standard errors,
c
A B
35.88 I_ 2.34
:
%
C
2
95.29 1 4.63 106.5 (97- 120)
195.0 * 21.9
83.0 f 4.3
LO.8
120.3 f
82.1
41.7
A
Vurunus greseus Dand.
Vuranus exlrnthematicus
Uromustix hardwicki
teguixin ilromostix uegyptia
Tupinambis
P~r~no~~ cortiurum Scebporus occidentalis
155 (132-195) 122 f 3.@ 131 142.5 104
54.25 (44-66)
109
Hemiductylus brooki Iguanu iguana Lacerto agilis chersonensis
%
Heloderwru suspecturn
A
91.4-113.9 93
;
% A
MO.5
74 181.6-212.8 173 93 94 192 (151-250) 600 150
45
--.
Glucase mg/lOO ml
Eumeces fascia&s Eumeces obsoletus Gekko gecko Heioderma horridum
Ctenosuura ucunthuru Eyerniu Cunninghami (Gray)
C~~u~l~~ Chumaefeo Cnemidophorus Sexlineutus Coleonyx variegutus
Anolis crrroktcnsis
Species
May September January August November
February July December September April January
November August-September
August-September March-April August
Periods of ecophysiological cycle
Zain-UL-Abedin & Qazi (1965) Jahangeer et uf. (1973) Jahangeer VI al., 1973 Jahangeer et al., 1973 Kumar & Kbanna (1975) Cisse & Demaille (1975) Cisse & Demaille (1975) Haggag et ul., (1966) Haggag et al., (1966) Zain-ULhAbedin & Qazi (1965)
Dessauer ( 1952) Milker & Wurster (t956, 1958) Dawson (1960) Dessauer (1952) Zarafonetis % Kalas (1960) review in Dessauer, Edwards & Dil (1935); Dessauer (1952) Rapoport & Guest (1941) review in Dessauer (1970) Zain-UL-Abedin & Qazi (1965) Hernandez & Co&on (1951) Vfadescu (1965) WoLfe (1939) Dawson & Foulson (I962) Miller & Wurster (1956) Penhos et al. (1965) Khalil & Yanni (1959)
Miller & Wurster (1956) Milter & Wurster (1956) Dessauer ( 1952) Dessauer (1952) Goin & J&son (1965); review in Dessauer 1970 Hernandez & Caulson (1951) Moore (1967)
References
Table 4. Data reported by different authors concerning blood glucose levels in various species of hzards
535
Evolution of glucose induced hyperglycemia
than the glucose level recorded in the fasting period (Di Maggio & Dessauer, 1963). In these experiments the injected dose of glucose is the same (4 mg in 0.4 ml of water) whatever the lizards’ weight (3.5 to 7 g), that means 0.57 to 1.14 gjkg body weight. In Lacertu ugilis chersonensis the normal glucose level is reached 48 hr after glucose administration (1 g/kg body weight) (Vladescu, 1965). In U. hardwicki, induced hyperglycemia also lasts 48 hr before disappearing (Kumar & Khanna, 1975). We were able to notice that, in I/. rxanthematicus, the blood glucose level again turns close to normoglycemia about 48 hr after the intravenous injection of glucose (0.6 g/kg body weight). Unlike the mentioned authors we mainly concentrated our study on the immediate evolution of the induced hyperglycemia by referring to animals captured at various periods of their ecophysiological cycle. In C: exanrhematicus, glycemic regulation is low in nourishment period (October) while the insulin content of pancreas is high (Godet & Adjovi, 1980). This may seem abnormal when referring to mammalian physiology, but these results were previously observed in A. carolinensis, on perfused pancreatic islets (Rhoten, 1974). During starvation period (February), the glycemic regulation is better though the insulin content of pancreas is low. No study is known concerning animals in such a severe starvation state, thus we miss any comparison element. In other respects it apparently disagrees with the results obtained by Miller & Wurster (1958), Sabnis & Rangnekar (1968) and Kumar & Khanna (1975), who observe B-cells degranulation in pancreatic islets after glucose administration. More particularly in U. hardwicki, an important glucose loading (2 to 5 g/kg body weight) induces B-cells degranulation in 1 to 2 hr and an atrophy of these cells in 72 hr (Kumar & Khanna, 1975). Therefore
liberation of pancreatic insulin in lizards could be only stimulated by an important glucose overloading and a dose of 0.6/kg b.wt is not sufficient to determine an insulinic regulation during the nourishment period. In order to complete our study in V. exanthematicus. it seems necessary to study the relations between different doses of glucose overloading and the insulin output during the life cycle of the animal.
REFERENCES ACID
R.. DUGUYR. & SAINT&IRONSH. (1961) Variations de la glydmie, du glycogkne hkpatique et de I’aspect histologique du pancrtas. chez Vipera aspis, au tours du cycle annuel. J. Physiol., Land. 53, 807-824. CISSEM. (1980) Ecophysiologie cornparke de deux Varans en milieu sahtlien (S&gal). Thtse doctorat d’Etat. Universitk de Nice. CISSE M. and DEMAILLE(1975) Le cycle annuel glucidolipidique chez un Varan du Stntgal. C.r. SPanc. Sot. Biol. 169, 1084-1089. CONARDV. (1955) Mesure de I’assimilation du glucose. Acru mrd. /w/y.. Br~rurlks. 46-66. DAW~CINW. R. (1960) Review in Dessauer. Biology o( rhr Rrpfilitr (1970). DESSAUERH. C. (1953) Hibernation of the lizard, Anolis curolinmsis. Proc. Sm. cup. Biol. Med. 82, 351-353.
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DESSAUER H. C. (1970) Blood chemistry of reptiles: physiological and evolutionary aspects. In Biology of the Reptilia. Vol. 3. pp. 1-72. DESSAUERH. C. & COUL~~N R. A. (1952) Biochemical studies on the lizard An&s carolinensis. Proc. SOC. exp. Biol. Med.
80, 742-744.
DI MAGGIO A. III & DESSAUERH. C. (1963) Seasonal
changes in glucose tolerance and glycogen disposition in a lizard. Am. J. Physiol. 204, 677-680. DUP~GODET M. & ADJOVIY. (1980) Seasonal variations of immunoreactive insulin contents in pancreatic extracts of a sahelian lizard (Varanus exanthematicus). Camp. Biothem. Physiol. 69A, in press. EDWARDS& DIL (1935) Review in Dessauer. Biology of the Reptilia
(1970).
GOIN & JAKSON(1965) Review in Dessauer, Reptilia
Biology
of the
of rhe Reptilia
(1970).
HUTTONK. E. (1960) Seasonal physiological changes in the red-eared
turtle.
Pseudemys
scripta
elegans.
Copeia
4,
JAHANGEER S. K., LONE P. & ASLANM. (1973) Studies on plasma glucose levels of the spinay tailed lizard, Uromasfix hardwicki (Gray). Biologica 19, 85. KHALILF. & YANNIM. (1959a) Studies on carbohydrates in reptiles--I. Glucose in body fluids of Uromastix aegyptia. Z. oeryl. Physiol. 42, 192-198. KHANNAS. S. & KUMAR S. (1975) Blood glucose in the indian
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lizard.
Ur0masti.u
hardnicki.
U.S.A., 767-771. KUMARS. & KHANNAS. S. (1979) Inducement of hyperglycemia by glucose loading and its effects on the pancreatic islets in the lizard. Uromasrix hardwkki. Zoo/. Beitr. Deu. 24, 405415. Copeia
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MOBERLYW. R. (1968) Hibernation in the desert iguana, Dipsosaurus dorsalis. Physiol. Zool. 36, 152-160. MOOREG. P. (1967) Seasonal variations in blood glucose lizard, Egernia
cunninghami
(Gray) 1832. Physiol. Zoo1 40, 261-272. PENHOSJ. C.. HOUSSAYB. A. & LUJAN M. A. (1965) Total pancreatectomy in lizards. Effects of several hormones. Endocrinology
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RHOTENW. B. (1974) Sensitivity of Saurian pancreatic islets to glucose. Am. J. Physiol. 227, 993-997. SABNISP. B. & RANGNEKAR P. V. (1968) Effects of administration of hyperglycemic substances and tolbutamide on the morphology of the pancreatic islet cells and glycemic level in the lizard. Varanus monitor. J. Unit. Bombay 37, 51-55.
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bignyi.
MALAIS~EW. J., MALAI~~E- LAGAE F. & WRIGHT P. H. (1967) Effect of fasting upon insulin secretion in the rat.
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HAGGAGG., RAHEEMK. & KHALIL F. (1966) Hibernation in Reptiles-II. Changes in blood glucose, hemoglobin. red blood cell count, protein and non protein nitrogen. Comn. Biochem. Phvsiol 17. 335-339. HERNANDEZT. & COUL& R. A. (1951) Biochemical studies on the iguana (black iguana-Ctenosaura acantura-and common iguana-Iguana iguana rhinolopha) Proc. Sot. exp. Biol. Med. 76, 175-177. HOU~~AYB. A. & PENHOSJ. C. (1960) review in Dessauer. Biology
LOPESN., WAGNERE., BARROSM. & MARQUESM. (1954) Glucose. insulin and epinephrin tolerance tests in the normal and hypophysectomised turtle, Pseudemys d’or-
SKOCZYLASR. & SIDORKIEWICZ E. (1964) Studies on the blood sugar level in the grass snake (Nafrix nutris L.) Camp. Eiochem.
Phvsiol.
48. 439-456.
VL.ADE& C. (1965) kesearches induced hyperglycemia in Rep. Roumaine
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YVETTEADIOVI FacultC des Sciences et Techniques, Universitt de Nice, 06100 France (Received 13 October 1980)
Abstract--During the ecophysiological cycle of Varanus exanthematicus: 1. Normoglycemia can be defined only by referring to each period of the ecophysiological cycle. 2. Intravenous injection of glucose (0.6 g/kg body weight) induces an immediate and durable hyperglycemia. (a) V. exanthematicus shows a glucose tolerance which can be compared to that reported in other lizards (for a duration of 48 hr). (b) Between the two characteristic periods of the ecophysiological cycle, significant differences have been found at the beginning of the response to induced hyperglycemia: the lizards captured in October (nourishment period) present a glucose cellular assimilation slower and less efficient than that of animals captured in December (beginning of the starvation period) or February (middle period of starvation). (c) The lizards captured in June (transition between starvation and nourishment period) show variables responses. 3. Our results concernine the elvcemic reeulation in V. exanthemaricus are compared to those reported by different authors in othir Ligaids.
INTRODUCTION Numerous studies concerning the blood glucose levels in reptiles exhibited important variations according to different species. The minimal values were observed in turtles, crocodiles and snakes (Dessauer, 1970; Skoczylas & Sidorkiewicz, 1974); the maximal ones were found in lizards (see Dessauer, 1970, and data in Discussion, Table 4). These variations of blood glycemic levels characterize the reptilian species from mammals where glycemia is rather stable from one species to another. In addition, seasonal’ variations of climatic types such as: temperature, sunlight and hygrometry, determine modifications in the biological activity of many reptiles. The biorhythm rest/fasting induces changes in blood chemical constituents and specially variations in blood glucose level. This was observed in alligators (Coulson et al., 1950; Hernandez & Coulson, 1952) turtles: Pseudemys scripta elegans (Hutton, 1960) Emys orbicularis (Vladescu, 1964) and less frequently in snakes: Vipera aspis (Agid et al., 1961), Natrix natris (Skoczylas & Sidorkiewicz, 1974). Among lizards these variations were particularly pointed out in Anolis carolinensis (Dessauer, 1952, 1953) vat-anus greseus (Haggag et al., 1966), Egernia cunninghami (Moore, 1967) and more precisely in Vromastix aegyptia (Khalil & Yanni, 1959) Vromastix hardwicki (Jahangeer et al., 1973) and V. exanthematicus (Cisse & Demaille, 1975) (see data in Discussion Table 4). In all the reptiles, the response to hyperglycemia induced by oral or intraperitoneal administration of glucose indicates a glucose tolerance really lower than that of mammals. This was pointed out in snakes: Bothrops jararaca (Prado, 1946). Xenodon merremii *Supported by a grant research from D.G.R.S.T. of Senegal (No. 2.806 art. 4031.1). 529
(Houssay & Penhos, 1960), turtles: Pseudemys sorbignyi (Lopes et al., 1954), alligators (Coulson & Hernandez, 1953; Penhos er al., 1967) and lizards: Eumeces sp (Miller and Wurmster, 1958) Tupinambis sp (Penhos et al., 1965) V. hardwicki (Kumar & Khanna, 1975). After we have verified that V. exanthematicus exhibits the same characteristics, our study was limited to the short-time response to hyperglycemia in relation to the biorhythm. MATERIALS AND METHODS Lizards were captured in the Sahelian area of Senegal, 8 to 10 days prior to study. During their captivity in the laboratory, they were held under similar hygrothermic conditions to those of their biotope (Cisse, 1980), but they were fasting. The animals were studied at each characteristic period of their ecophysiological cycle: -in October. nourishment period and sexual post-activity -in December, beginning of starvation -in February. period of starvation and rest tdiapause) -in June. end of starvation. Each group was made of 4 to 5 animals of both sexes, weighing from 750 to 1500 g (usually, females were inferior to 1OOOgin weight). The experiments were performed at laboratory temperature, between 25 and 28°C. An influence of anaesthetics on plasmatic glucose level (approximate increase 27%) was noticed. Consequently, the hyperglycemia test was performed on awake animals after a local anaesthesia of abdominal teguments. Blood takings and glucose administration were made in the abdominal part of the posterior vena cava. The experiment took place as follows: (1) Taking of reference blood (2) Fast injection of a hypertonic solution of glucose _ (Aguettant Laboratory): 0.6 gikg body weight. (3) Samolinas of blood, at regular intervals: 10. 20. 30. 50, 70, 90 and-180 min after glucose administration (to). The blood samples were collected in sodium fluoride tubes and centrifuged at 5000 rev/min for 5 min.
530
YVETTE ADJW
Glucose was estimated by glucose oxydase and peroxydase method (kit Sigma) with a net-spectrometer “Labospat” (Jobin & Yvon).
The mean values are given f standard errors and i confidence limits for (N - 1) degrees of freedom. The level of significance was set at 0.05. Statistical comparisons were made using Student’s r-test.
RESULTS
Establishment animuls
of normoyl~cemiu
stcrndrrrd of cuptire
An induced hyperglycemia study cannot be signihcant without relation to the standard glycemia level for a considered population and its seasonal variations. We have called this parameter standard of normoglycemic evolution. The glycemic analysis of a population of lizards similar to ours was carried out by Cisse et d. (1975) immediately after capture. In our study. glycemia was estimated just before experiments (Table I). The standard curve obtained with captive animals is by all account inferior to that of animals in their own environment. This difference is particularly marked for the animals captured during nourishment period (September and October) and at the end of the starvation period (June). The mean values of normoglycemia estimated during October are statistically different from those of December and February (P = 0.05). Analysis
of induced h_vperylycemia
The analysis of response-curves to hyperglycemia was carried out in relation to three parameters. According to Conard (1955) “the most probable law which expresses the speed of disappearance of injected glucose is an exponential curve corresponding to the formula: C = A e- k’, in which C is the glucidic concentration at time t. e the basis of neperian logarithms, A and K, constants”. When sugar concentrations are plotted on a logarithmic ordinate. a straight line of regression is obtained which represents the temporal evolution of the hyperglycemia (Fig. I). In addition to the response-curves obtained, significant physiological values are given: (a) The constant K. angular coefficient of the straight line. which expresses the speed of decrease of the induced hyperglycemia following cellular assimilation of glucose; (b) The yield UhO. expresses the yield of the chemical reaction. basis of the assimilation process during the first hour of the experiment; (c) The residue of hyperglycemia. R. is determined for the time f = 180 min expressed in percentage of the glycemia for I = 20min. It gives information about glucose tolerance of k’. eurrrltltemlrtic,u.s. in our experimental conditions. Comparison of response-curves fdr the unimrds tested in October, December and Fehruur),. The results of
hyperglycemia tests reported in Table 2 and Fig. I. permit us to notice the following. During the first 20 min after the glucose administration, the plasmatic glucose level decreases more or less rapidly. This corresponds to glucose diffusion into extracellular space. We observe that. whatever the normoglycemia of the
531
Evolution of glucose induced hy~rgIy~emia
_
400-
r
z
?!
z‘:
tooA
--
100’
0
iz
n cl:
.R
I
5
Ix
_-___---I
1
10
20
t
30
SO
f
III
__-I-----
___----
I
i
-27
t 90
70
tlmrs
t mn.b
Fig. 3. Theoretical curve of induced hyperglycemia. A = Theoretical concentration, I : period of extracellular
K =
repartition;
1ogA - 1ogc t
,
U6p = t - emksa5 or
II: beginning of glucose assimilation;
A-C Us0 = -. A
III: period of glucose
tolerance.
the blood glucose levels are quite similar at I = 20 min from one group to another (no significant differences). Between t = 20 min and t = 90 min, the speeds of cellular assimilation of’ glucose, K, and Ihe yields, UeO, present significant differences (P = 0.05) between the mean values of October compared to the mean values of December, the mean values
animals,
of October present significant differences when compared to the mean values of February (UbO in February is twice as high as in October), but no significant differences are observed between December and February. If we relate these results to the ecophysio~ogi~al cycle of the animals, we can observe that all the ani-
Fig. 2. Hyperglycemic curves related to ecophysiological cycle of V. exanthernuticus. A-Blood glucose Ievels in normal ordinate. B-Blood glucose levefs in logarithmic ordinate. Mean values F standard errors.
* Standard
errors.
r=180min R (“, Hyperglycemia
GIucose assimilation K cl hO
t contidence
f = 20 min)
t & + + + + +
67.05 (t 106.69) 17.23(&27.41) 23.45 ( i: 37.31) 20.68(&32.91) 14.10( k22.44) 19.61 (k31.20) 58.91 (k93.73)
during
11.13.59)
limits of the mean values for (N) determinations
80.67 f 8.54
0.27 lo-’ i_ 0.02 lo-’ (kO.03 0.152 k 0.011 (+0.018)
398.12 311.50 299.25 287.12 271.50 256.00 239.62
Evolution of induced hyperglycemia in mg/lOO ml t= 10mm 20 30 50 70 90 180
12.1(*)(+7.3)(t)
(5)t
estimated
OCTOBER
hyperglycemia
83.8 f
of induced
r = 0 Normogly~mia in mg/lOO ml
Table 2. Evolution
and P: 0.05.
IO- “)
nourishment (5)
and starvation
66.15 7t 8.59(+13.66)
0.43 lo-> & 0,10~0-~(~0.14 0.228 i: 0.047 ( i: 0.075)
f 13.40(+21.32) f 43.37( &69.00) rf: 40.40 ( k64.27) I: 41.56 (f66.13) + 36.07 ( k 57.40) It 35.05 (k55.77) rf: 39.74 (k63.23)
57.7 c?: 10.7 (,8.2)
DECEMBER
(October)
448.25 317.37 296.87 274.37 255.25 234.25 209.87
period
10 2)
period
k & & + It + t:
71.47(+113.71) 48.39 ( If:77.00) 40.81(*64.94) 27.44 (k43.65) 30.27(*48.17) 30.22 (k48.08) 26.28 (k41.82)
12.9)
(5)
64.93 + 5.11
I k8.14)
0.49 IO- ’ f 0.07~~0.12) 0.320 F 0.042 f kO.067)
338.00 281 SO 253.00 216.25 204.00 198.25 195.75
44.8 f 10.4(*
FEBRUARY
(December-February)
533
Evolution of glucose induced hyperglycemia
mals in starvation exhibit the same reaction to hyperglycemia (K and Uho present no significant differences) while those captured during the nourishment period (October) indicate a lower cellular assimilation of glucose (K and UhO significantly inferior to the values of December and February) [Fig. 21. For t = 180min. R is higher in October than in December or February (P = 0.05). The results for the 3 hr following the administration of glucose show that the disappearance of blood glucose is slower during the activity and nourishment period than during starvation. Response-curre.s of the unimtrls in June. During June. period of transition between starvation and the food-intake period. the variability of the responses do not permit us a statistical treatment of the data. At the beginning of the experiment (t = 20min), the rapid drop in glucose level induces values of glycemia similar to those reported for the animals of December and February. Yet, this glycemia is high and significantly different from the glycemia of the animals of October (P = 0.05). Between t = 20 min and t = 90 min. the individual differences are markedly important. specially for K and Lie,, (see Table 3). For t = I80 min. R is not very different from one animal to another. The variability of these responses is probably due to modifications in climatic conditions of the biotope which determine physiological modifications in the lizards and prepare them to enter into the nourishment period (CissC, 1980). DISCUSSION
Tables 4 and 5 show values of normoglycemia expressed in mg/lOOml in different species of lizards. Within these results a great disparity can be noticed which may come from different origins such as: methods of blood sampling or analysis, use or nonuse of anaesthesia, sex, ecophysiological period of the animal when captured or tested.
When ecophysiological period has been mentioned the highest values concern the period of nourishment and metabolic activity; the lowest values are in relation to the period without food-intake (fasting and starvation). This can also be found in V. exanthematicus, even in captivity (see Table 1). When normoglycemia has been estimated all over the year (Table 5) we can notice again that, firstly, some lizards keep all over the year a low normoglycemia compared to the others, and in this case, this could be in relation to the place of blood-sampling (dorsal aorta for Uromastix aegyptia, inferior vena cava for U. hardwicki) or to the assay methods (Gawadi method or ortho-toluidine photometric method). Secondly, the values of glycemia are markedly different between January-February-March and JulyAugust, or, according to the biorhythms, between rest period and activity period. For the three cases reported in this table the increase of normoglycemia during March-June could be partly due to the elevation of hygrometric and thermal conditions of the biotope (Moore, 1967; Cisse, 1980). In the experiments of induced-hyperglycemia which were carried out in various species of lizards (Di Maggio & Dessauer, 1963; Vladescu, 1965; Kumar & Khanna, 1975), the glucose administration was done by oral or intraperitoneal route. In our study, oral administration of glucose provides a great diversity in the hyperglycemia test results. This could come from individual differences in intestinal absorption. The amounts of glucose administrated in the different experiments reported herewith vary between 1 and 5 g/kg body weight. The dose we used (0.6 g/kg) allowed us to go beyond the maximal normoglycemia and to remain close to physiological limits. From the point of view of glucose tolerance in lizards, in A. carolinensis the glucose level stays high during less than 36 hr in a period lasting from March to July; on the other hand, during Autumn and Winter, this level remains for more than 48 hr higher
Table 3. Induced hyperglycemia estimated in June, phase of transition between starvation and nourishment period I
2
3
92.5
89.5
84.0
Evolution of induced hyperglycemia (mg/tOO ml) f = 10min 20 30 50 70 90 180
369.0 352.5 365.0 357.0 343.5 362.0 274.5
387.0 353.5 319.0 295.0 282.0 284.0 261.0
427.0 359.0 346.5 336.5 328.5 305.0 258.0
Glucose assimilation K UhO
0.053 lo-’ 0.020
0.312 lo-’ 0.248
0.177 lo-’ 0.107
77.87
73.83
71.86
r = 0 normoglycemia in mg/lOO ml
r=180min R (“d Hyperglycemia t = 20)
11 30 7 14.46
265 f 236 & 11O_t 136.5 $:
Vuranus monitor
A-Metabolic activity and nourishment period. B---Fasting state. C-Starvation period. *-Mean values + standard errors,
c
A B
35.88 I_ 2.34
:
%
C
2
95.29 1 4.63 106.5 (97- 120)
195.0 * 21.9
83.0 f 4.3
LO.8
120.3 f
82.1
41.7
A
Vurunus greseus Dand.
Vuranus exlrnthematicus
Uromustix hardwicki
teguixin ilromostix uegyptia
Tupinambis
P~r~no~~ cortiurum Scebporus occidentalis
155 (132-195) 122 f 3.@ 131 142.5 104
54.25 (44-66)
109
Hemiductylus brooki Iguanu iguana Lacerto agilis chersonensis
%
Heloderwru suspecturn
A
91.4-113.9 93
;
% A
MO.5
74 181.6-212.8 173 93 94 192 (151-250) 600 150
45
--.
Glucase mg/lOO ml
Eumeces fascia&s Eumeces obsoletus Gekko gecko Heioderma horridum
Ctenosuura ucunthuru Eyerniu Cunninghami (Gray)
C~~u~l~~ Chumaefeo Cnemidophorus Sexlineutus Coleonyx variegutus
Anolis crrroktcnsis
Species
May September January August November
February July December September April January
November August-September
August-September March-April August
Periods of ecophysiological cycle
Zain-UL-Abedin & Qazi (1965) Jahangeer et uf. (1973) Jahangeer VI al., 1973 Jahangeer et al., 1973 Kumar & Kbanna (1975) Cisse & Demaille (1975) Cisse & Demaille (1975) Haggag et ul., (1966) Haggag et al., (1966) Zain-ULhAbedin & Qazi (1965)
Dessauer ( 1952) Milker & Wurster (t956, 1958) Dawson (1960) Dessauer (1952) Zarafonetis % Kalas (1960) review in Dessauer, Edwards & Dil (1935); Dessauer (1952) Rapoport & Guest (1941) review in Dessauer (1970) Zain-UL-Abedin & Qazi (1965) Hernandez & Co&on (1951) Vfadescu (1965) WoLfe (1939) Dawson & Foulson (I962) Miller & Wurster (1956) Penhos et al. (1965) Khalil & Yanni (1959)
Miller & Wurster (1956) Milter & Wurster (1956) Dessauer ( 1952) Dessauer (1952) Goin & J&son (1965); review in Dessauer 1970 Hernandez & Caulson (1951) Moore (1967)
References
Table 4. Data reported by different authors concerning blood glucose levels in various species of hzards
535
Evolution of glucose induced hyperglycemia
than the glucose level recorded in the fasting period (Di Maggio & Dessauer, 1963). In these experiments the injected dose of glucose is the same (4 mg in 0.4 ml of water) whatever the lizards’ weight (3.5 to 7 g), that means 0.57 to 1.14 gjkg body weight. In Lacertu ugilis chersonensis the normal glucose level is reached 48 hr after glucose administration (1 g/kg body weight) (Vladescu, 1965). In U. hardwicki, induced hyperglycemia also lasts 48 hr before disappearing (Kumar & Khanna, 1975). We were able to notice that, in I/. rxanthematicus, the blood glucose level again turns close to normoglycemia about 48 hr after the intravenous injection of glucose (0.6 g/kg body weight). Unlike the mentioned authors we mainly concentrated our study on the immediate evolution of the induced hyperglycemia by referring to animals captured at various periods of their ecophysiological cycle. In C: exanrhematicus, glycemic regulation is low in nourishment period (October) while the insulin content of pancreas is high (Godet & Adjovi, 1980). This may seem abnormal when referring to mammalian physiology, but these results were previously observed in A. carolinensis, on perfused pancreatic islets (Rhoten, 1974). During starvation period (February), the glycemic regulation is better though the insulin content of pancreas is low. No study is known concerning animals in such a severe starvation state, thus we miss any comparison element. In other respects it apparently disagrees with the results obtained by Miller & Wurster (1958), Sabnis & Rangnekar (1968) and Kumar & Khanna (1975), who observe B-cells degranulation in pancreatic islets after glucose administration. More particularly in U. hardwicki, an important glucose loading (2 to 5 g/kg body weight) induces B-cells degranulation in 1 to 2 hr and an atrophy of these cells in 72 hr (Kumar & Khanna, 1975). Therefore
liberation of pancreatic insulin in lizards could be only stimulated by an important glucose overloading and a dose of 0.6/kg b.wt is not sufficient to determine an insulinic regulation during the nourishment period. In order to complete our study in V. exanthematicus. it seems necessary to study the relations between different doses of glucose overloading and the insulin output during the life cycle of the animal.
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YVETTEADJOVI
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