Amino acid modulation of glucose-induced insulin and glucagon release in diabetic patients

Amino Acid Modulation of Glucose-induced Insulin and Glucagon Release in Diabetic Patients Yutaka Seino, Masaki

Ikeda, Kenichi Nakane,

Susumu

Hiroshi

Nakahara,

Seino, and Hiroo lmura

In order to determine whether amino acids have a beneficial effect on glucose tolerance in diabetes, the effect of intravenous infusion of mixed amino acids on plasma insulin, glucagon, and blood glucose responses to oml glucose loading was studied in patients with mild to moderate diabetes. lntmvenous infusion of mixed amino acids over a period of 30 min which was started 30 min or immediately before oml glucose loading significantly aug-

mented the insulin response but did not improve the blood glucose curve, probably due to excessive glucagon response. However, amino acid infusion over a period of 60 min started immediately before oml glucose loading evoked a sustained rise of plasma insulin associated with a lesser degree of glucagon secretion, thus causing significant improvement of the blood glucose curve.

D

IABETIC PATIENTS are characterized by an impaired initial plasma insulin response to glucose. Since amino acid-induced insulin response is relatively well retained,im3 an intrinsic defect in glucose receptors has been postulated in diabetes mellitus. Floyd et al4 first reported that arginine or a mixture of certain amino acids acted synergistically with glucose on insulin release. Efendic et al5 and our previous study6 have demonstrated that in mild and moderate diabetics, pretreatment with arginine infusion improves plasma insulin response to intravenous glucose loading. Recently, through the development of a specific glucagon radioimmunoassay, some investigators7-9 have demonstrated that excessive glucagon secretion is associated with diabetes mellitus and may play an important role in the pathogenesis of diabetes mellitus. lo In the present study, we investigated the effect of an amino acid mixture given in different time schedules on plasma insulin, glucagon, and blood glucose responses to oral glucose loading in diabetic patients. MATERIALS

AND

METHODS

Subjects The study involved 21 diabetic subjects (14 males and 7 females), aged 28-56 (mean age 45) who had no specific therapy whatsoever for at least the previous 6 mo and had never received an insulin injection. The pertinent data on the 21 diabetic subjects, including duration of diabetes, severity of diabetes, and diabetic complications, are shown in Table I. They had fasting blood glucose levels ranging from I04 to 254 mg/dl, with diabetic glucose tolerance curves as estimated by the criteria of the Japan Diabetic Society.” The controls were I4 normal subjects (8 males and 6 females), aged 21-56 (mean age 41). They

From the Third Division. Department of Medicine. Kobe University School of Medicine and Ikeda Clinic, Kobe. Japan. Received for publication August 23. 1976. Reprint requests should be addressed 10 Dr. Yutaka Seine. Third Division, Department of Medicine. Kobe University School of Medicine. 7-13 Kusunoki-cho. Ikuta-Ku-Kobe. Japan. 0 1977 by Grune & Stratton. Inc. ISSN 0026-0495. Metctbolism, Vol. 26, No. 8 (August), 1977

911

912

SEINO ET Al. Table I.

Relevant Data on the Diabetic Subjects Studied Weight

FBS

Patients

(vr)

Sex

(kg)

(mg/dl)

N.M.

49

104

49

63

204

G.F.

51

F M M M M F M M M F F

60

M.Y.

58

198

54

184

63

190

Age

W.M.

38

LA.

46

N.Y.

47

O.Y.

41

M.T.

34

M.T.

54

N.K.

40

H.Y.

56

T.Y.

28

Y.S.

36

S.S.

40

H.T.

49

S.T.

55

I.K.

54

MM.

49

M.F.

51

S.Y.

41

Y.M.

37

M M F F M M F M M M

Duration

of

Diabetes (yr)

1

Complication With Diabetes

-

(+)

3

Retinopathy -

3 6

Retinopathy -

(+)


-


62

182

51

225

4

-

54

179

1

-

65

147

1

60

204

2 7

43

184

65

208


57

220


60

242

3

63

187

2

60

179

56

206

56

123,

62

155

65 58

-

4
Retinopothy

(+)

Retinopathy -

(+)

2

-

196

2

-

174

3

-

-


were nonobese and had no family history of diabetes. In addition, during experiment3 all diabetic and normal subjects were encouraged to eat a weight-maintaining diet with at least 40% of the calories as carbohydrate. In all cases, the 50 g of oral glucose tolerance test was performed without any pretreatment. Some of the normal subjects (experiment I) and diabetic patients (experiments l-3) were then retested 3-14 days later in order to investigate the effect of pretreatment with the mixed amino acids. The mixed amino acids used are shown in Table 2.

Experiment

1

Seven normal subjects and seven diabetic patients received oral glucose loading following the intravenous infusion of mixed amino acids (45.5 g) over a period of 30 min. Blood was withdrawn 30 min. I5 min. and immediately before and 30,60,90, 120, and 180 min after the glucose loading.

Table 2. Constituent

of a Mixture

Present Experiments-45.5 Amino Acid

Quantity (e)

Tyrosine

0.2

Cyst&e

0.1

Arginine

4.9

Histidine

2.1

Alanine

3.3

Asparogine

0.8

Glutamine

0.4

Glycine

6.3

Tryptophan

0.7

of Gamin0 Acids in the R (400 ml) Amino Acid

Proline Serine lsoleucine Leucine Lysine Phenylalanine Methionine Threonine Wine

Quantity (e)

4.3 1.9 2.4 4.6 3.1 3.9 1.7 2.0 2.0

913

AMINO ACID MODULATION

Experiment 2 In nine diabetic patients, 45.5 g of mixed amino acids was infused intravenously over a period of 30 min and 50 g of glucose was given orally at the same time when the infusion was started. Blood was withdrawn immediately before and then again 15, 30, 45, 60, 90, 120, and 180 min after the start of the infusion.

Experiment 3 In five diabetic patients, 45.5 g of mixed amino acids was infused intravenously over a period of 60 min, beginning at the time of oral glucose loading. Blood was withdrawn at same intervals as in experiment 2. All experiments were performed between 7:OO a.m. and 1O:OO a.m. after overnight fasting and absolute bed rest for at least 30 min. Blood was withdrawn from an indwelling needle placed in the antecubital vein with plastic syringes. An aliquot of the blood was used for the determination of Another 2-ml aliquot of blood for glucagon deblood glucose by the Technicon AutoAnalyzer.‘* termination was promptly placed into chilled tubes containing Zoo0 units of Trasylol in a volume of 0.2 ml; the mixture was immediately centrifuged at 4’C and plasma was separated and frozen at -20°C. The remaining portion was centrifuged and plasma was separated, frozen, and stored at -20°C for the measurement of insulin. Plasma insulin was measured by the double antibody was determined by the immunoprecipitation technique of Hales and Randle.t3 Plasma glucagon radioimmunoassay method of Sakurai and Imura,14 utilizing antiserum 30 K purchased from the Diabetes Research Fund, University of Texas Southwestern Medical School, Dallas, Tex. The insulin and glucagon assays were run simultaneously for all samples of each experiment. Statistical analysis was performed by either the Student’s t test or the paired t test. RESULTS

Plasma Insulin and Glucagon Responses to Oral Glucose Loading in Normal Subjects and Diabetic Patients

In normal subjects, the mean blood glucose level rose from the mean basal level of 80 i 3.2 mg/dl (&SE) to the mean peak level of 123 f 2.0 mg/dl at 30 min after the oral glucose loading. In diabetic patients, oral glucose loading elicited a significantly greater rise in blood glucose from the basal level of 185 f 14.2 mg/dl to the peak value of 337 f 20 mg/dl, which occurred 60 min after the glucose loading. The mean (&SE) basal insulin level in normal subjects was 14 f 2.4 &/ml, which rose significantly, reaching the peak value of 79 + 9.3 pU/ml at 30 min after glucose administration. Plasma insulin response was lower in diabetics than in normal subjects in spite of higher blood glucose levels, with the mean peak value of 36 A 6 pU/dl occurring 90 min after glucose loading. The plasma insulin response in diabetics was particularly impaired at 30 min, as compared with that in normal subjects (p < 0.01). The mean (‘t SE) fasting glucagon levels in normal and diabetic subjects were 87 f 7 pg/ml and 103 f 5 pg/ml, respectively. In normal subjects, plasma glucagon levels decreased substantially following the oral glucose loading, reaching the nadir of 60 f 6 pg/ml at 120 min, which was significantly lower than the basal level (p < 0.01). In diabetic patients, plasma glucagon was not suppressed and rather tended to rise despite pronounced hyperglycemia. Eflect of Pretreatment With Mixed Amino Acids on Plasma Insulin and Glucagon Responses to Oral Glucose Loading in Normal Subjects (Fig. 1)

Plasma insulin rose significantly within the first 15 min of amino acid infusion, reaching the mean peak level of 118 f 19.2 pU/ml at 0 min, that is, at the

SEINO ET Al.

91A

11

I

I

1

-30

0

30

60

90

,

120 Time kMin

1

Fig. 1. Effect of mixed amino acid infusion on plasma insulin, glucagon, and blood glucose responses to oml glucose administration in seven normal subjects. Amino acids were infused from -30 to 0 min. Means f. SE are shown. *p < 0.05; **p < 0.01.

end of infusion. Plasma insulin levels decreased following the subsequent glucose loading, but the values at 30,60, and 90 min (86 f 13.6,64.0 & 1.7, and 32 * 2.5 ,uU/ml, respectively) were slightly, but not significantly, higher than those after glucose loading alone. Plasma glucagon also rose significantly from the mean (&SE) basal level of 93 f 5 pg/ml to 436 f 19 pg/ml at the end of amino acid infusion. Although plasma glucagon declined gradually after oral glucose loading, it still remained high at 30 min, as compared with the value after glucose loading alone (p < 0.01). The amino acid infusion raised blood glucose from the baseline of 86 f 1.7 mg/dl to 104 f 4.1 mg/dl at 0 min. However, blood glucose concentrations at 30 and 60 min after glucose loading (104 st 5.0 and 112 & 3.4 mg/dl, respectively) were significantly lower than the corresponding control values (p < 0.05). Eflect of Pretreatment With Mixed Amino Acids on Plasma Insulin Responses to Oral GIucose Loading in Diabetic Patients (Fig. 2) When mixed amino acids were infused into diabetic patients, plasma insulin also rose significantly to the levels of 87 f 13.2 and 105 =+=17.0 pU/ml at - I5 and 0 min, respectively; these values were not significantly different from the values in normal subjects. Plasma insulin levels at 30, 60, and 90 min after the

AMINO

915

ACID MODULATION

Amino Arid* (45.51rli.v.

Fig. 2. Effect of mixed amino acid infusion on plasma insulin, glucagon. and blood glucose responses to oral glucose adminic tration in seven diabetic patients. Amino acids were infused from -30 to 0 min. Means f SE am shown. lp < 0.05; ‘*p < 0.01.

1001 b -30

0

30

60

90

120 Time Min

1

subsequent glucose loading (66.5 A 10 pU/ml, 82.8 + 20 PI-J/ml, and 77.5 f 22.4 pU/ml, respectively) were significantly higher than those produced by glucose loading alone. These values were not greatly different from those in normal subjects, although the 30-min value was lower in diabetics. Despite almost normal plasma insulin levels achieved by the pretreatment with amino acids in diabetics, blood glucose concentrations were not significantly ameliorated. They were 158 f 6.4 mg/dl, 187 f 34.5 mg/dl, and 218 + 32 mg/dl at -30, 0, and 30 min, respectively, after the oral glucose loading, the latter of which was only slightly lower than the value obtained in the control experiments, where glucose alone was given. Plasma glucagon levels rose more remarkably in diabetic patients than in normal subjects following the infusion of amino acids, with the peak value of 812 A 75 pg/ml at 0 min (p < 0.01 versus normal controls). They gradually declined thereafter. E$ect of Concomitant Infusion of Mixed Amino Acids Over a Period of 30 min on Plasma Insulin and Glucagon Responses to Oral Glucose Loading in Diabetic Patients (Fig. 3)

Following the oral administration of glucose combined with amino acid infusion, plasma insulin showed a prompt rise from the baseline of 16.7 f 2.8 pU/ml to the peak value of 72 A 17.8 flu/ml at 30 min, which coincided with the end of the infusion. This plasma insulin rise was significantly greater than

SEINO

916

600

t----w

-

ET Al.

Clvcorc

z. z I 4 3 1 B e

300-

-‘-Y-----__t_

__.._

____I t_____--__.._._.~

Tmw

Vin

Fig. 3. Effect of mixed amino acid infusion on plasma insulin, glucagon, and blood glucose responsesto oml glucose administmtion in nine diabetic patients. Amino acids were infused from 0 to 30 min. Means f SE are shown. *p < 0.05; **p < 0.01.

that produced by glucose loading alone. Plasma glucagon also rose abruptly, reaching the mean level of 622 & 105.5 pg/ml at the end of the infusion; this value was markedly higher than that in the control experiment where glucose alone was given (p < 0.01). A gradual rise of blood glucose was elicited by the administration of glucose and amino acids, reaching the levels of 212 f 10.7 mg/dl and 233 f 12.2 mg/dl at 30 and 60 min, respectively; both of these values were significantly lower than the corresponding values produced by glucose alone (p < 0.01). However, blood glucose levels at 90, 120, and 180 min were not significantly different between two groups, Effect of Concomitant Infusion of Mixed Amino Acids Over a Period of 60 min on Plasma Insulin and Glucagon Responses to Oral Glucose Loading in Diabetic Patients (Fig. 4) When amino acids oral glucose loading, and 73 f 12.0 pU/ml high even thereafter. those in the control whereas the values

were infused over a 60-min period starting at the time of plasma insulin rose from the baseline to 74 f 15.6 pU/ml at 30 and 60 min, respectively, and remained relatively The values at 30 and 60 min were significantly higher than experiments where glucose alone was given (p < 0.05). at 90 min were very similar. Plasma glucagon showed

AMINO

917

ACID MODULATION

Amino Aridr’45.5ali.r.

Fig. 4. Effect of mixed amino acid infusion on plasma insulin, glucagon, and blood glucose responsesto oral glucose administmtion in five diabetic patients. Amino acids were infused from 0 to 60 min. Means f SE are shown. ‘p < 0.05; l=p < 0.01.

9

g ; 2

300

200 1 m

,oo

L

0

,

30

60

90

120 Timr

180 .Min

high levels, 397 f 11.3 pg/ml and 398 f 16.3 pg/ml, at 30 and 60 min, respectively, the latter of which coincided with the end of amino acid infusion. These values were significantly higher than those in the control experiments (p < 0.01). The mean blood glucose concentrations after the glucose load were 220 f 27.9 mg/dl (GE), 211 A 28.0 mg/dl, and 221 f 18.4 mg/dl at 30, 60, and 90 min, respectively. These values were significantly lower than those in the control studies (p < 0.05) and also lower, though not significantly, than those in experiments 1 and 2. DISCUSSION

In spite of extensive studies on plasma insulin response to amino acids in normal subjects, relatively little is known about amino acid-induced insulin release in diabetic patients.le3 Floyd et al.’ and Cremer et al.* have reported that both insulin releases induced by glucose and amino acids decrease in proportion to the severity of diabetes. Fajans et al. Is have shown that arginine-induced insulin release in diabetes is preserved better in its first (early) phase than its second (late) phase. Efendic et al.s have reported that arginine-induced insulin release is only slightly impaired in prediabetics or diabetics as compared with agree with the obthat in normal subjects. Our previous studies3s6 substantially

918

SEINO

ET Al.

servation of Efendic et al.,5 indicating that arginine infusion elicits an almost normal insulin response in mild or moderate diabetics when compared on the basis of the absolute plasma insulin levels. These results are in contrast with the fact that plasma insulin response to glucose, especially in its early phase, is significantly impaired in diabetic patients. Furthermore, pretreatment with arginine infusion could improve impaired plasma insulin response to intravenous glucose loading in mild diabetics, as shown by our previous studies6 and by Efendic et a1.5 Present studies further demonstrate that intravenous infusion of the amino acid mixture started 30 min before or immediately before the oral glucose administration can improve the plasma insulin response significantly. Unexpectedly, however, the glucose tolerance curve was not significantly improved in those experiments in spite of a better insulin response. This might be accounted for by our observation that the infusion of mixed amino acids significantly raised plasma glucagon, which*m turn raised blood glucose, possibly through its glycogenolytic action. The responses of plasma glucagon and blood glucose to the infusion of mixed amino acids were both significantly greater than those in normal subjects. These results agree with the previous observations7@*‘7 that arginine infusion provokes a greater response of plasma glucagon in diabetic patients. In order to find the method of administration of amino acids which minimizes the rise of blood glucose, we then prolonged the infusion period from 30 min to 60 min in experiment 3, which started immediately before the oral glucose loading. This produced a similar increase of plasma insulin and a smaller increase of plasma glucagon than those found in the other two experiments, which might result in the improvement of the glucose tolerance curve. These results suggest that amino acids have a beneficial effect on glucose tolerance in diabetic subjects under certain conditions. REFERENCES 1. Floyd JC Jr, Fajans SS, Conn JW, et al: Secretion of insulin induced by amino acids and glucose in diabetes mellitus. J Clin Endocrinol Metab 28:266-276, 1968 2. Cremer GM, Molnar GD, Taylor WF, et al: Studies of diabetic instability. II. Tests of insulinogenic reserve with infusions of arginine, glucagon, epinephrine and saline. Metabolism 20:108331098, 1971 3. Seino Y, Kurahachi H, Goto Y, et al: Comparative insulinogenic effects of glucose, arginine and glucagon in patients with diabetes mellitus, endocrine disorders and liver disease. Acta Diabetol Lat 12:89-99, 1975 4. Floyd JC Jr, Fajans SS, Pek S, et al: Synergistic effect of certain amino acids and glucose upon insulin secretion in man. Diabetes 19:109115,1970 5. Efendic S, Cerasi E, Luft R: Quantitative study on the potentiating effect of arginine on

glucose-induced insulin responses in healthy, prediabetic, and diabetic subjects. Diabetes 23: 161. 171, 1974 6. Seino Y: Insulin secretion in patients with diabetes mellitus-Enhancement of insulin in adult onset diabetics by several drugs a,.d amino acid. J Jpn Diabetes Sot 17:221-231, 1974 7. Unger RH, Aguilar-Parada E, Mtiller WA. et al: Studies of pancreatic alpha cell function in normal and diabetic subjects. J Clin Invest 49:837-848, 1970 8. Heding LG. Rasmussen SM: Determination of pancreatic and gut glucagon like immunoreactivity (GLI) in normal and diabetic subjects. Diabetologia 8:4088411, 1972 9. Buchanan KD, McCaroll AM: Abnormalities of glucagon metabolism in untreated diabetes mellitus. Lancet 2: 1394-1395, 1972 10. Unger RH, Orci L: The essential role of

AMINO

ACID MODULATION

glucagon in the pathogenesis of diabetes mellitus. Lancet 2:14-16, 1975 11. Kuzuya N: The criteria of glucose tolerance curve by the Committee of Diagnostic Criteria for Diabetes Mellitus of Japan Diabetes Association. J Jpn Diabetes Sot 13: 1-7, 1970 12. Hoffman WS: Rapid photoelectric method for determination of glucose in blood and urine. J Biol Chem 120:51-55, 1937 13. Hales CN, Randle PJ: Immunoassay of insulin with antibody precipitate. Biochem J 88:137-146, 1963 14. Sakurai H, Imura H: A sensitive radio-

919

immunoassay for glucagon using talc method. Jpn J Nucl Med 10:135-136, 1973 15. Fajans SS, Floyd JC Jr, Knopf RF, et al: Amino acids and insulin release in viva. Isr J Med Sci 8:233-242, 1972 16. Ohneda A, Ishii S, Horigome K, et al: Glucagon response to arginine after. treatment of diabetes mellitus. Diabetes 24:81 l-819, 1975 17. Seino Y, Goto Y, Kurahachi H, et al: Alteration of plasma glucagon responses to arginine in patients with diabetes mellitus, Cushing’s syndrome and hypothyroidism after treatment.

Horm

Metab

Res 9:28-32,

1977