A role for endogenous prostaglandins in defective glucose potentiation of nonglucose insulin secretagogues in diabetics

A Role for Endogenous Prostaglandins in Defective Glucose Potentiation of Nonglucose Insulin Secretagogues in Diabetics

Noninsulin glucose

dependent levels.

diabetics, insulin

the (SSL

glucose on the

of the

subjects control

nonglucose

= 39

different

ratio

significantly nonglucose reversible

improved

These

secretagogues by an inhibitor

(SS

control

that

resulting

No.

and diabetic = 34

normal PG’s may in impaired

i

PGE, inhibited subjects

an insulin

was

significantly

values

in diabetics

4 AU/ml,

studied

responses

less

or sodium and on the

AIR to arginine

SS, however,

Similarly,

by measuring

with

glucose

in diabetics glucose

to these

in the insulin 2 5

14@/ml,

the AIR to arginine defined

in normals

glucose This

= 28

= 80 i

and

as SS

potentiation

potentiation

stimuli.

SS

but not in

(PGE

potentiation than

but did not change defective

defect

in diabetics

was

had no effect

n = 6, p == ns).

a SS-sensitive

in

defective

E, (PGE,)

= 74 k 7 /.d_Jlml. control

was

in the

isoproterenol The

plasma

to glucose

= 18 t 3, n = 9, p < .05)

infusion,

play a role in the insulin

and

the AIR to arginine

(PGE

a role

prostaglandin subjects.

control

for their

responses

play

n = 11, p < .Ol).

f 9 /.dJlml.

of the AIR to arginine

dependent diabetes mellitus ONINSULIN (NIDDM) is characterized by defective insulin responses to both glucose and nonglucose secretagogues. While the acute insulin response to an intravenous glucose pulse in NIDDM is either totally absent’ or paradoxical,’ there is relative preservation of the acute insulin response to arginine, isoproterenol, or secretin.‘,‘.” However, it has been recently demonstrated that the magnitude of the insulin response to nonglucose stimuli is dependent upon the circulating plasma glucose level in both normal and diabetic subjects, with elevations in plasma glucose level potentiating the acute insulin responses to these stimuli.6 Thus, when the diabetics’ apparently normal insulin responses to nonglucose secretagogues are examined in the light of the expected potentiating effect of their elevated plasma glucose levels, they are seen to be subnormal. This defect can be further evaluated by examining the ability of glucose to potentiate nonglucase stimuli. Glucose potentiation can be quantified by measuring the effects of induced changes in prestimulus plasma glucose levels on the insulin responses to nonglucose stimuli. With this technique, it has been shown that the ability of glucose to potentiate nonglucase signals is impaired in diabetics.’ Defective recognition of glucose signals may therefore be responsible for the abnormal insulin responses to both glucose and nonglucose insulin secretagogues in diabetics. Previous studies from this laboratory have suggested that endogenous prostaglandins may be involved in mediating defective glucose recognition in diabetics. Infusion of PGEz in normal human subjects 30.

control

also

to arginine

n = 8, p = ns), suggesting

and one after

potentiation

of either

(AIR’s)

are subnormal insulin

defect

in

of the AIR to is partially

of PG synthesis.

N

Metabokm, Vol.

might

k 5 AU/ml,

(SS = 38

but not in normal

toward

endogenous

prostaglandins

normal

4 /.dJ/ml,

Conversely,

potentiation

Glucose

in diabetics

f

= 21 + 4 pU/ml,

one before

potentiation

suggest

= 39

abnormal

of infusions

= 37

that

in the

responses

in both

control

in diabetics

in diabetics.

glucose.

effects

insulin

secretagogues

implicated

whether

to arginine

pulse

glucose,

been the

acute

12 ~U/ml.

of SS on glucose

glucose

findings

i

n = 7, p < .05),

of plasma

AAIRlAprestimulus

examined

on the

subjects

stimuli

+ 7 AU/ml,

levels

We

and R. Paul Robertson

to nonglucose

have

to explore

response

(SS = 61

(SS = 19 + 4 /.dJ/ml,

to these

normals.

insulin

in normal

n = 5, p = nsj. The effect the

stimuli. inhibitor,

A. Metz,

responses

performed

the AIR to an isoproterenol

response

at two

insulin

Stewart

prostaglandins

to nonglucose a PG synthesis

AIR to arginine

/,&j/ml.

have was

by SS in diabetics

augmented normal

study

potentiation

augmented

R. McRae,

endogenous

present

responses

salicylate

diabetics

Since

John

11 (November).

1981

inhibits the acute insulin response to an intravenous glucose pulse and impairs glucose disposal.’ Conversely, in diabetics, infusion of sodium salicylate. a prostaglandin synthesis inhibitor,x augments basal insulin levels, partially restores the acute insulin response to glucose in a glucose dose-dependent manner, and improves subnormal glucose disposal.‘.’ It is important to emphasize, however, that many in vitro studies have shown stimulation of insulin secretion by prostaglandins.“.“,” It is not yet clear whether these divergent findings in vivo and in vitro are necessarily contradictory or, if so, which ones are more relevant to human beta cell function. The present study investigates whether endogenous prostaglandins also play a role in the defective glucose potentiation of insulin responses in diabetics to two nonglucose insulin secretagogues, arginine and isoproterenol, by examining the insulin responses to these From the Division of Clinical Pharmacology, University of Washington School of Medicine, and the L’eteruns .-ldministration Keceivedfir publication December 23. I 9X0. Supported in part bv the U.S. Veterans .4dmtnistration Ke.tearch and Education Program. This work was presented in part at the Wc,.stern Swiet! Jtir Clinical Research, Curmel. CalfJornia. February Y, I97Y. ut the Fourth International Prostaglandin Conferewe, Washington, D-C... Mall 31, 1979. and at the 40th .4nnual Meeting qf the American Diabetes Association. Washington. D.C., June 16. I YNO. Address reprint requests to R. Paul Robertson. ,M.D.. Dir$vrorr 01 Clinical Pharmacology University of Washington .Medical School. Seattle, Washington 98195. c 1981 by Grune & Stratton, Inc. 0026 ~049_5/8//301 I -000.5$02.00~0

1065

1066

McRAE, METZ, AND ROBERTSON

secretagogues during infusions of a prostaglandin synthesis inhibitor in both normal and diabetic subjects. The relative sensitivity of arginine-induced insulin secretion to the inhibitory effects of an exogenous prostaglandin E, infusion is compared in normal and diabetic subjects. Possible alterations in prostaglandin metabolism in diabetics are evaluated by measurements of plasma levels of PGE and the primary circulating metabolite of PGE, (13,14-dihydro,l5 keto-PGE,) in normal and diabetic subjects, both in the control state and during infusions of PGE,. Finally, this study investigates whether endogenous prostaglandins may be involved in the abnormal glucose potentiation of these nonglucose insulin secretagogues by examining the ability of a prostaglandin synthesis inhibitor, sodium salicylate, to improve glucose potentiation in both diabetics and normal subjects. MATERIALS

AND

METHODS

Subiects _I

Fourteen normal human male volunteers were studied. These subjects had no personal or immediate family history of diabetes mellitus and were using no medications. Their ages ranged from 1940 yr (mean e SE = 27 + 2 yr). Their weight, expressed as percent of ideal body weight (Metropolitan Life Insurance Co. Tables, 1959). ranged from 96%-142% of ideal body weight (mean = 108 f 5%). Fasting plasma glucose ranged from 81 to 108 mg/dl (mean 93 t 3 mg/dl). Twenty-two male diabetic subjects were studied who had no history of ketoacidosis and who managed their diabetes with either diet or diet plus an oral hypoglycemic agent. All oral hypoglycemic agents were discontinued for l-2 wk prior to any study. All diabetics had fasting hyperglycemia at the time of study (fasting plasma glucose levels = 200 + 15 mg/dl). Ages of the diabetic subjects ranged from 37-66 yr (mean = 57 + 2 yr) and weight from 102%156% of ideal body weight (mean = 13 1 t 3%).

Study Protocol Studies were conducted with subjects in the supine position after a I2 hr overnight fast. After informed consent was obtained, a scalp vein needle was placed in the antecubital vein of each arm and kept patent with a slow infusion of 0.9% sodium chloride. Blood samples were obtained through a three-way stopcock from one intravenous line and drugs were administered through the other IV line. After a 30 min wait, blood samples for plasma glucose and insulin determinations were obtained every 5 min over a 15 min period. Basal insulin was calculated as the mean of these four values. Insulin secretion was then stimulated by a rapid intravenous pulse of 2 grams of arginine hydrochloride, IO%, or 2 mcg of isoproterenol, both given in less than 5 sec. These doses have been previously shown to provide a half-maximal stimulus for insulin secretion in both normal and diabetic subjects. ‘J’,” Blood samples were obtained at 2, 3, 4, 5, 7, 10, and 15 min after the arginine or isoproterenol pulse and assayed for plasma levels of glucose and insulin. The acute insulin response (AIR) was defined as the mean of the three peak plasma insulin increments over the prestimulus level of insulin, obtained between 2 and 5 min after the pulse and expressed as percent of basal insulin. The prestimulus plasma insulin level for the initial stimulus pulse was the basal insulin level, calculated as described above. For subsequent pulses, the last plasma insulin level

obtained less than two minutes before the pulse was used as the prestimulus plasma insulin level for determining the AIR. The total insulin response to each stimulus pulse was also determined by calculating the area under the insulin curve above the prestimulus insulin level from t&l5 min after the stimulus pulse. The effect of a prostaglandin synthesis inhibitor on arginine or isoproterenol induced insulin secretion was assessed by comparing the acute insulin responses to each of these stimuli before and during an infusion of sodium salicylate. Fifteen min after the first stimulus, an infusion of sodium salicylate was begun at the rate of 40 mg/min and was continued for 1 hr prior to and during the second stimulus pulse. In previous studies, this infusion rate of sodium salicylate for one hour resulted in serum salicylate levels of 19 t I mg per dl.9 Blood samples for plasma insulin and glucose determination were obtained during a control period, every 15 min during the salicylate infusion, and at 2, 3, 4, 5, 7. 10, and I5 min after each stimulus. It has been previously shown that sequential pulses of arginine or isoproterenol given as often as every 30 min during a saline infusion elicited AIR’s that are not significantly different.‘J*‘4 The effect of exogenous PGE, on arginine-induced insulin release was assessed by infusing PGE, at the rate of 10 mcg per min intravenously for 30 min prior to a subsequent arginine pulse. In previous studies, we observed that this is the maximum infusion rate that can be given without producing symptoms or blood pressure changes but which achieves a significant rise in systemic plasma PGE levels.” Blood samples for plasma insulin and glucose determination were obtained during a control period, at 5. 10, 15. and 30 min during the PGE, infusion. and at 2, 3, 4, 5, 7, 10. and 15 min after each stimulus pulse. In additional studies, to compare the plasma levels of PGEl and the primary circulating PGE, metabolite, 13,14-dihydro,l5-keto PGEz(PGE,) achieved by PGE, infusion, the PGE, infusion rate was adjusted to 6 mcg per M2 body surface area for 35 min in both normal and diabetic subjects and plasma levels of PGE, and PGE, were measured. Glucose potentiation of the AIR to arginine was determined by measuring the AIR to a 2 g IV arginine pulse as described above at ambient plasma glucose levels and again after the prestimulus plasma glucose had been lowered by an insulin infusion. Infusions of crystalline zinc insulin (Eli Lilly Corp., Indianapolis, IN) were administered to the normal subjects at the rate of 0.33 mLJ/kg/min by constant infusion pump for 20-30 min while bedside plasma glucose levels were measured by a Beckman glucose analyzer (Beckman Instrument Co., Irvine CA). A higher infusion rate of insulin (1 .O mU/kg/min) was used in diabetic subjects for a 30-60 min period. These infusion rates have been previously shown not to induce significant elevations in plasma catecholamine levels and do not inhibit insulin secretion when plasma glucose is maintained at a constant level by a glucose clamp technique.” After stopping the insulin infusion, a 30 min equilibration period was allowed to permit plasma insulin levels to fall to preinfusion levels before a second_ arginine pulse was administered at the lower plasma glucose level. Venous blood samples for determination of glucose and insulin levels were obtained at IO min intervals during the insulin infusion and recovery period, and plasma catecholamines were measured before and 30 min after the insulin infusion was discontinued. Glucose potentiation (GP) was calculated as AAIR/AG, or the ratio between the difference in the AIR to arginine at basal and at lowered prestimulus plasma glucose levels divided by the change in the prestimulus plasma glucose level.6 This relationship has been shown previously by Halter, et al. to be linear over the range of plasma glucose observed in these studies for isoproterenol-induced insulin secretion.6 The effect of sodium salicylate on glucose potentiation in both diabetic and normal subjects was assessed by comparing glucose potentiation measured in the above fashion on a control study day to

PGE, GLUCOSE POTENTIATION

1067

AND DIABETES

DIABETICS

glucose potentiation

measured on a second study day after a sodium salicylate infusion had been given. On the second study day, venous blood samples for plasma insulin and glucose determination were measured over a IS min basal period and every 15 min during the I

“=

11

ARGININE. 20 I”

s f

hr infusion of sodium salicylate (40 mg/min) prior to measuring glucose potentiation in the above described manner. The glucose potentiation ratios (aAlR/AG) in the presence and absence of sodium sallcylate were compared. Because of the relatively long serum half-life (15-30 hr) of salicylate at the concentrations achieved in this study. it was not necessary to infuse salicylate continuously thrl)ughout the study to maintain effective serum levels.

ti 8

225

0 3 0

20°

1

m@aa

175

; Y d

125 1

Analytical

Methods

Blood samples for glucose and insulin assay were collected in tubes containing EDTA (I mg/ml) and kept on ice until centrifuged at 4°C. and plasma was frozen for later analysis. Plasma glucose was measured by an AutoAnalyzer ferricyanide method.” Plasma insulin was measured by a modification of the method of Morgan and Lazarow.” Serum salicylate was determined by a commercial kit.‘* Plasma PGE was measured by radioimmunoassay after ethyl acetate extraction and silicic acid column chromatography. as described previously.9 Plasma levels of the major circulating metabolite of PGE,; 13.14-dihydro-15.keto-PGE, (PGE,), were measured by a modification of the radioimmunoassay of Levine20 with modifications and characteristics as previously described.” Blood samples for plasma catecholamines were collected in EGTA and glutathione and were measured in the laboratory of Dr. Jeffrey Halter by the single isotope enzymatic assay of Peuler and Johnsor? with characteristics as previously described.13

Statistical

0

Fig. 1.

40

40

mS/mln

60

I”

SO

100

The

response

intravenous

pulses

of

intravenous

infusion

of plasma

arginine

of sodium

(2

insulin

and plasma

grams)

salicylate

before

and

glucose

to

during

an

in diabetics.

Methods 0.7 mg/dl, plasma insulin levels rose significantly (p < .Ol) to 41 I 8 FU/ml in association with a fall in plasma glucose to I78 + I9 mg/dl ( p -. .OI ). The AIR above the new prestimulus insulin level to a second arginine pulse was significantly augmented (AIR = 61 + 12 pU/ml, n = II, p ‘- .Ol). despite the significant (p -z .Ol) fall in prestimulus plasma glucose level of 15 -t 4 mg/dl. The total plasma insulin response to the arginine pulse (insulin area above prestimulus insulin, 0-l 5 min was also augmented by salicylate (208 + 30 pU/ml - min control versus 354 2 65 pU/ml - min salicylate. p -_ .OOS). In contrast, in normal subjects. a salicylate infusion failed to augment the insulin response to arginine (Table I). After 60 min of salicylate infusion, the

RESULTS

Thr Eflect o$Sodium to Arginine

Salicylate

Infusion

on the AIR

The effect of a sodium salicylate infusion on the insulin response to arginine in II noninsulin dependent diabetics is shown in Fig. I. Mean fasting plasma glucose was 193 +- 20 mg/dl and mean basal plasma insulin level was I9 ir 3 pU/ml. The control arginine pulse elicited an acute insulin response (AIR) of 37 c 5 pU/ml. During a 60 min infusion of sodium salicylate, which resulted in serum salicylate levels of 15.4 + 1.

SALICVLATE,

20

0

MINUTES

Statistical analysis was performed by Student’s t test, paired t test. and Wilcoxon rank sum test. Results are reported as mean i standard error of the mean.

Table

SODIUM

I -20

Comparison

of the Acute

Insulin

Responses

to Arginine

and lsoproterenol

of Sodium

in Normal

Before Sodwm Salwlate Prestlmulus mgldl

Before

and During

During Sodurn Sahcylate

Basal

GlUCOSe St3mulus

Subjects

an Infusion

Salicylate

Prestimulus

~-

Prestlmulus

IRI

AIR,

Glucose

IRI

@U/ml

*U/ml

mg/dl

kLU/Fll

34 + 4

97 t 5

19 f 2

39 i- 4

21

97

16

19

AIR, &/ml -__

Argmme 2q IV n =. 6

lsoproterenol 2 me9 IV n-8

99

* 5

12

i

95 + 4

15

k 2

2

?4

+ 5

i

2

t4

1068

McRAE,

rise in plasma insulin from 12 k 2 to 19 ct 2 pU/ml was significantly less than that seen in the diabetics (p < .05). The accompanying small (2 mg/dl) but significant (p < .05) fall in plasma glucose was also less than that seen in diabetics (p < .05). The AIR to a second arginine pulse (39 k 4 pU/ml) given after 60 min of the salicylate infusion was not significantly different from the control AIR (34 * 4 pU/ml) to arginine in these normal subjects. The total plasma insulin response was also not augmented by salicylate in normal subjects (232 k 22 pU/ml . min control vs. 257 k 30 pU/ml . min salicylate, p = ns). The EJect of Sodium Salicylate Infusion Acute Insulin Response to Isoproterenol

on the

In the diabetic subjects, sodium salicylate also augmented the insulin response to a second nonglucose secretagogue, isoproterenol (Fig. 2). Nine diabetic subjects had a mean fasting plasma glucose of 206 * 21 mg/dl and a mean basal insulin level of 19 * 4 pU/ml. A 2 mcg isoproterenol pulse elicited on AIR of 18 k 3 pU/ml. After 60 min of salicylate infusion, plasma insulin rose to 43 -t 1 1 &/ml (p < .Ol) while plasma glucose fell to 195 * 18 mg/dl (p < .05). The second AIR above the new prestimulus insulin level to a second isoproterenol pulse was significantly augmented (AIR = 38 t 9 pU/ml, n = 9, p < .05) during the salicylate infusion. A significant (p < .Ol) increase in the total insulin response to isoproterenol also occurred during salicylate infusion (I 20 + 19 DIABETICS n.9 ISOPROTERENOL

ISOPROTERENOL

2PWlV

2PS IV ii 250,

0

SODIUM -20

0

SALICVLATE,

20

40

40

SO

mS/mln

IV

80

100

MINUTES

Fig. 2. intravenous during

The

response

isoproterenol

a sodium

salicylate

of plasma pulses infusion

insulin (2

and plasma

micrograms)

in diabetics.

glucose before

to and

METZ,

AND

ROBERTSON

~U/ml- min control vs. 257 f 62 ~U/ml- min salicylate). In normal subjects, as seen with arginine, sodium salicylate failed to augment the acute insulin response to isoproterenol (Table 1). During the salicylate infusion, the small increase in plasma insulin level was again significantly less (p < .02) than that seen in the diabetics, and there was no significant change in the prestimulus plasma glucose level after the salicylate infusion. The AIR to an isoproterenol pulse during the salicylate infusion (19 + 4 pU/ml) was not significantly different from the insulin response during the control period (21 + 4 pU/ml). Similarly, there was no augmentation of the total insulin response to isoproterenol during salicylate in normal subjects (134 +- 29 pU/ml.min control versus 125 t 27 ~U/ml~min salicylate, p = ns). Thus, sodium salicylate augmented the insulin responses to arginine and isoproterenol specifically in diabetics but not in normal subjects. The Eflect of a PGEz Infusion Response to Arginine

on the Acute Insulin

To evaluate whether these differences in salicylate sensitivity between diabetics and normals might reflect an underlying increased sensitivity of the beta cell in diabetics to endogenous prostaglandins, we compared the ability of a PGE, infusion to modify the insulin response to arginine in these two groups. The insulin responses to 2 gram arginine pulses were measured before and during an intravenous infusion of PGE, at the rate of 10 mcg/min, in seven diabetic subjects (Fig. 3). There were no significant changes after 30 min of PGE, infusion in plasma insulin levels (preinfusion = 13 -t 2 pU/ml, post infusion = 13 * 2 pU/ml, n = 7, p = ns) or in plasma glucose levels (preinfusion = 21 1 -t 38, post infusion = 200 i 43 mg/dl). There was significant inhibition of the AIR to arginine during the PGE, infusion (AIR-control = 39 + 7 pU/ml, AIR-PGE2 infusion = 28 + 5 pU/ml, n = 7, p -C .025) in these diabetic subjects. Total insulin areas were reduced during PGE, in 6 of 7 diabetics, but the differences for the entire group were not significant (222 * 41 pU/ml.min control, 169 2 23 PGEz, p = ns). We have previously shown’ that a 10 mcg/min infusion of PGEz in normal subjects failed to significantly inhibit the insulin response to a 2 gram arginine pulse (AIR-control = 80 * 14 &J/ml. AIR-PGE = 74 + 7 pU/ml, n = 5, p = ns). Similarly, total insulin response to arginine was not diminished during PGEz infusion in normal subjects (480 +_ 92 pU/ml. min control, 448 + 55 pU/ml- min PGE,, p = ns). In additional studies, plasma levels of PGE,, the major circulating metabolite of PGE, (13,14-dihy-

PGE, GLUCOSE POTENTIATION

1069

AND DIABETES

DIABETICS

(normals = 12,000 + 4,577, 1,547 pg/ml, n = 4, p = ns). peripheral venous plasma PGE infusion were not significantly and diabetics (normals = 213 ics= 192t42pg/ml,n=4,p=ns).

n=7

ARGININE

ARGININE

2g IV ?

275,

E

2g IV

diabetics = I 1,094 2 Additionally, the peak levels achieved by the different in normals + 108 pg/ml, diabet-

1

Thr Effect of Sodium Salicylate on Glucose

Potentiation of the Acute Insulin Response lo Arginine To assess whether the augmentation of the insulin response to nonglucose secretagogues in diabetics by sodium salicylate might be mediated by improvements in defective glucose potentiation of these secretagogues, glucose potentiation of the insulin response to arginine was measured in the presence and in the absence of a salicylate infusion in both normal and diabetic subjects. The glucose potentiation study in six diabetic subjects is shown in Fig. 5. On the control study day, shown in the upper portion of the figure. the AIR to an arginine pulse was measured before and after the plasma glucose was lowered by an insulin infusion. Mean basal plasma insulin levels were 14 + 3 pU/ml and mean fasting plasma glucose levels were 204 t 36 mg/dl. The control argininc pulse elicited an AIR of 32 * 4 FU/ml. After the insulin infusion and equilibration period. the plasma glucose level before the second arginine pulse fell to I29 t 30 mg/dl, with a A prestimulus plasma glucose level of 74 i 24 mg/dl. The second arginine pulse at this lowered plasma glucose level evoked an AIR which was signihcantly smaller than the control AIR (AIR = 22 ‘-c 4 dJ/ml, p c .Ol). The decrement in the acute insulin response, AAIR, divided by the decrement in the prestimulus plasma glucose level, Xi, provides a ratio,

MINUTES Fig. 3. intravenous

The

rasponse

arginine

of plasma

pulses

before

insulin

and plasma

and during

glucose

an infusion

to

of PGE,

in diabetics.

dro, 1Sketo-PGE,), were measured in the control state and during an infusion of PGE, at 6 mcg/M* body surface area/min for 35 min. There were no significant differences in basal levels of PGE, between normal and diabetic subjects as shown in Fig. 4 (PGE, = 104 _t 76 pg/ml normals, 90 t 36 pg/ml diabetics. n = 4. p = ns). Peak PGE, levels during PGEz infusion were also not significantly different I6000 z j$l4000 ; i

12000

0 &

,’

10000

,’ ,

A *

r’

8000

,’

P e I

6000

E P n2

4000

2000

9 a’ Fig.

4.

Plasma

tabolite

of PGE,.

infusion

of PGE,

levels

of the

13,14-dihydro.15 in diabetics

major keto

and normal

circulating PGE,.

during

subjects.

f

--=-A

0

mean

-20

-10

0

IO

20

30

MINUTES

40

50

60

70

80

McRAE, METZ, AND ROBERTSON

1070

DIAIIIETICS n=5 ANGININE 22 I”

s

250

s_ 00 SE

150

3y

200 I

u__mm

0 INSULIN I” -20

0

20

40

40

MINUTES

4,RGININE 20 I”

00

, 100

AROININE 25 I”

+

4

25-

oOOIUY

40

0

,NOULIN I”

5ALKxLAlE.4Dnym

20

40

50

50

100

120

YINUTES

AAIR/AG, which is an index of glucose potentiation of the AIR to arginine. For these diabetic subjects, mean AAIR/AG = 0.18 + 0.07. On a second study day, shown in the lower portion of the figure, mean basal plasma insulin was 13 + 4 VU/ml and mean fasting plasma glucose level was 211 + 44 mg/dl. An infusion of sodium salicylate resulted in a mean serum salicylate level of 16.2 * 1 .O mg/dl and was accompanied by a significant rise (p < .05) in the plasma insulin level to 33 k 11 pU/ml and a small but insignificant fall in mean plasma glucose level to 202 + 44 mg/dl. The AIR to an arginine pulse after the salicylate infusion was 47 k 8 &J/ml. After an insulin infusion and an equilibration period, the AIR to arginine was again measured at the lowered prestimulus plasma glucose level (mean prestimulus

140

I $50

Fig. 5. The effect of sodium salicylate on glucose potentiation of the acute insulin response (AIR) to arginine in diabetics. On the control study day (upper portion of the figure), the insulin response (solid line) to arginine is measured before and after plasma glucose (dashed line) was lowered with an insulin infusion. and the glucose potentiation ratio, AAIRI AG. was determined. On a second study day Bower portion of the figure), an infusion of sodium salicylate was given (serum salicylate levels = 16.2 + 1 mg/dl end 12.6 ? 1 mg/dl respectively. before the two subsequent arginine pulses). The glucose potentiation ratio, AAIRIAG, was again determined by measuring the AIR to arginine before and after plasma glucose was lowered with an insulin infusion.

plasma glucose = 126 + 31 mg/dl, AG = 72 k23 mg/dl) and was significantly smaller than the control AIR (mean AIR = 27 + 4 pU/ml, p < .Ol). The mean glucose potentiation ratio, AAIR/AG, after salicylate was 0.42 t 0.14, significantly greater than the control glucose potentiation ratio (p < .OS). Serum salicylate level before the second arginine pulse was 12.6 + 1 mg/dl. Similar glucose potentiation studies in the absence and in the presence of sodium salicylate were conducted in four normal subjects (Fig. 6). On the control study day shown in the upper portion of the figure, basal plasma insulin was 10 -+ 3 pU/ml and fasting plasma glucose level was 89 + 5 mg/dl. The control AIR to arginine was 32 + 5 pU/ml. An insulin infusion lowered the prestimulus plasma glucose level

PGE, GLUCOSE POTENTIATION

1071

AND DIABETES

20

0

-20

.O “MUTES

Fig.

6.

The

glucose response

(AIR)

Plasma nine

glucose

mg/dl. quent

levels

arginine

tion

ratio

the

AIR

glucose

was

determined.

was to

was

of

salicylate _

17

arginine lowered

before

figure),

given and

the

two

glucose

determined with

an

On a second

3 mgldl The

plasma with

potentiation

the

was

before

pulses). again

f

to argi-

after

glucose

portion

respectively,

subjects.

lowered

the

of sodium

salicylate

was

and

on

insulin

responses

and

(lower

salicylate acute

in normal

before

line)

AAWAG. day

the

lines)

measured

infusion,

infusion

of

(solid

(dashed

insulin study

of sodium

to arginine

insulin

were

ratio.

effect

potentiation

and

an

(serum 14

+ 2

subse-

potentia-

by measuring after

an insulin

plasma

infusion.

to 71 + 5 mg/dl (LIG = 18 + 4 mg/dl). The second arginine pulse at the reduced plasma glucose level elicited an AIR of 17 + 4 pU/ml, significantly less than the control AIR (p < .05). The mean glucose potentiation ratio, IAIR/AG, was 0.83 t 0.14, significantly greater than in the diabetics (p < .025). On the second study day, shown in the lower part of the figure, mean basal insulin was 8 + 4 FLU/ml and mean fasting plasma glucose was 87 2 2 mg/dl. The salicylate infusion produced a mean serum salicylate level of I7 k 2 mg/dl and was accompanied by an elevation of mean plasma insulin (12 t_ 4 pU/ml) and a minimal change in mean plasma glucose level (86 -e 2 mg/dl). The initial AIR to arginine was 26 * 8 pU/ml. After an insulin infusion, the mean prestimulus plasma glucose level fell to 71 i 4 mg/dl (AG = 15 i 4 mg/dl), and a second AIR to arginine at the reduced plasma glucose level was 17 f 3 /*U/ml. Glucose potentiation, AAIR/AG, in the presence of sodium salicylate was 0.67 -c 0.15, which was not significantly different from control. The serum salicylate level was 14 + 2 mg/dl before the second arginine pulse. The insulin infusions and the subsequent falls in

YINUTES

plasma glucose did not produce significant rises in plasma norepinephrine (NE) and epinephrine (E) (Diabetics: basal NE = 273 -+ 42 pg/ml, NE after insulin = 349 -t 34 pg/ml. n =- 12,~) = ns; basal E = 45 + 9 pg/ml, E after insulin = 55 i- 7, n = 12. p = ns. Normals: basal NE = 200 1 25 pg/ml, NE after insulin = 239 + 39 pg/ml. n = 8. p = ns: basal E =

I

I

=ns

,.i PC.05

Fig.

7.

Comparison

of

the

effect

glucose

potentiation

(AAIRIAG)

of the

arginine

in diabetics

and normal

subjects.

of

sodium

acute

insulin

/

salicylata response

on to

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McRAE. METZ, AND ROBERTSON

30 + 3 pg/ml, E after insulin = 76 k 31 pg/ml, p = ns). The results of the glucose potentiation studies are summarized in Fig. 7. Glucose potentiation was markedly reduced in the diabetic subjects (0.18 + 0.07) compared to normal (0.83 +- 0.14, p < .025). Moreover, sodium salicylate significantly augmented glucose potentiation in the diabetics (0.42 f 0.14 salicylate versus 0.18 + 0.07 control, p < .05) but had no effect on glucose potentiation in the normal subjects (0.67 f 0.15 salicylate vs. 0.83 -c 0.14 control, p = ns).

DISCUSSION

These studies demonstrate that in NIDDM insulin responses to the nonglucose secretagogues, arginine and isoproterenol, which are subnormal relative to the elevated plasma glucose levels, can be restored toward normal by a prostaglandin synthesis inhibitor,’ sodium salicylate. Salicylate was ineffective, on the other hand, in augmenting insulin secretion to these secretagogues in normal subjects. Basal insulin secretion was augmented by sodium salicylate in both diabetics and normal subjects, although to a significantly greater extent in the diabetics. These findings are in accord with those of Giugliano et a1.,24 who found that oral aspirin therapy for three days in normal subjects increased basal insulin levels and the insulin levels during a 30 min arginine infusion but did not increase the arginine-induced increments in plasma insulin over the elevated basal insulin levels. In the present study, salicylate’s ability to augment the insulin responses to arginine and isoproterenol in diabetics, but not in normal subjects, suggests that endogenous prostaglandins may be contributing toward the subnormal insulin responses to these stimuli in diabetics. We have previously demonstrated that sodium salicylate, at concentrations similar to those achieved in the current study, potently inhibits PGE synthesis in monolayer cultures of neonatal rat pancreas. Concomitantly, sodium salicylate appeared to augment glucose recognition and increase insulin secretion in these pancreatic cultures.25 In the human, Hamburg has reported decreased urine PGE, metabolite levels following oral sodium salicylate administration.8 Because basal levels of circulating PGE, and PGE, metabolites are low and approach the sensitivity of most assays, it has not been possible to date to show lowering of circulating levels of these compounds by prostaglandin synthesis inhibitors. However, the findings that another prostaglandin synthesis inhibitor, ibuprofen, reproduces these salicylate effects in the rat pancreatic cultures26 and in vivo in the human27

suggest that salicylate’s effects are related to prostaglandin synthesis inhibition rather than due to a prostaglandin-unrelated effect of the drug. Also supporting the contention that the salicylate effect is prostaglandin-related are the present studies showing that an exogenous infusion of PGE, produced a reciprocal inhibitory effect on arginine-induced insulin release, specifically in the diabetics and not in the normal subjects. These studies have also demonstrated that diabetics have an increased susceptibility of the stimulated beta cell to the inhibitory effects of an exogenous PGE, infusion, compared to normal subjects. The present studies demonstrate no evidence for a generalized overproduction of PGE, by diabetics in the basal state. Basal plasma levels of the major circulating PGE, metabolite, 13,14-dihydro- 15-keto-PGE,, in the diabetics were no different from those of normals. Moreover, during the PGE, infusions, there were no differences in the plasma levels of PGE, or PGE, achieved or in the rates of disappearance of PGE, to suggest either defective degradation of PGE, or impairment of the further metabolism or excretion of PGE, in the diabetics. These studies do not, however, rule out a local overproduction of prostaglandins by the diabetic which might not produce measurable pancreas, changes in peripheral venous prostaglandin levels. On the other hand, the selective effects of both infused PGE, and sodium salicylate on arginine-induced insulin secretion in the diabetics, suggest an increased sensitivity of the stimulated beta cell in diabetics to both exogenous and endogenous prostaglandins. This increased sensitivity may be islet-specific as there were no significant differences in the hemodynamic responses to the PGE, infusions in diabetics compared to normals (data not shown). It is possible that some of the differences found in the responses of the diabetics and normals to sodium salicylate and PGE2 may relate to differences between the two groups in age and weight. The diabetic group was significantly heavier and older than the normals. Against this interpretation of the data is the qualitative nature of the differences between the normal and diabetic groups (no effect of PGE, or salicylate in the normals; a significant effect in the diabetics). It should be noted that previous studies of the effects of prostaglandins and prostaglandin synthesis inhibitors on islet function have produced conflicting data, as we have discussed in a recent review.** All in vivo studies have consistently shown that exogenous PGE inhibits glucose-induced insulin secretion.7.“9-35 Augmentation of insulin responses in vivo has been consistently found for all prostaglandin synthesis inhibitors 7.24.27.33.36.37 except for indomethacin, which is

PGE. GLUCOSE POTENTIATION

usua]]y27.3”-41

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AND DIABETES

but not always4’,43 inhibitory. Indomethacin has other, non-prostaglandin related effects on cyclic AMP-dependent protein kinase44 and on calcium flux across membranes4’ which may be expected to alTect insulin secretion. In contrast, most in vitro studies have shown that exogenous PGE stimulates insulin secretion.“-” Other in vitro studies, however, have found either no effect46A9 or an inhibitory effect’5.r0 of PGE on insulin secretion. The reasons for these discrepant findings are unclear. Differences in tissue preparation (isolated perfused pancreas vs. perifused isolated islets, for example), differences in glucose concentration in the media, and differences in the types and concentrations of prostaglandins and prostaglandin synthesis inhibitors are probably important factors. This present study does demonstrate that a likely mechanism by which sodium salicylate augments, and presumably by which PGE, inhibits, the insulin response to nonglucose secretagogues in diabetics is through changes in the ability of circulating glucose to potentiate the insulin response to these stimuli. This glucose potentiating effect normally results in increased insulin responses to nonglucose secretagogues when plasma glucose is raised and decreased insulin responses when plasma glucose is lowered. In the present study, glucose potentiation of arginineinduced insulin release, estimated by measuring the change in the acute insulin response to arginine at two ditrerent levels of plasma glucose, was significantly less in diabetics, similar to that previously shown for the glucose potentiation of insulin responses to isoproterenol.h Defective glucose potentiation may therefore account for previous findings3,‘3,‘4 of similar insulin responses to nonglucose secretagogues in diabetics and normal subjects despite the diabetics’ elevated plasma glucose levels. which would be expected to produce augmented insulin responses to these non-glucose stimuli. A 1 hr infusion of sodium salicylate improved glucose potentiation of arginine-induced insulin secretion toward normal in the diabetic subjects. Sodium salicylate, perhaps by improving beta cell recognition of glucose signals, improved glucose potentiation, allowing the elevated plasma glucose levels in the diabetics to augment more effectively the insulin responses to these nonglucose stimuli. In contrast, in the normal subjects, a salicylate infusion was ineffective in augmenting insulin release to arginine or isoproterenol and did not change glucose potentiation of these aecretagogues. The lack of effect of sodium salicylate on glucose potentiation in the normal subjects suggests that glucose potentiation of nonglucase signals may be already maximally operative in nondiabctics. Although the sodium salicylate infusion

was discontinued before the arginine pulses were given to avoid salicylate toxicity. the circulating levels of salicylate remained at a plateau of 13-l 7 mg/dl throughout the experiments due to its long circulating half-life. The changes in the prestimulus plasma insulin levels during the salicylate infusions are also consistent with the changes in glucose potentiation of arginineinduced insulin secretion. Diabetics, with elevated plasma glucose levels, had a greater increase in prestimulus plasma insulin levels during the salicylate infusion than did normals. If salicylate is augmenting the sensitivity of the beta cell to glucose signals, then these elevated prestimulus plasma insulin levels during the salicylate infusion may in fact be a stimulated state of insulin secretion during which the diabetic beta cell may have an increased ability to recognize and respond to the elevated plasma glucose levels. This increased sensitivity to glucose may then allow the elevated plasma glucose levels to more normally potentiate the insulin responses to nonglucose secretagogues such as arginine and isoproterenol. In normal subjects, the salicylate infusion had a smaller effect upon prestimulus plasma insulin levels and did not augment glucose potentiation, perhaps reflecting near-maximal sensitivity of the normal beta cell to the normal plasma glucose levels. It seems likely then that defective recognition of glucose signals by the diabetic beta cell may be responsible for both the absolute inability of glucose and the diminished ability of nonglucose stimuli to elicit acute insulin secretion. These findings are compatible with the “glucoreceptor hypothesis” which postulates a specific defect in a glucoreceptor on the diabetic pancreatic beta cell.” Previous studies suggest a role for endogenous prostaglandins as mediators of defective glucose recognition in diabetics. 7.9,28,33S36.37 The present study provides evidence that cndogenous prostaglandins may also mediate defective insulin responses to nonglucose secretagogues by reducing the beta cell’s ability to recognize or process glucose signals resulting in impaired glucose potentiation of nonglucose signals. Thus. a single prostaglandin-mediated defect in glucose recognition may be involved in the defective insulin responses to both glucose and nonglucose signals in diabetics,” a defect that can be ameliorated by an inhibitor of endogenous prostalandin synthesis.

ACKNOWLEDGMENT The authurs thank Dr. Jeffrey Halter fur measurements plasma catecholamines and Lylian Fuller. Catherine Pfeifcr. Barbara O’Neill for technical assistance.

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reverse n-adrenergic inhibition of acute insulin response to glucose. Am J Physiol239:E49&E500. 1980 44. Kantor HI), Hampton M: lndomethacin in submicromolar concentrations inhibits cyclic AMP-dependent protein kinase. Nature (Land) 276:841-842, 1978 45. Radomirov R. Petkov V: lndomethacin and aspirin influences on the contractile effects of prostaglandin E, on guinea pig ileum at different Ca concentrations. CR Acad Bulg Sci 30:775-777, 1977 46. Vance JE. Buchanan KD. Williams RH: Glucagon and insulin release. Intluencc of drugs affecting the autonomic nervous system. Diabetes 20:78-82. I97 I 47. Saunders RN, Moser CA: Changes in vascular resistance induced by prostaglandins Ez and F,, in the isolated rat pancreas. Arch Int Pharmacodyn I97:86-92. 1972

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48. Landgraft R. Landgraft-Leurs MMC: The prostaglandin system and insulin release studies with the isolated perfused rat pancreas. Prostaglandins 17:599-6 13, 1979 49. Rossini AA. Lee JB, Frawley TF: An unpredictable lack of effect of prostaglandins on insulin release in isolated rat islets. Diabetes 20:374. 197 I 50. Burr IM, Sharp R: Effects of prostaglandin E, and of epinephrine on the dynamics of insulin release in virm. Endocrinol 94:835-839, 1974 51. Robertson RP, Metz SA: Prostaglandins. the glucoreceptor. and diabetes. N Engl J Med 30 I : I446- 1447, 1979 52. Metz SA, McRae JR. Robertson RP: Hypothesis: prostaglandins mediate defective glucose recognition in diabetes mellitus. Prostaglandins and Medicine 4:247-- 254, 1980