602

Specialia

EXPERIENTIA 31/5

The Effect of P r o g e s t e r o n e on P l a s m a Insulin in the Rabbit Sex steroids have been shown to influence carbohydrate m e t a b o l i s m in m a n y i n s t a n c e s in b o t h a n i m a l a n d m a n ~ Treatment with progesterone increased pancreatic insulin s e c r e t i o n in t h e r a t 2 a n d in t h e m o n k e y K I n m a n , t h e insulin response during an oral glucose test was increased a f t e r p r o g e s t e r o n e t r e a t m e n t 4, b u t t h i s c o u l d n o t b e c o n f i r m e d d u r i n g a n i.v. g l u c o s e t e s t ~. I n t h e r a b b i t , t h e e f f e c t of e s t r a d i o l o n g l u c o s e t o l e r a n c e h a s b e e n m e a s u r e d b u t p l a s m a i n s u l i n w a s n o t d e t e r m i n e d 6. W e s t u d i e d t h e e f f e c t of p r o g e s t e r o n e t r e a t m e n t o n t h e i n s u l i n r e s p o n s e a f t e r a n i.v. g l u c o s e t e s t in r a b b i t s . T h e t e s t s w e r e p e r f o r m e d d u r i n g , as well as a f t e r , t h e p r o g e s t e r o n e t r e a t m e n t . W e s h o w e d t h a t in t h e r a b b i t - as in o t h e r s p e c i e s s t u d i e d so far - p r o g e s t e r o n e t r e a t m e n t c a u s e d i n c r e a s e d levels of p l a s m a insulin. Materials and methods. T w o r a n d o m i z e d g r o u p s of 6 N e w Z e a l a n d w h i t e r a b b i t s (2-2.5 kg) w e r e u s e d . 1 g r o u p r e c e i v e d 5 m g p r o g e s t e r o n e / k g b o d y w e i g h t s.c. in 0.6 m l a r a c h i s oil d a i l y . T h e c o n t r o l g r o u p w a s i n j e c t e d w i t h a r a c h i s oil a c c o r d i n g t o t h e s a m e s c h e m e . A f t e r a 2 h f a s t , a 3 5 % s o l u t i o n of g l u c o s e w a s i n j e c t e d r a p i d l y i n t o t h e e a r v e i n in a d o s a g e of 1 g/kg. B l o o d s a m p l e s f r o m t h e e a r v e i n s w e r e c o l l e c t e d in h e p a r i n i z e d t u b e s a t 0, 5, 15, 30 a n d 45 m i n a n d p l a c e d o n ice. P l a s m a g l u c o s e was determined with the glucose oxidase method (Boehr i n g e r t e s t kit) a n d p l a s m a i n s u l i n w i t h t h e d o u b l e a n t i b o d y solid p h a s e ( D A S P ) m e t h o d ~ w i t h p i g i n s u l i n as s t a n d a r d s. Plasma progesterone was determined with a competitive p r o t e i n b i n d i n g a s s a y 9. Glucose disappearance rates were calculated with the l e a s t s q u a r e m e t h o d f r o m t h e l o g a r i t h m i c v a l u e s of t h e p l a s m a g l u c o s e c o n c e n t r a t i o n s ~~ T h e i n t e g r a t e d i n s u l i n

curves were determined by calculating the total area u n d e r t h e r e s p o n s e c u r v e a b o v e zero level. Results and discussion. I n t r a v e n o u s g l u c o s e t o l e r a n c e tests were performed after 5 weeks treatment with progesterone and 1 and 7 weeks after treatment had been stopped. Figure 1 shows the plasma glucose and insulin r e s p o n s e c u r v e s of t r e a t e d a n d c o n t r o l r a b b i t s a f t e r 5 weeks treatment. Natural progesterone produced no s i g n i f i c a n t c h a n g e in t h e r a t e of g l u c o s e d i s a p p e a r a n c e ( T a b l e I), b u t t h e m e a n i n t e g r a t e d i n s u l i n r e s p o n s e w a s s i g n i f i c a n t l y h i g h e r in t h e g r o u p t r e a t e d w i t h p r o g e s t e r o n e t h a n in t h e c o n t r o l g r o u p ( F i g u r e 2). T h e s a m e r e s u l t was obtained 1 week after progesterone treatment had b e e n s t o p p e d . T h e p l a s m a p r o g e s t e r o n e levels w e r e still i n c r e a s e d 1 w e e k a f t e r t r e a t m e n t (Table II).

1 W. N. SPELLACY, Am. J. Obstet. Gynec. lOd, 448 (1969). 2 N. V. COSTRINI and R. K. KALKHOFF,J. clin. Invest. 50, 992 (1971). P. BECK, Diabetes 18, 146 (1969). M. JACOBSON and R. K. KALKHOFF, Clin. Res. ?7, 287 (1969). 5 1~. GOBERNA, F. GARCIA ALBERTOS, J. TAMARIT RODRIGUEZ,

E. DEE RIO and R. Roco, Diabetologia 9, 69 (1973). 6 M. TALAAT, Y. A. HABIT, S. ABDEL NABY, H. HAMDI, A. Y. MALEK,

Z. A. IBRAmM and A. F. SAAD, Arch. int. Pharmacodyn Th@r. 753, 290 (1965). T F. C. DEN HOLLANDER, A. H. W. M. SCHUURS and H. VAN H~LL, J. immun. Methods 1, 247 (1972). s D. 12. PORTER, J. MORATINOS and S. ELLIS, Experientia 29, 1144 (1973). 9 B. T. MARTIN, B. A. COOK a n d W. P. BLACK, J. Endoer. 46, 369

(1970). 18 J. B. O'SULLIVAN, P. J. SNYDER, A. C. SPORER, R. V, DANDROW

and D. CHARLES, J. clin. Endocr. 31, 33 (1970).

Table I. Mean (-4- SEM) glucose disappearance rates (K) following intravenous glucose

Table II. Mean 4- SEM plasma progesterone concentrations

Group

Group

Placebo Progesterone

n

6 6

K (rag/100 ml/min)

Progesterone (ng/ml)

During treatment

After treatment

During treatment

After treatment

(5 weeks)

1 week

7 weeks

(5 weeks)

1 week

7 weeks

3.00 4- 0.23 3.35 :~= 0.20 N.S.

2.56 i 0.21 2.94 ~ 0.36 N.S.

2.65 -t- 0.07 2.94 i 0.37 N.S.

4• 1 67 4- 25 p < 0.005

8• 26 ~ 7 p < 0.005

4• 3 -4- 1 N.S.

Treatment of groups and calculation of K-values as described in Materials and methods.

E

n

Placebo Progesterone

6 6

Progesterone was determined as described in Materials and methods. The P-values were calculated with the Wilcoxon test.

-5O

400-

0

o

-40

E =L

E

v

-30

oo

~ c

200-20

~0

m

K

10 0

100" 10

2'0

3'0

4'0

()

Time in minutes

1;

2'0

3'0

4'0

Fig. i. Plasma glucose and insulin responses to i.v. glucose after 5 weeks progesterone treatment. O-- 9 placebo group; 9 progesterone group.

15.5. 1975

Specialia A1

A2

B1

B2

C1

C2

18~ 1 6 84

14-

5:L

8 m r

-

6

603

T h e s e r e s u l t s agree w i t h t h e r e p o r t e d effects of prog e s t e r o n e o n i n s u l i n responses d u r i n g glucose t o l e r a n c e tests2,3. I t h a s b e e n s h o w n t h a t p r o g e s t e r o n e t r e a t m e n t augments the plasma insulin response without having any a p p a r e n t effect on glucose tolerance. One e x p l a n a t i o n is t h a t p r o g e s t e r o n e i n d u c e s t-cell h y p e r p l a s i a while a t t h e s a m e t i m e it is a n i n s u l i n a n t a g o n i s t i c a u s i n g i m p a i r e d p e r i p h e r a l tissue u t i l i z a t i o n of glucose z. T h e f a c t t h a t p r o g e s t e r o n e i n h i b i t s t h e i n s u l i n effect o n glucose u p t a k e of r a t d i a p h r a g m in v i t r o n s u p p o r t s t h i s suggestion. S e v e n weeks a f t e r p r o g e s t e r o n e t r e a t m e n t h a d b e e n s t o p p e d , t h e r e was no s i g n i f i c a n t difference b e t w e e n t h e i n t e g r a t e d i n s u l i n r e s p o n s e s of t r e a t e d a n d c o n t r o l r a b b i t s . T h i s shows t h a t t h e p r o g e s t e r o n e effect is fully reversible. T h e p r o g e s t e r o n e levels were also n o r m a l i z e d a f t e r 7 weeks (Table II). T h e r e could b e a c o r r e l a t i o n b e t w e e n t h e r a t e in d r o p of p r o g e s t e r o n e levels a n d t h e n o r m a l i z a t i o n of t h e i n s u l i n r e s p o n s e s b u t n o a t t e m p t was m a d e to s t u d y t h i s q u e s t i o n .

-I-

Fig. 2. Mean ( • SEM) integrated plasma insulin responses during an i.v. glucose tolerance test in rabbits. A) after 5 weeks treatment. 1. Placebo group. 2. Progesterone group (p < 0.05). B) 1 week after treatment had been stopped. 1. Placebo group. 2. Progesterone group (p < 0.01). C) 7 weeks after treatment had been stopped. 1. Placebo group. 2. Progesterone group (N.S.). The P-values were calculated with the permutation test xe. 11 B. C. J. SUTTER, M. T. gUTTER DUB, R. LECLERQ and R. JACQUOT, D{abetologia 9, 92 (1973).

Rdsumd. U n t r a i t e m e n t p e n d a n t 5 s e m a i n e s avec de la progesterone a provoqu6 une augmentation significative de la r6ponse i n s u l i n i q u e ~ l ' i n j e c t i o n i.v. de glucose chez la lapine. U n e s e m a i n e apr6s la fin d u t r a i t e m e n t , les t a u x de p r o g e s t e r o n e et la r6ponse i n s u l i n i q u e 6 t a i e n t encore 61ev6s. 7 s e m a i n e s apr6s le t r a i t e m e n t la r @ o n s e i n s u I i n i q u e 6taft de n o u v e a u n o r m a l e , m o n t r a n t q u e l'effet de la p r o g e s t e r o n e 6 t a f t e n t i ~ r e m e n t r6versible. A. I). HAMBURGER, A. C. J. I~UIPERS a n d J. VAN DER VIES

Organon International B. V., Endocrinological R ~ D Labs, P.O. Box 20, Oss (The Netherlands), ] 1 December 7974. 12 S. SIEGEL in Non Parametric Statistics/or the Behavioural Sciences (McGraw-Hill, New York 1956), p. 152.

Water Deprivation inRats: Elevated Plasma Neurophysin Levels W a t e r d e p r i v a t i o n in r a t s is a p o t e n t s t i m u l u s to t h e h y p o t h a l a m o - n e u r o h y p o p h y s i a l s y s t e m a n d leads t o release of b o t h o x y t o c i n a n d v a s o p r e s s i n 1. T h e a v a i l a b i l i t y of specific r a d i o i m m u n o a s s a y s for o x y t o c i n , v a s o p r e s s i n a n d t h e i r c a r r i e r p r o t e i n s , t h e n e u r o p h y s i n s , led us t o r e i n v e s t i g a t e t h e effects of p r o l o n g e d w a t e r d e p r i v a t i o n o n t h e a m o u n t s of t h e s e s u b s t a n c e s p r e s e n t in t h e r a t n e u r a l lobe. P l a s m a n e u r o p h y s i n levels were also det e r m i n e d in t h e s a m e a n i m a l s in o r d e r to a s c e r t a i n w h e t h e r t h e y were e l e v a t e d b y t h e s t i m u l u s a n d in o r d e r to s t u d y , in a n i n d i r e c t way, t h e k i n e t i c s of n e u r o h y p o p h y s i a l h o r m o n e secretion. A d u l t , m a l e r a t s of t h e S I V 50 s t r a t a ( S p r a g u e - D a w l e y derived) w e i g h i n g b e t w e e n 230-310 g were k e p t 5 t o a cage a n d allowed food a d l i b i t u m (Nafag 850 pellets). 28 a n i m a l s used as c o n t r o l s h a d , in a d d i t i o n , free access t o t a p water. 10 g r o u p s of 10 a n i m a l s e a c h were d e p r i v e d of d r i n k i n g w a t e r for periods r a n g i n g f r o m i to 10 days. A t t h e e n d of t h e p e r i o d of w a t e r d e p r i v a t i o n , t h e a n i m a l s were d e c a p i t a t e d . E a c h n e u r o h y p o p h y s i s was rapidly removed, separated from adjacent tissue and t r a n s f e r r e d to a P y r e x t u b e c o n t a i n i n g 2 m l of distilled w a t e r for h o m o g e n i z a t i o n . I n s o l u b l e m a t e r i a l in t h e homogenate was removed by centrifugation and the s u p e r n a t a n t used for r a d i o i m m u n o a s s a y . B l o o d w a s

collected f r o m t h e bodies i m m e d i a t e l y a f t e r d e c a p i t a t i o n a n d c e n t r i f u g e d for b l o o d h a e m a t o c r i t d e t e r m i n a t i o n . T h e p l a s m a was used for m e a s u r e m e n t of o s m o t i c p r e s s u r e a n d chloride c o n c e n t r a t i o n , as well as for d e t e r m i n a t i o n of i m m u n o r e a c t i v e n e u r o p h y s i n levels. N e u r o p h y s i n levels were d e t e r m i n e d b y r a d i o i m m u n o a s s a y u s i n g a cross-species r e a c t i v e a n t i b o d y (AhlV) raised against bovine neurophysins; purified bovine n e u r o p h y s i n I I s e r v e d as s t a n d a r d 2. O x y t o c i n a n d v a s o p r e s s i n were a s s a y e d u s i n g specific a n t i b o d i e s a c c o r d i n g t o p r e v i o u s l y d e s c r i b e d m e t h o d s a. A f t e r 1 d a y of w a t e r d e p r i v a t i o n , t h e a m o u n t s of immunoreactive oxytocin and vasopressin present in the n e u r a l lobe d i d n o t differ g r e a t l y f r o m t h o s e of c o n t r o l rats. A s l i g h t increase in v a s o p r e s s i n c o n t e n t a n d a decrease in o x y t o c i n c o n t e n t were seen, b u t n e i t h e r were s t a t i s t i c a l l y significant. W i t h longer periods of r e m o v a l of water, a p r o g r e s s i v e fall i n t h e g l a n d c o n t e n t of b o t h I C. \u JONES (1969).

and B. T. PICKERINO,

J. Physiol., Lond. 227, 553

z j. j. LEGROS, P. FRANCHIMONTand J. C. HENDRICK, C. r. S6anc. Soc. Biol., Paris 163, 2773 (1969). J. J. LEOROS, U. STEWART, J. J. NORDSIAN~'I,J. J. DREI~USS and P. FRANCHI~iONT, C. r. S6anc. Soc. Biol., Paris 165, 2443 (1971).