Seasonal variations of immunoreactive glucagon contents in pancreatic extracts of a sahelian lizard (Varanus exanthematicus)

0300-9629:81/690031-12$02.00/O Copyright Q 1981 Perpamon Press Ltd

Cenp Bwthem. Phwel. Vol. 69A. pp. 31 10 42. 1981 Printed in Creak Britarn. All rights reserved

SEASONAL VARIATIONS OF IMMUNOREACTIVE GLUCAGON CONTENTS IN PANCREATIC EXTRACTS OF A SAHELIAN LIZARD (K4RANUS EXANTHEMATICUS)* MWEILLE DUPGGODET

Laboratoire de Neurophysiologie,

and YVETTEADIOVI Facultt des Sciences et Techniques, Universitt de Nice, 06100 France (Received 4

August

1980)

Abstract-I. The immunorea~ive glucagon contents were estimated in the whole pancreas as well as in the cranial, medial and caudal parts throughout the year. 2. We have determined if there is any relationship between the evolution of the glucagon content and the annual ecophysiological cycle of the animal. We found: A bimodal profile of the pancreatic glucagon levels: the maximal values observed during starvation, the minimal ones during the food intake period. A cephalo-caudal gradient of concentration of the pancreatic glucagon content. A difference in the evolution of dynamic transfers between cranial and medio-caudal parts. 3. The results were compared with those reported for different species and the accent is on the anatomical and functional disparity between the different parts of the pancreas, suggesting a possible interaction between these different parts.

were immediately removed, carefully dissected out of surrounding tissues and divided in three parts: cranial, medial and caudal parts. The cranial part was separated from the medial part by an artery which leads from the spleen to the gall-bladder as well as the pancreatic vein pouring in the sub-hepatic circulation. The boundary between the second and third parts is more arbitrary. Only the caudal oart of the nancreas undergoes growth every year and we can observe the lastformed endocrine islets (Godet er al., unpublished data). Each part was weighed, wrapped in aluminium foil and frozen in liquid nitrogen. The pancreatic samples were homogenized in a buffer medium pH 7.4 (TRISt IO-’ M, EDTA$ 10m3M, DTT$ 10e3 M; Glycerol 10% v/v). After ~ntrifugation (30,~rpm for 45min) the extracts were decanted and frozen at -2O”C. Pancreatic proteins were estimated by Lowry’s method on diluted (1:20) ahquots (Lowry et al., 1951). Pancreatic glucagon was determined by radioimmunoassay using a glucagon kit supplied by Biodata products, the main components of which are antiserum 30 K (dilution 1:1800)(Luyckx, 1974) and [iZ51]labelled glucagon. The sensitivity limit of the system is of about 60 pg/ml. The inhibition of the antibody-bound [‘251]glucagon was studied for different concentrations of standards ranging from 125-2000 Pa/ml and the diluted lizard oancreatic extracts . -. (1:400-I :2WO)exhibited a good parall&sm with the standard curve, indicating a satisfactory cross-reactivity between K e~anc~e~~fjcus and beef-pork t&carton. All samples were assayed in triplicate. ‘Glucagon concentrations are expressed as pg equivafents of beef-pork glucagon per mg of proteins, per mg of wet tissue and per 1OOg body weight.

While the hormonology of pancreatic glucagon has been well studied in Man and in higher vertebrates (for review see Lefebvre et al., 1972), either for thera-

peutic reasons or for the adjustment of methods of analysis, numerous gaps in our knowledge still exist in the lower vertebrates (see references in Table 9 in Discussion). These gaps concern the topographic distribution of glucagon contents and their possible relation to the ecophysiological cycle. The choice of a sahelian reptile is not fortuitous. The determinism of the animal cycle has permitted us to establish a parallelism between the evolution of the pancreatic glucagon content and the physiological state of the animal throu~out the year. MATERIALS Capture

AND METHODS

and maintenance

Adults lizards (Varanus exanthematicus) of both sexes, weighing 75&165Og (75trlOOOg for females and IOOO-1650g for males) were captured in the sahelian region of Senegal at each characteristic period of their tropical life cycle. In the laboratorv. thev were held. for at least 1wk prior to sacrifice in a fasting-state, at adequate environmental temperature and humidity (see Cisse 1980 and our ecophysiological data). Lizards were killed after anesthesia (iv. administration of 10mg pentobarbital~g body weight), the pancreases

Sratisticai

* Supported by a grant from D.G.R.S.T of Senegal (No. 2. 806 art. 4031.1). t TRIS: aminomethylidinetrimethanol. $ EDTA: ethylenediaminetetracetic acid. $ DTT: 1,4dithioerythritol. C.B.P.

69,A.--r

treatment

of the data

The mean values are given f standard errors and + 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. 31

MIRE~LLE DUPE-GODET and YVETTE ADJOVI

32

Table 1. Seasonal variations of p/P and p’/P ratio

1

J

F

M

IA

M

J

JY

A j

0

s

1 p/P

82.73

82.74

’

l

+1.17 (5)

8. 55

6.66

+O. 44 (7)

20.83 (7)

92.36 t 15.20 - (4)

t 12.7’ - (6)

5.82

6.11

%82I

94. OE

t10.25 - (7)

t24.79+11.7e - (7) - (5)

l

4.42 “7 P

81.38

N

I

k 2.29 (5)

+ 0.87

(6)

1 I

D

1 86.50 t 18. 07

-I-

-

(4)

6-68

4.86

1.37 (5)

+ 0.69 (4)

p/P = pancreas weight mg/lOOg body weight. p’/P = protein content of pancreas mg/lOO g body weight * = mean values * confidence limits for P = 0.05 and (N) determinations.

(mg)/lOOg body weight, Table 1). Figure 1 shows a significant augmentation (P > 0.001) of p/P during the period of nourishlaent (July to October) compared to the period of starvation (November to June). November and June constitute a period of transition with a significant differentiation of both phase of nourishment and phase of starvation, P > 0.001. This increase in weight depends on the pancreatic protein content. The ratio p’/P (protein content of pancreatic tissue (mg)/lOOg body weight) runs parallel to the former. There is also the possibility of tissue hydration, though this hydration affects the pancreas in the same manner as the whole body. The period of nourishment corresponds to a modification of exocrine cellular contents (enfolding of granular endoplasmic reticulum, development of Golgian secretions and abundant zymogen grains, Mattei et al. 1980) as well as endocrine modifications (development

Ecoph~sioloyical data

From a climatic point of view, Senegal belongs to the sahelian regions characterised by an alternance between a dry season (November to June) and a rainy season (July to October). The temperature cycle follows approximately the hygrometric cycle and the highest monthly values (4045’C) can be observed from April to October. Adapted to the biotope. the physiological cycle of V. urar~thematicus is schematically divided in two periods: (a) an active period. from the nutritional and endocrinologic points of view (July to October): reproduction takes place in October. (b) a rest period during which the catatonic lizard hides in tree cavities. the metabolic needs of the animal are satisfied by the mesenteric fat reserves. RESULTS Amtd

derelopn~et~t of the pancreatic

This is expressed

weight

by the ratio p/P (pancreas

weight

JFMAM,

JY

I

A

s

CJ

*

0

c--+--e

%

Fig.

1. Seasonal

OF! P

of p/P and p’/P ratio. p/P = pancreas weight mg/lOOg body weight. p’/P = protein content of pancreas mg/lOO g body weight.

variations

I

219

I

9 646 41

I

error, ***confidence

2622

+ 4 562

1

+ 12,115

1 I

1 60,174

095

I

ci,

216,265

23,288

M

140,294

*Mean values, **standard

organ

content of

Total Glucagon

141,364

f77400

F

+13 127fl5 (4,

256

+8

i65

PART

+9

104,087

I

21,360

-C

+

J

CAUDAL

PART

MEDIAL

PART

CRANIAL

Glucagon content of A

variations

46777

I

I

+2 292

14,742

+2 613 (4)

22,396

J

+ 3 650 hl

+3480

1 I

I

:

+

I

I

JY

glucagon

A

’

5096

655 Ifi)

I

I

f5

+4

675 51

552

111,614

+3

3 484

9 189

+ 5

t-

S

777

I

I

f3

038 41

+2 437

1 15,394

+l 236 (A\

+

10,865

+14600

24,456

0

of proteins)

16,662

(pg equiv/mg

P = 0.05 and (N) determinations.

826 4)

520

15,293

1

extractable

3 646 +3 644 CL__&)

1 20,204

+

+ 3 476

16,270

+

32,280

M

on pancreas

limits of the mean values for

Table 2. Seasonal

318 ill,635 fA\

+7

27,186

I

(41

+7 223

+4 543

133,622 I

39,651

D

I

I

+

+

41

861

542

122,809

&_

+ 4 163

+ 3 968

10,707

+lO, 028 + 4 514 4) 4)

37,670

N

I

I

1

Clucago

*

t*

*

I-

t-

1

390

t 449

765

t 1216 ---&a-d

t

2 204

t 828 !4!

1195 --. : 521 307

+

,+ 939 .&a

291 (61

591 :

+ 277

t

1415

_ -ed.__

293

1140

370

+

+

1166

+ 616 _lirl__ =__I__

+ 664

--.4801

;r 672 --f 838 ...-.kk

1901

b 621 t536 =.&L._:__(5)_

:

--_-

-.-

2005

I

1

934

A

t

:

411 (5)

1220

701 (4)

I

2

)+

?

!

:

5) ,t

399

320

I

+

158 I51

1 $ 127

893

(5)

!

+ 266 (6)

!: 213

787

+ 176 (5)

?: 141

776

245 (5)

196

209

199

620

1001

1 _..

23b

790

294

.~~.~~ __!5)

/

I

t

t

1158

1260 178

lOINID

s

2 284 t’“_

I I

t

_

---.------it

_ __.~~_

I

;t

~_. --

~_ -

JY

2 441

-t -

--.

1078

..__ &I

!: 106 “._ t 170

-

2 512 fi_,____.-._ ..zz-__

,t 410 _--

1493

J

Mean values, **standard error, ***confidence limits of the mean values for P = 0.05 and (N) determinations.

organ

content of

Total

c

MEDIAL PART

CRANIAL PART

GLUCACON CONTENT OF

Table 3. Seasonal variations on pancreas extractable glucagon (pg equiv/mg wet tissue)

-

449 (4)

(5)

+ 670

+ 538

1673 ~.._

:

282

103

1289

(4)

248

156

f165 141

I

,+ 298 L4)

I

I

!:

+

662 _...... ..-

_.~~

,+ 351 (4)

?; 221

1058

%!J”

1:

_....

2739 : 884

1 ,+ 187

I

1 2271

.

344 (4)

2174 :

t

2 216

2960

I

I I

1

..-

-_.

__ tl5,875-.

f15,133

~_ _.__..-.

M

~-__-

35,130

942

31, 142 +5

,

M

+10,483 _ _. ___._.__ ____ tlo,997

---.

A

254

27,516

:5 a61 _ ..__ _. .~ _~..-.__--~ t7 306

+7

--”

429

25,342

6

S

34,362

A

+5

I

JY

+a 237

34: 957

J

* Mean values. **standard errors, ***confidence limits of the mean values for P = 0.05 and (N) determinations.

of organ

content

Total Glucagor

f48,974

___.-..

978

f4

PART

____

33.199

29,005

PART

3897

44,411

CAUDAL

2a,46 1

F

!:

J

+6407

MEDIAL

PART

CRANIAL

Glucagon content of _~

/

_

+5

57,516

:14,500

77, a41

N

645 +10,895

30,809

+ 1 374

42, a30

0

Table 4. Seasonal variations on pancreas extractable glucagon (pg equiv/lOO g body weight)

s

t 2 702

22,017

!: 2 694

25,230

D

+

a

36

MIREILLEDUPE-GODETand

YVETTE ADJOVI

pq.drq

l

**.10’rq./t00*. 0.z

I

In*

I.f

~

Fig. 2. Seasonal evolution of total pancreatic glucagon contents, expressed as: -f---t-+pg pg equiv/mg wt. ---+--o---+-pg equiv/lNg b. wt. (mean values; see standard errors in tables 2, 3,4).

equiv/mg proteins. _

of B granules from which comes the insulin material in the B pancreatic cells, unpublished data). Annual development of pancreatic immunoreactive glucagon content

Our results are expressed in terms of pg equiv/mg wet tissue and pg equiv/mg proteins, which enable us to compare these results with those given by other authors. The metabolic aspect is indicated (pg equiv/iOO g body weight). These results in Tables 2, 3, 4 and Figs 2, 3, 4 and 5, show the same profile of general development. They will be considered from the point of view of total glucagon content of the organ as well as of the different parts of the pancreas. Total content of the organ

Figure 2 shows a bimodal evolution including: -a spike during November, at the beginning of the starvation period (mean values: 33,622 f 4543 pg equiv/mg proteins, 2271 _t 187 pg equivjmg wt., 209,287 3_ 22,485 pg equiv/fOO g b. wt). -a more important spike during February-March, at the middle of the starvation period (mean values: 60,174 f 4562 pg equiv/mg proteins, 4801 + 591 pg equivfmg wt., 354,331 f 42,851 pg equiv/lOO g b. wt.).

- two periods of minimal values, during which the quantities of immunoreactive glucagon are far from negligible (for example, mean values of September, during the nourishment period: 11,614 f 4552 pg equiv/mg proteins, 1001 + 320pg equiv/mg wt., 97,996 & 31,675 pg ~uiv/l~ g b. wt.). Contents

of d@erent fractions

of the organ

The same bimodal evolution

was observed clearly in the cephalic part, while that in the two other parts was less obvious. Cephalic part. The analysis of Figs 2,3 and 4, shows a bimodal pattern. The spikes suggest that the immunorea~ive glucagon of this part of pancreas is responsible for the annual profile of the whole organ. Table 5 gives the high values observed during February-March as compared to minimal values of September-October. This indicates that in the cranial part during the medial period of starvation, there is 8-10 times more glucagon than during the food intake period. Figure 6A shows the same results in another way: the values of giucagon content are expressed in per cent of the maximal values of Februa~-March. These

Pancreatic

Fig. 3. Seasonal

evolution

of pancreatic

extracts

of a sahelian

37

lizard

glucagon contents expressed as pg equiv/mg values f standard errors.

results are important, since they demonstrate the existence of a dynamic transfer in the organ as well as between the organ and the body. Further researches on this and the hormonal output seem to be necessary. Me&-cauda/ part. The statistical analysis of the results on the glucagon content do not indicate significant difference for the annual development

proteins.

Mean

between these two parts of pancreas, except during a limited period of the cycle, where the differences are dependent on the method of presentation (see Table 6). This means that there is a parallelism in glucagon content of the medial and caudal parts during the cyclic evolution. No distinction will be made between the medial and caudal parts, and accordingly the term medio-caudal part will be used.

Table 5. Glucagon contents in:

pg equiv/mg proteins

pg equiv/mg

wt.

pg equiv/lOO g b. wt.

February-March September-October

141,364 * 21,051 16,622 + 6370

11,733 k 2790 1,158 + 196

250.649 + 17,466 3 1,960 + 9045

Central starvation

8.48

10.13

7.84

Food

intake

period

38

MIREILLEDIJPU~ODET and YVETTEADJOVI

10 00

500

400’0 : 1 L L i

J

e ,o p” 3001 f s z ;

i 2ooa

1000

Fig. 4. Seasonal evolution of pancreatic glucagon contents expressed as pg equiv/mg wt. Mean values + standard errors.

The quantitive analysis of glucagon is shown by histograms (Figs 3,4 and 5) indicating a bimodal evolution less important than that concerning the cephalic part. The principal spike is situated during November, at the beginning of the period of starvation and not in February as is the case with the cranial part, showing a significant difference in the time of the dynamic transfer. We can see that in the medio-caudal part, a wide series of low values near to

one another slope gradually from March to December. The ratio “beginning of starvation/food intake period” is 2.5 (see Table 7). This means that the transfer in this region of the pancreas is low during the greatest part of the cycle. The results can be given in an other way (Fig. 6C), where the levels of glucagon are expressed in percentages of the maximal value of November. According to the unities utilised, the modality of transfer during

Table 6. Significant difference observed in :

pg equiv/mg proteins

October + December December October

0.05 < P < 0.02 N.S 0.01 < P < 0.001

pg equiv/mg wt.

pg equiv/lOO g b. wt.

NS 0.05 < P < 0.02

N.S N.S

N.S

N.S

39

Pancreatic extracts of a sahelian lizard

Fig. 5. Seasonal evolution of pancreatic glucagon contents expressed as pg equiv/lOOg b. wt. Mean values + standard errors.

February-March when the term pg can be observed. maximal one of bimodal pattern.

seems to be different. Accordingly, equiv/mg wt. is used, a second spike The value of this spike reaches the November. giving to this curve a

Comparison between cephalic and ~edi~ca~dal purrs. There is a significant difference between the im-

munoreactive

glucagon contents of the cranial part

and the mediocaudal parts practically during the whole cycle (Table 8). The greatest amount of glucagon can be found in the cephalic part of the pancreas. showing a cephalo caudal gradient of distribution of the pancreatic glucagon. The important transfer can be observed in the cephalic part (ratio from 8 to 10).

Table 7. Glucagon contents in: November September beginning of starvation Food intake period

pg equivimg proteins

pg equiv/mg wt.

pg equiv/lOO g b. wt.

27.180 + 9586 11.317 +_3721

2013 + 369 732 f 203

67,678 + 16.093 26.429 f 6173

2.40

2.75

2.56

MIREILLEDUP&GODETand YVETTEADJOVI The quantity of glucagon content in the medio caudal part is much less important and more stable (ratio 2.5). The transfers are not done during the same period of the cycle: in the cephalic region, they take place principally at the middle of the starvation period (February-March), while in the mediocaudal part, they are done once starvation has begun (November). DISCUSSION

01

m

,FMAYJ

Fig. 6. Comparative seasonal variations between pancreatic glucagon contents and liver glycogen. The arrow indicates the maximal annual y0 mean values, in February for cranial part (A), and in November for mediocaudal parts (C). (B) Mean values of liver glycogen + standard errors (From Cisse 1980). -+-+-+pg equiv/mg proteins. m pg equiv/mg wt. ---O---O---*--pg equiv/lOOg b. wt. C.P: cranial part. M.P + CLP: mediocaucal part.

The results given in Table 9 for different species of vertebrates, show a great disparity of values. It is difficult to draw definitive conclusions and to give the characteristics relative to species, or even class. Some authors have suggested important values concerning Sauropsida and particularly in reptiles (Assan ef al., 1969, Rhoten, 1976). The values given for the frog, the cow and even for man are sometimes as important. Is the variation due to the time of extraction of the pancreas in animals showing a cyclic ecophysiological evolution? Our observations show the variations between the period of nourishment and that of starvation. These values are specific characteristics to V. exanthematicus and a single value cannot have any significance. Is the variation due to the region of extraction when the whole pancreas is not taken in consideration? Our observations show variations (from 1 to 6) between the cephalic and the mediocaudal regions at the same period of the cycle. Is it due to sex? The sex of animals utilised in studies is not always mentioned. In reptiles, the female tortoises (Chrysemys d’orbignyi) have higher values than the males, when the glucagon contents are reported in grams of wet tissue (Marques et al., 1968). Due to the fact that a great heterogeneity was observed in females of V. exanthemaricus in our research work, we have been obliged to ignore them. It is important to establish a parallelism between the development of the glucagon content shown and that of the glycogen content of the liver (Fig. 6, A, B, C). It is essentially the medio-caudal part of the pancreas which shows the mobilization of hepatic glycogen at the beginning of starvation in November. The cephalic part shows a slow mobilization of glycogen during the remaining part of starvation period (from January to June). Different parts of the pancreas do not possess the same function in addition to the topo-

Table 8. Statistical analysis--P values for the different glucagon contents (r-test). The cranial part (C.P) compared to medial and caudal parts (C P + Cl. P) of pancreas, in terms of: Annual cycle J F M-A M J JY-A s 0 N D

pg equiv/mg Proteins C P/MP + Cl. P

pg equiv/mg wet tissue CP/MP + Cl. P

pg equiv/lOO g body wt CP/MP + Cl. P

>O.ool >O.ool

>0.001

>O.ool >O.ool

>O.OOl

> 0.001

>O.OOl >O.OOl

0.05 4 P < 0.02

0.05 < P < 0.02 0.05

0.05 >O.OOl 0.05 >O.OOl

0.01 < P < 0.001 0.01< P < 0.001 0.01< P < 0.001 >0.001

N.S’I’ 0.05 < P < 0.02 N.S P > 0.001

(*) NS: not significant. A P value less than 0.05 was considered significant.

Pancreatic extracts of a sahelian lizard Table 9. A, B, C-Data

reported by Moody (1972) and other authors. expressed in the same unit. D and E-Typical mean values + standard error of present study

A-Glucagon

content

~~puncreasfrom

wrrtrhrare

cold-adored

species

Glucagon in k equiv*/g wet tissue

Species Amphibian

41

References

species

Axolotl species (Tesrudo hermanni) (Chrysemys d’orhignyi)

et al. (I 969) et al. (1969)

Assan Assan

1.05 7.40

Frog (Rana esculenta) Reptilian

Tortoise

Male Female Lizard (Agama

1.81 + 0.14 0.174 0.046 3390.0 + 340 0.007 0.026

agama)

(Anolis

curo~jnensis) ~~sfeo~oernus retraspis)

B-Glucagon

content

of pancreas from

warm-blooded

Grill0 PI al. (1976) Rhoten (1976) Grille et a!. (1976)

certehrate

species

Glucagon pg equiv/g wet tissue

Species

al. (1969)

Marques et al. (1968)

1.29 + 0.11

Crocodile

Asian

Assan et

43.00

References

species

Chicken Duck Mammalian

I .40 2.50

Assan et Assan et

0.40 1.20 2.40 0.275 0.746 0.46 5.00 1.50 - 5 4.50 2.20 0. IO 8.00 1.50 0.10 - 0.90 3.70 9.00

Assan er at. (1969) Assan et al. (1969) Unger et ai. (1966) Buchanan er af. (1967) Assan et al. (1969) Assan rt al. (1969) Assan et al. (1969) Assan et al. (I 969) Unger rr al. (1966) Buchanan er al. (1967) Assan ef al. (1969) Unger (1972) Assan et al. (1969) Unger et al. (1966) Buchanan rr al. (1967) Unger (1972)

Rat Guinea pig Rabbit Cat Dog Sheep Beef Man

C-Torah

glucagon

content

Man cranial part caudal part

D-G/wagon

ofpan~reas

(1969)

@g equio)

2.0t 127.0t 22.4 0.84t 3.7 8.8 - 13.4 26 - 36.3

Rat Dog

contents

of Varanus

exanthemaricus

pancreas

expressed

November 2.27 + 2.96 + 2.17 F 1.67 + E-Total

g/wagon

2.41 + 0.28

* equiv = porcine equivalent. t = mean values.

February-March

0.18 0.21 0.28 0.53

4.80 + 11.73 + I.87 * 1.90 + content

Unger Unger Unger Unger Unger Unger Unger

et al. (1967) rt al. (1967) et al. (1961) et al. (1961) et al. (1961) et al. (1961 et al. (1961)

as pg equil;/g

wet

tissue

Food intake period

Starvation

Total organ

(1969)

species

Mouse

Total organ Cranial part Medial part Caudal part

al. al.

0.59 2.70 0.44 0.67

of pancreas

3.63 k 0.87

September-October 0.89 + 0.12 1.15 f 0.19 0.77 & 0.14 0.62 + 0.19

(pg equk)

1.39 + 0.38

MIREILLE DUPE-GODE T and Y~ETTZ ADIOVI

42

graphic disparity of their contents. This double disparity suggests the existence of regulative interpancreatic phenomena, acting at a distance. that is to say getting beyond the limits of the Langerhans’ islets. Further experiments are necessary to study this problem. The existence of spikes separated by regions of low variations seems to mean possible local modifications of glucagon contents. The ascending phase of the spike observed during the starvation period, may correspond to an increase in volume and density of s( granulations and to the augmentation of the number of x granules with or without a multiplication of A cells. Acknowledgements-We are grateful to Dr B. Krebs for helpful discussions and we express our appreciation to Mr W. Gruner for his excellent technical assistance. (R.1.A Laboratory, centre Lacassagne, Nice).

REFERENCES

ASSAN R., TCHOBROUTSKYG. & RO~SELING. (1969). Caracterisation radioimmunologique de glucagon dans les tissus digestifs de diverses exptces animales. Path. Biol. 17, 747-755. CISSE M. & DEMAILLE J. (1975) Cycle annuel glucido Lipidique chez un varan du Senegal. C.r. Sdanc. Sot. Biol. 169, 4. 10841089. CI~SE M. (1980) Ecophysiologie comparee de deux varans en milieu sahelien. These doctorat d’Etat. Universite de Nice.

GRILLKJ T. A. I., ITO R. & WATANABE K. (1976) The endo-

crine pancreas of some West African reptile. Electron microscopy, histochemistry and hormone content in The

evolution ofpancreaticislers (Edited by GRILLO, LEIBSON 8~ EPPLE), pp. 189-249. Pergamon Press. LEFEBVRE J. & UNGER R. H. (1972) G/wagon. Pergamon Press, Oxford. LOWRY 0. M.. RO~EBROUGHTN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the folin phenol reagent J. hiol. Chem. 193, 265-275. LUYCKX A. (1974) Secretion de I’insuline et du glucagonEtude clinique et exp&imentale, Chap. IX. pp. 82-99. Masson Edit. MARQUE.SM. & KRAEMER A. (1968) Extractable insulin and glucagon from turtle’s (Chrysemys d’0rbign.H) pancreas. Comp. Biochem.

Physiol.

27, 439-446.

MATTEI X. & GODET R. (1980) Etude pancreas exocrine non fonctionnel

ultrastructurale du chez le Varan (V. exanrhemaficus). Ultrasrruc. Research (in press). MCODY A. J. (1972) Gastrointestinal glucagon-like immunoreactivity. In Glucagon, Chap. 21. (Edited by LEFEBVRE & UNGER), pp. 319-341. Pergamon Press. Oxford. RHOTEN W. B. (1976) Glucagon levels in pancreatic extracts and olasma of the lizard. Am. J. Anar. 147, I. 131-137. UNGER-R. H. (1972) Glucagon and glucagon immunoreactivity in plasma and pancreatic tissues. In Glucagon Chap. IV. (Edited by LEFEBVRE& UNGER) pp. 205-21 I. Pergamon Press, Oxford. UNGEK R. H.. EISENTRAUT A. M.. MACCALL M. S. & MADISON L. L. (1961) Glucagon anti bodies and an immunoassay for glucagon. J. c/in. Incesr. 40. 1280-1289. UNGER R. M., KETTERER H. & EISENTRAUT A. M. (1966) Distribution of immunoassayable glucagon in gastrointestinal tissues. Mrfaholism 15, 865-867. UNGER R. H. & EISENTRAUT A. M. (1967) In Hormones in Blood Vol. 1. Chap. V. pp. 83-128.