Effects of acute sodium salicylate on the abnormal counterregulatory glucagon and epinephrine responses to insulin hypoglycemia in diabetic rats

Life Sciences, Vol. 44, pp. 301-310 Printed in the U.S.A.

Pergamon Press

EFFECTS OF ACUTE SODIDlq SALICYLATE ON THE ABNORMAL COUNTERREGULATOEY GLUCAGON AND EPINEPHRINE RESPONSES TO INSULIN HYPOGLYCEMIA IN DIABETIC RATS

D.G. PATEL

D i v i s i o n o f Pharmacology and M e d i c i n a l Chemistry C o l l e g e o f Pharmacy, U n i v e r s i t y o f C i n c i n n a t i Cincinnati, OH 45267 (Received i n f i n a l

form December 2, 1988)

SUgaRY

E f f e c t s o f a c u t e sodium s a l i c y l a t e i n f u s i o n on g l u c a g o n and epinephrine responses to insulin hypoglycemia were studied in streptozotocln diabetic and age-matched control rats. Sodium sallcylate (50 mg/kg/h) was infused intravenously alone for 90 minutes and then with insulin in short-term (i0-15 days post-streptozotocin) and long-term (80-100 days post-streptozotocln) diabetic as well as age-matched control rats to produce hypoglycemia. Sodium salicylate decreased basal plasma glucose in control and diabetic rats but increased basal plasma glucagon levels only in control rats. The infusion of sodium sallcylate during Insulin-hypoglycemla in control and short-term diabetic rats caused a significant increase in glucagon secretion. Long-termdlabetlc rats have impaired glucagon and epinephrine secretory responses to Insulin-hypoglycemla. This defect was normalized by acute sodium sallcylate infusion during insulinhypoglycemia. However, Indomethacln (5 mg/kg i.p.; twice at 18 hr intervals) improved, but failed to completely normalize the abnormal glucagon and epinephrine secretory responses to Insulln-hypoglycemla in long-term diabetic rats. These results suggest that endogenous prostaglandins may play a partial role in the impairment of glucagon and epinephrine secretion in response to insulin-hypoglycemla in longterm diabetic rats. Glucagon and e p i n e p h r i n e s e c r e t i o n s i n r e s p o n s e to i n s u l i n - h y p o g l y c e m i a have been reported to be impaired in diabetics with autonomic neuropat/~y as well as in experimental diabetic rats (1-3). It is unlikely that the sympathetic branch of the autonomic nervous system is related to the impaired glucagon secretion (4-7); however, there are indications that chollnerglc parasympathetic nerves may be involved. Truncal vagotomy in man significantly impairs the glucagon response to Insulln-hypoglycemia, but does not alter basal glucagon levels (8). Atropine blocks the glucagon response to Insulin-hypoglycemla in both normal and short-term diabetic rats (9). The lack of glucagon and epinephrine secretion in response to Insulln-hypoglycemla in long-term diabetic rats is reversed with a single subcutaneous injection of carbachol (9). However, other investigators have disputed the importance of cholinerglc nerves in glucagon secretion during insulln-hypoglycemia (10-12). Prostaglandlns have been reported to modulate cholinergic transmission (13,14) as well as various metabolic functions including the endocrine system responsible for glucose homeostasis (15). P r o s t a g l a n d i n s i n h i b i t b o t h b a s a l and g l u c o s e - s t i e n a l a t e d i n s u l i n r e l e a s e i n 0024--3205189 $3.00 + .00 Copyright (c) 1989 Pergamon Press plc

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normal and diabetic subjects (16-18). Acute infusion of sodium sallcylate significantly increases glucagon and epinephrine responses to insulinhypoglycemia in normal subjects ( 1 9 ) a n d long-ta_~a ~iabetlc rats (20). The aim of this study was to investigate the role of sodium sallcylate, an inhibitor of prostaglandln synthesis, in the blunted glucagon and epinephrine secretions during Insulln-hypoglycemla in diabetic rats. Acute infusion of sodium sallcylate normalized the altered glucagon and epinephrine responses to insulln-hypoglycemla in long-term diabetic rats. However, Indomethacln, another prostaglandln synthesis inhibitor, only partially improved the abnormal glucagon and epinephrine responses to insulln-hypoglycemla in long-term diabetic rats. The results suggest the possibility that endogenous prostaglandlns m a y b e partlally involved in the lack of glucagon and epinephrine responses to insulinhypoglycemia in long-term diabetic rats.

MATERIALS AND METHODS Animals. Sprague-Dawley male rats weighing 275-300 g were divided into two welght-matched groups - control and experimental. Diabetes was induced in the experimental group by i.v. injection of streptozotocln as previously described (211. Groups of rats were studied for glucagon and epinephrine release during insulln-hypoglycemla 10-15 days (short-term diabetic) or 80-100 days (long-term diabetic) after the induction of diabetes. Age-matched rats were used as controls. Anlmals were housed three per plastic cage in animal quarters controlled for temperature, humidity, and light cycle and were fed Purina Rat Chow a n d w a t e r ad llbltum. Experimental Protocol. I n s u l i n - h y p o g l y c e m i a was i n d u c e d a s d e s c r i b e d previously (3) with the following changes: In experiments pertaining to the acute effects of sodium salicylate on glucagon and epinephrine secretion during Insulln-hypoglycemla, both short-term and long-term diabetic and respective agematched control rats were used. The Jugular vein (for infusion) and the carotid artery (for blood sampling) were cannulated under chloral hydrate anesthesia (350 mg/kg i.p.). A fasting blood sample (2.0 ml) was collected in a heparinlzed syringe; 1.0 ml (for glucagon) was transferred into an ice-cold tube containing 50 ~i of Trasylol (10,000 U/m1) and EDTA (1.2 mg/tube) and the remaining 1.0 ml (for epinephrine) was placed into the second ice-cold plain tube. All blood samples were kept at 4"C until centrifuged. Sodium sallcylate (50 mg/kg/h) in normal sallne was infused for 90 mln. A second blood sample (2.0 ml) was collected for glucagon and epinephrine measurements and plasma glucose (PG) levels were determined. Insulin was injected i.v. as a bolus according to the following formula: 2.0 + (PG-IO0) x 0.02 U/kg. I,--edlately after the bolus injection, insulin (5 U/kE/h), chloral hydrate (57.5 mg/kg/h) and sodium salicylate (50 mg/kg/h) were infused at a rate of 2.3 ml/h. Approximately 50 ~i blood samples were collected perlodlcally (15 times) to determine plasma glucose levels to monitor hypoglycemia. When the plasma glucose level dropped to between 30 and 40 mg/dl a third blood sample (2.0 ml) was collected for glucagon and epinephrine estimations. An equal amount of normal saline was injected after each blood collection to avoid hypovolemla. All blood samples were centrifuged at 1500 x g (4°C) for 15 mln. Plasma samples were stored at -20°C for glucagon and at -75°C for epinephrine determinations at a later date.

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In a second series of experiments, long-term diabetic and age-matched control rats were studied. Indomethacln (5 mg/kg i.p.) was injected twice in both diabetic and control rats, first a t t h e time of food withdrawal and second after an lg h fast Just before the induction of anesthesia with chloral hydrate. As described earller, a fasting blood sample (2.0 ml) was collected from a carotid artery and divided into two Ice-cold tubes for glucagon and epinephrine measurements. Insulln hypoglycemia was induced as described above except that sodium salleylate was deleted from the infusion. Analytical technio~Qs. Plasma glucose concentration was d e t e r m i n e d b y t h e glucose oxidase method, using a glucose autoanalyzer (Beckman I n s t r u m e n t s , Fullerton, CA). P l a s m a g l u c a g o n was m e a s u r e d b y r a d i o i - - , u n o a s s a y using cyrstalline Porcine Pancreatic glucagon as standard (Eli Lily and Co., Indianapolis, IN) ( 2 2 ) . Both standards and samples were extracted with ice-cold acetone to eliminate "big plasma glucagon" (23). Plasma epinephrine levels were determined by high performance liquid chromatography with the electrochemical detection technique of Hallman et al. (24) as modified by Patel (3). Statistical stEnificance was c o m p u t e d b y e i t h e r S t u d e n t s ' t test or Duncan multiple range test. Differences between means were considered statistically significant w h e n p was l e s s t h a n 0 . 0 5 .

TABLE I Plasma Glucose Levels Pre and Post Sodium Salicylatz Infusion in Control and Diabetic

Animals

Rats

Glucose mg/dl before

after drug

Control (8)

133±27

110±25,

Short-term Diabetic (8)

325±62

261±54-

Long-term Diabetic (12)

421±57

309±59*

R e s u l t s a r e e x p r e s s e d a s mean ± S . D . ( ) - number of obsewations. Sodium sallcylate 50 mg/kg/h was infused intravenously for 90 mln. * p < 0.05 compared with respective before sodium sallcylate infusion.

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A c u t e sodium s a l i c y l a t e i n f u s i o n s i g n i f i c a n t l y r e d u c e d plasma g l u c o s e l e v e l s i n a l l groups o f r a t s ( T a b l e I ) . In addition, salicylate infusion accelerated the rate of decline of plasma glucose durln s Insulln-hypoglycemla and reduced the total amount o f insulin needed t o achieve the target hypoglycemia value of 30-40 mg glucose/dl of plasma (Table II). Due to the initial hyperglycemla, the total amount of insulin needed to effect hypoglycemia in diabetic rats was considerably more than in controls. However, even in these diabetic rate (both short-term and long-term) the hypoglycemia was achieved with less insulin than in the respective non-sallcylate treated rats.

TABLE II

T o t a l I n s u l i n U n i t s I n f u s e d and R a t e o f F a l l o f G l u co se During I n s u l i n I n f u s i o n With and W it h o u t Sodium S a l i c y l a t e i n C o n t r o l and D i a b e t i c R a t s

Animals

Control without sodium s a l i c y l a t e

Total insulin required to obtain h y p o g l y c e m i a (U)

R a t e of fall of glucose (mE/sin)

3.2_+0.9

2 .9 ±0.4

2.1_+0.2.

2.22±0.4*

6.7±1.6

3.3 _+0.6

4.8±0.4*

2.0 ±0.I*

6.4+1.9

2 .9 +_0.6

5.2±1.1"

2.2 _+0.2*

(9)

Control with sodium sallcylate (8) Short-term diabetic w i t h o u t sodium s a l i c y l a t e Short*term diabetic w i t h sodium s a l i c y l a t e

(9)

Long-term d i a b e t i c w i t h o u t sodium s a l l c y l a t e Long-termdtabetic w i t h sodium s a l i c y l a t e

(8)

(9)

(12)

R e s u l t s a r e e x p r e s s e d as mean ± S.D. ( ) - number o f o b s e r v a t i o n s . Sodium e a l i c y l a t e 50 m s / k ~ n w a e i n f u s e d i n t r a v e n o u s l y f o r 90 min. * p < 0 . 0 5 compared w i t h u n t r e a t e d a n i m e l s .

929 ± 272*

-

28 + 10

±

99*

1258 ± 236 *+

124

39 ± 13

765 ± 192"

-

72 ± 36

Short-term Diabetic with Saline (8)

1075 ± 197 *+

60 ± 11

53 ± 13

(8)

Short-term Diabetic with Sodium Salicylate

167 ± 67*

I00 ± 35

Long-term Diabetic with Saline (9)

1003 ± 167*+

65 ± 22

61 ± 18

(12)

Diabetic with Sodium Sallcylate

Long-term

Pre and Post

All results are expressed as means ± S.D. ( ) - number of observations. Sodium sallcylate (50 mS/kg/h) was infused for 90 minutes before insulin infusion and with insulin (50/kg/h) until plasma glucose levels dropped to 30-40 ms/dl. p < 0.01 compared with respective basal. + p < 0.01 compared with respective without sodium sallcylate infusion.

Hypoglycemic

After 90 min of Sodium Sallcylate Pre-hypoglycemia

Basal

(8)

Control with Sodium Salicylate

(pg/ml) A f t e r A c u t e I n f u s i o n o f Sodium S a l i c y l a t e Insulin-Hypoglycemia in Normal and Diabetic Eats

Control with Saline (9)

Plasma Glucagon

TABLE III

u~ O tn

0

OQ

o

m

m

0

~o 00

w

0

0

37 ~ 13"

2 + 2

47 _+ 17 *+

3+1

2 + i

37 _+ 6*+

4_+2

3 + 2

ii ~ 8*

1 ± 1

w i t h Saline (9)

Long-term Diabetic

( ) - number of observations,

28 _+ 5*

1 + 1

(8)

w i t h Sodium Salicylate

Short-term Diabetic

39 + 8*+

4±2

3 + 2

(12)

with Sodium Salicylate

Long-term Diabetic

p < 0.01 compared with respective basal. + p < 0.01 compared wi~h respective wlthou~ s o d i ~ s~llcylate infusion.

Sodium s a l i c y l a t e (50 m~/kg/h) was infused f o r 90 mtn. b e f o r e i n s u l i n i n f u s i o n and i n s u l i n (50/kg/h) u n t i l plasma glucose l e v e l s dropped ~o 30-40 mg/dl.

All results ere expressed as mean ± S,D.

Hypoglycemic

After 90 mln of Sodium Salicylate Pre-hypoglycemia

Basal

(8)

Sodium w i t h Saline Sallcylate (8)

Saline (9)

Short-term Diabetic

Control with

(pmol/ml) Levels After Acute Infuslon of Sodium Salicylate Pre and Post Insulin-Hypoglycemia i n Normal a n d D i a b e t i c R a t s

Control with

Plasma Epinephrine

TABLE IV

o0

o=

o

o

b~

O e~ w

O O~

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Salicylate infusion caused a slgniflcant increase in the basal plasma glucagon levels of control rats, but no such increase in either short-term or long-term diabetic rats (Table IIl). Sallcylate infusion also caused a significant (p increased hypoglycemlc plasma epinephrine secretion in control, short-term and long-term diabetic rats (Table IV). In long-term diabetic rats this increase in epinephrine response to hypoglycemia was essentlally identical to that in control rats in absence of sodium sallcylate. This suggested that acute infusion of sodium sallcylate normalize the impaired epinephrine response to hypoglycemia in long-term diabetic rats. TABLE V E f f e c t s o f I n d o m e t h a c i n on Plasma G l u c o s e , Glucagon and E p i n e p h r i n e L e v e l s D u r i n g I n t r a v e n o u s I n s u l i n I n f u s i o n i n Normal a n d L o n g - T e r m D i a b e t i c R a t s

(80-100 Days Following Streptozotocin Injection) Glucose mg/dl I

Control

(9)

Glucagon pg/ml H

I

Epinephrine pmol/ml H

I

H

163±26

33±3

28+10

929±272

2+2

37±13

134±23

34±3

31+_ 3

1003± 87

3+2

41± 4

34±7

100±35"

167±67"

I±i

11± 8*

32±3

86±37*

399±190.§

3±2

21± 7*§

without Indomethacin

Control (8) with Indomethacin

Long-term (9) 545±57* Diabetic without Indomethactn

Long-term (5)

466±56*6

Diabetic

with Indomethacin

All results I - Initial.

a r e e x p r e s s e d a s means ±SD. ( ) - number o f o b s e r v a t i o n s . H - Hypoglycemic state. I n d o m e t h a c i n (5 m g / k g i . p . was i n j e c t e d

twice at the time of food withdrawal and again after 18 h of fast Just before intravenous infusion of insulin (5 U/kg/h). * p < 0.01 compared with control. § p < 0.01 compared with diabetic without indomethacin.

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I n d o m e t h a c i n t r e a t m e n t s i g n i f i c a n t l y lowered f a s t i n g plasma g l u c o s e l e v e l s i n l o n g - t e r m d i a b e t i c r a t s , b u t n o t i n c o n t r o l r a t s (Table V). I n d o m e t h a c t n d i d n o t a l t e r f a s t i n g and hypoglycemic plasma g l u c a g o n and e p i n e p h r i n e v a l u e s i n control rats. However, s i g n i f i c a n t l y h i g h e r g l u c a g o n and e p i n e p h r i n e r e s p o n s e s t o h y p o g l y c e m i a were o b s e r v e d i n l o n g - t e r m d i a b e t i c r a t s t r e a t e d w i t h Indomethacin. Albeit, unlike sodium sallcylate, Indomethacin did not completely normalize the impaired glucagon and epinephrine responses to hypoglycemia in long-term diabetic rats. DISCUSSION

The a c u t e i n f u s i o n o f sodium s a l i c y l a t e s i g n i f i c a n t l y d e c r e a s e d b a s a l plasma glucose levels in control and diabetic rats. This may be due to the effects of sodium sallcylate on insulin release from pancreatic islet cells or from extrapancreatlc sources such as parotld glands (25). This was not confirmed as plasma insulin levels were not measured in this study. Furthermore, the total amount of insulin required to achieve an identical hypoglycemia was significantly lower in all groups of rats in the presence of sodium sallcylate when compared to those in the absence of sodium sallcylate. This may indirectly suggest that sodium sallcylate could have released endogenous insulin in these rats. The basal plasma glucose levels in control animals were significantly lower than those in diabetic rats at all ages. Therefore, the amount of insulin required to attain an almost identical nadir plasma glucose value in diabetic rats was considerably higher than that for age-matched control rats. This raises a valid question of whether the greater amount of infused insulin in diabetic rats may have suppressed the a-cells, and thereby glucagon release, in the response to hypoglycemia in long term d i a b e t i c rats. However, this is unlikely as the total amounts of insulin required to achieve an identical hypoglycemia in both shortterm and long-term diabetic rats were not different, but there was a significant difference in the glucagon response to Insulln-hypoglycemia. The short-term diabetic rats had normal glucagon response to insulln-hypoglycemia. Furthermore, Unger et al. (26) have suggested that insulln-induced hypoglycemia is a very powerful stimulus to glucagon secretion despite pharmacological levels of circulating insulin. Sodium salicylate, when acutely infused with insulin into normal and shortterm diabetic rats, induced significantly higher glucagon and epinephrine responses to hypoglycemia. The results confirm the findings by Metz et al. (19) in normal human subjects. Acute infusion of sodium sallcylate completely reversed the abnormal glucagon and epinephrine responses to insulin hypoglycemia in longterm diabetic rats. Glugliano et al. (27) have reported similar observations in insulln-dependent diabetic subjects. The modulation, by prostaglandins, of the release of noreplnephrlne from adrenergic nerve endings during stimulation of sympathetic nerves is well documented (26,29). The role of prostaglandlns in the release of acetylcholine is still controversial. In the isolated ileum or colon, prostaglandlns of the E series produce contractions which are reduced by tetrodotoxln or muscarlnlc receptor antagonists, while cholinesterase inhlbitors augment these responses (30-32). Prostaglandln°E reversibly antagonizes negative chronotroplc responses to regal stimulation in the isolated perfused heart, but does not alter similar myocardial effects produced by exogenous acetylchollne (33). These observations

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s u g g e s t t h a t p r o s t a g l a n d i n s i n t e r a c t w i t h soma c h o l i n s r g i c n e u r o n s t o e i t h e r facilitate or inhibit acetylcholine release. I t has b e e n r e p o r t e d t h a t t h e p a r a s y m p a t h e t i c n e r v o u s system p l a y s an i m p o r t a n t r o l e i n t h e g l u c a g o n r e l e a s e I n r e s p o n s e t o i n s u l i n - h y p o g l y c e m i a i n r a t s ( 9 ) . The l a c k o f g l u c a g o n r e s p o n s e to i n s u l i n - h y p o g l y c e m i a o b s e r v e d i n l o n g - t e r m d i a b e t i c r a t a c o u l d be due t o t h e possible deterioration of the parasympathetic nervous system, since acute carbachol infusion could normalize the altered glucagon and epinephrine responses t o i n s u l i n - h y p o g l y c e m i a i n l o n K - t e r m d i a b e t i c r a t s ( 9 ) . T h e r e f o r e , sodium salicylate, perhaps, augments ehollnergic discharge and thereby normalizes the b l u n t e d g l u c a g o n and e p i n e p h r i n e r e s p o n s e s t o i n s u l i n - h y p o g l y c e m i a i n l o n g - t e r m diabetic rats. However, i n d o m e t h a c i n , u n l i k e sodium s a l i c y l a t e , does n o t c o m p l e t e l y n o r m a l i z e t h e g l u c a g o n and e p i n e p h r i n e r e s p o n s e s t o i n s u l i n hypoglycemia i n long-term d i a b e t i c r a t s . T h i s i s n o t u n u s u a l as i n d o m e t h a c i n c a u s e s a d e c r e a s e i n g l u c o s e - s t i e m l a t e d i n s u l i n s e c r e t i o n i n normal human s u b j e c t s ; whereas sodium s a l i c y l a t e , a c e t y l s a l i c y l i c a c i d and i b u p r o f e n have stimulatory effects (34). I n c o n c l u s i o n , a c u t e sodium s a l i e y l a t e a d m i n i s t r a t i o n n o r m a l i z e s t h e abnormal g l u c a g o n and e p i n e p h r i n e r e s p o n s e s t o i n s u l i n - h y p o g l y c e m i a i n l o n g - t e r m d i a b e t i c rats. However, i n d o m e t h a c i n o n l y improves b u t does n o t c o m p l e t e l y n o r m a l i z e t h e s e a l t e r e d responses to i n s u l i n - h y p o g l y c e m i a i n long-term d i a b e t i c r a t s . This s u g g e s t s t h a t endogenous p r o s t a g l a n d i n s may p l a y a p a r t i a l r o l e i n t h e i m p a i r e d counterreEulatory hormones secretion in response to insulin hypoglycemia in longterm diabetic rats.

Sincere thanks are due to Dr. Kenneth Skau for his critical evaluation in the preparation of this manuscript, to David Bruch for his expert technical assistance, and to Ms. Donna Taylor for secretarial help. This work was supported by the Kroc Foundation.

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(1979). 12. J.C. MCLOUGHLIN, J.R. HAYES, K.D. BUCHANAN, J.G. KELLY, GuC 19 632-39 (1978). 13. J. GRIPENBERG, S.-E. JANSSON, V. HEINANEN, E. HEINONEN, J. HYVARINEN, and E.-M. TOLPPANEN, Br. J. Pharmacol. 33 387-93 (1976). 14. B. DELBARRE, E. ARON, G. DUMAS, and A. CHATELUA, Experlentia 27 920-22 (1971). 15. NAKANO, J.: Prostaglandlns, M.F. Cuthbert ed.; H. HernemannMed. Books. London, 1973, p.23. 16. D. GUIGLIANO, R. TORELLA, L. IMPROTA, F. D'ONOFRIO, Diabetic Metab. 189-191 (1978).

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Prostaglandlns and Glucagon

Vol. 44, No. 4, 1989

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