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Early insulin responses to glucose and to tolbutamide in maturity-onset diabetes
Early Insulin Responses and to Tolbutamide
to Glucose
in Maturity-onset
Diabetes
By NORA VARSANO-AHARON, ELSA ECHEMENDIA, ROSALYN S. YALOW AND SOLOMON A. BERSON
Intravenous glucose and tolbutamide tolerance tests were performed in six nonobese, nondiabetic control subjects, in six untreated nonobese maturity-onset diabetic patients without pancreatic diabetes and in one insulin-non-requiring patient with diabetes secondary to chronic panereatitis. Following injection of glucose the control group responded with a sharp rise in plasma insulin from a fasting level of 8 ? 2 ~&J/ml. to a peak value of 216 ? 29 &U/ml. at 2 minutes whereas the maturity-onset diabetics showed a very slight increase in plasma insulin from a fasting level of 19 f 4 &J/ml. to 24 f 10 au/ml. at 2 minutes and a peak value of 35 -C 9 &U/ml. at 30 minutes. In contrast initial insulin secretory responses of both groups to intravenous tolbutamide were essentially the same with peak values of 69 & 1 J&J/ ml. and 72 + 20 &U/ml. being obtained at 2 minutes from fasting levels of 6 ? 1 pUlm1. and 13 + 5 au/ml. in the control and diabetic groups, respectively. The
response of the maturity-onset diabetics was biphasic and there appeared to be a sustained insulin release for over 30 minutes. The subject with pancreatic diabetes had virtually no response to either glucose or tolbutamide. The rate of disappearance of crystalline beef insulin from the plasma of two subjects with maturity-onset diabetes, two control subjects and the subject with pancreatic diabetes was about the same for all, with half-times ranging from 9-11 minutes after the initial distribution phase. The differences in plasma insulin concentration therefore reflect differences in secretory rate rather than differences in metabolic turnover. The possibility is suggested that the early lesion in p cell function in the maturity-onset diabetics studied here is expressed specifically in a failure of insulin release to be triggered promptly by glucose rather than in a generally defective mechanism for synthesizing and storing insulin. (Metabolism 19: No. 6, June, 409-417, 1970)
N
ONDIABETIC ADULT HUMAN SUBJECTS respond to an orally administered glucose feeding with a brisk rise in the concentration of plasma insulin, which reaches a peak value within 30-60 minutes and then declines within the next two hours to or below fasting control levels.lz” In contrast, subjects with mild maturity-onset diabetes, who are not insulin requiring, characteristicahy exhibit a delayed initial rise in plasma insulin; however, in response to persistent hyperglycemia, plasma insulin may continue to increase for 2-3 From the Radioisotope Service, Veterans Administration Hospital, Bronx, N. Y., and the Department of Medicine, Mount Sinai School of Medicine, New York, N. Y. Received for publication November 17, 1969. NORA VARSANO-AHARON, M.D., ELSA ECHEMENDIA, M.D., ROSALYN S. YALOW, PH.D., AND SOLOMON A. BERSON, M.D.: Radioisotope Service, Veterans Administration Hospital, Bronx, N. Y., and Department of Medicine, Moant Sinai School of Medicine, New York, N. Y. METABOLISM, VOL. 19, No. 6 (JUNE), 1970
409
410
VARSANO-AHARON ET AL.
hours, reaching levels higher than the peak levels shown in non-diabetic controls.l,g Although obese, nondiabetic subjects may show elevated fasting plasma insulin concentrations” and increased insulin-secretory responses to glucose loading,4 the late high plasma insulin levels seen in maturity-onset diabetes during the course of an oral glucose tolerance test are exhibited by lean as well as by obese patients.“-l” The failure of diabetic subjects to respond to hyperglycemia with a rapid outpouring of insulin is more evident on intravenous than on oral administration of glucose. When an intravenous dose of glucose is given as a single rapid injection, the nondiabetic subject displays a peak insulin response in a few minutes whereas in the diabetic, the plasma insulin concentrations rise only slowly to a relatively low plateau .6*7 That the islet /3 cell of the diabetic subject has greater insulin-secretory reserve than is revealed by the response to intravenously administered glucose is suggested, first by the high plasma insulin levels frequently achieved 2-3 hours after oral administration of glucose,‘,” and second by the observation of Simpson et al. I:{ that a greater initial insulin response can be obtained with glucagon than with glucose in maturity-onset diabetics. The present study was initiated to compare the very early plasma insulin responses to intravenously administered glucose and sodium tolbutamide in normal subjects and in patients with maturity-onset diabetes. MATERIALS AND METHODS Intravenous glucose tolerance (IVGTT) and tolbutamide tests were performed in six untreated, nonobese maturity-onset, mild diabetics, six nonobese, nondiabetic controls (hospital personnel or patients with minor illness) and one subject with pancreatic diabetes secondary to chronic pancreatitis who did not require insulin. All subjects were within 100 + 10 per cent of the mean ideal weight. After an overnight fast, subjects were kept recumbent and venous blood samples were drawn a few minutes before and 2, 5. 10, 20, 30, 40, 50. 60, 90, 120 minutes after rapid injection of 25 Gm. glucose in 50 ml. water, or 1 Gm. sodium tolbutamide in 10 ml. water. Both glucose and tolbutamide were given within 30 seconds and timing was started from the onset of injection. Blood samples taken into heparinized syringes were immediately centrifuged under refrigerated conditions: plasma was separated and stored frozen at - 15’ C until assay. The IVGTT preceded the tolbutamide test by two or three days. The IVGTT was repeated in two of the diabetics several days following the tolbutamide test. Plasma glucose concentrations were determined by the method of Hoffmanr-’ in the Technicon autoanalyser. Plasma insulin concentrations were determined by radioimmunoassay1 using crystalline human insulin (Novo) as standard. The rate of disappearance of insulin from plasma was studied in two subjects with maturity-onset diabetes, two control subjects and in one subject with pancreatic diabetes. Following injection of crystalline beef insulin 0.1 U/Kg. body weight, plasma insulin using the injected material as a concentrations were measured by radioimmunoassay’ standard. RESULTS The results of the mean plasma intravenous glucose are given in Fig. glucose in the control group was 95 mean of 129 k 10 mg./lOO ml. in
glucose and insulin concentrations after 1 and Table 1. The mean fasting plasma i- 4 mg. (SEM)/lOO ml., compared to a the maturity-onset diabetic group. Corre-
EARLY
INSULIN
411
RESPONSES
AVERAGE CONTROL
OF 6 LEAN SUBJECTS
AVERAGE OF 6 LEAN MATURITY -ONSET DIABETICS
‘%L~~ 220
Fig l.-Plasma insulin and glucose concentrations during intravenous glucose tolerance tests in nondiabetic and in maturity-onset diabetic subjects.
TIME IN MINUTES
Table 1 .-Plasma
Time (min.)
0 2 5 10 20 30 40 SO 60 90 120
Glucose and Insulin Concentrations During Intravenous Glucose Tolerance Tests (25 Gm.)
Controls Plasma Glucose ‘p&g ‘$$I
95 280 247 220 179 163 137 117 116 100 92
+ 4 f 18 k 14 r 19 f 14 k 11 f 11 f 11 -I- 12 -c 6 ?I 4 ~-
(6)
Plas;s,I;3yh Mean + SkM
8r 216 76 42 31 36 27 22 24 14 14
f +-c k f k f f f -c
2 29 13 7 4 7 4 6 6 5 5
Diabetics Plasma Glucose (ggl$&j
129 318 270 245 245 237 203 187 188 146 127
4 -t 2 -t r k 2 k f zz +-
10 74 35 28 30 1.5 15 15 14 20 17
(6) Plasma Insulin (N/ml. 1 Mean 2 SEM
19 24 32 32 26 3.5 26 24 33 32 28
t 2 _’ i t k -c zk + k k
4 10 9 9 4 9 9 9 8 8 7
sponding plasma insulin concentrations were 8 + 2 pU/ml. and 19 rt 4 pU/ml., respectively. Following injection of glucose the control group responded with a sharp rise in plasma insulin to a peak value of 216 4 29 pU/ml. at 2 minutes (the earliest sample drawn) which was followed by a precipitous drop to 76 + 13 ,&J/ml. at 5 minutes and to 42 + 7 pU/ml. at 10 minutes after the injection. By the end of 20 minutes plasma insulin had declined further to 31 +- 4 @/ml. and after a small, but statistically insignificant, secondary rise at 30 minutes, continued to fall as the glucose concentration returned to near control levels. In contrast to the controls the maturity-onset diabetics showed only a small initial insulin-secretory response to intravenous glucose. At 2 minutes the circulating insulin level had risen only 5 $J/ml. and continued to increase only slightly. The maximal increase was reached at 30 minutes which coincided with the small second peak exhibited by the normal control group. Thereafter, from
VARSANO-AHARON
412
ET AL.
60 to 120 minutes insulin concentrations changed little, remaining above control levels and plasma glucose concentrations fell slowly. The insulin-secretory response to intravenous tolbutamide and the mean plasma glucose concentrations in control and diabetic groups are shown in Fig. 2 and Table 2. The responses in both groups were almost instantaneous; the peak plasma insulin levels were obtained in the first samples drawn at 2 minutes. The mean increments in control and diabetic groups were 63 and 59 $J/m.l., respectively. The response of the normals was characterized by a single pulse with rapid, smooth decline of the circulating insulin to the preinjection levels. In contrast, the response of the maturity-onset diabetics was biphasic. After the initial pulse, there appeared to be a sustained release of insulin, continuing for about 30 minutes, which was then followed by a slow decline in plasma insulin concentration.
Fig. Z.-Plasma insulin and glucose concentrations during intravenous tolbutamide tests in nondiabetic and in maturity-onset diabetic subjects.
Table 2.-Plasma
Time (min.)
0 2 5 10 20 30 40 50 60 90
Glucose and Insulin Concentrations Tolbutamide Test (1 Gm.)
Controls (6) Plasma Insulin Plasma Glucose (mg./loo ml.) NJ/d) Mean t SEM Mean -c SEM
98 f
8
6cl
98 98 83 70 66 65 70 79 82
8 5 4 3 3 4 3 4 4
69 k 43 k 31 * 19 -c 12 k 10 k 9k2 8&2 7+-7
-c +-c t 5 f +?I +-
1 3 3 2 3 2
During Intravenous
Diabetics 6) Plasma Glucose Plasma Insulin (mg./100 ml.) wJ/rnl.) Mean i SEM Mean t SEM
130 118 100 110 102 98 92 93 90 91
I? 18 r 4 k 16 r 16 k 14 c 16 k 13 -e 13 r13 r 8
13 72 42 38 39 36 22 19 18 16
t 5 t 20 t 12 t 7 rf- 9 t 6 ? 5 2 4 k 5 i- 7
413
EARLYINSULINRESPONSES
The integrated increment in plasma insulin concentration over the period O-90 minutes, averaged 40 per cent greater in diabetics than in the control group ( 1139 PU-min./ml. and 8 15 PU-min./ml., respectively). The mean maximum fall in plasma glucose was 31 per cent and occurred at 60 minutes in the maturity-onset diabetics compared to 34 per cent at 40 minutes in the control group. The subject with pancreatic diabetes (Fig. 3) had a fasting plasma insulin concentration of only 1.5 pU/ml. and showed virtually no insulin-secretory response to the IVGTT or to tolbutamide stimulation; the plasma glucose curve remained flat after tolbutamide. There were no significant differences in the rates of disappearance of injected insulin in the control group, the maturity-onset diabetics and the subject with pancreatic diabetes (Fig. 4). DISCUSSION That the initial insulin-secretory response to a glucose load is defective in insulin-non-requiring, maturity-onset diabetics has been recognized from the earliest studies on immunoreactive plasma insulin. Iv2 In itself the characteristic delay in insulin secretion might signify nothing but a diminished capacity to synthesize or release insulin as a consequence of a reduction either in the number of /3 cells or of the function of each of a normal number of p cells. However, the ability to achieve high plasma insulin levels late during the course of an oral GTT associated with an obvious defect in the early secretion of insulinlJ raises the possibility that the secretory capacity of the islet ,Q cell may not be generally depressed in maturity-onset diabetes but rather that there may be a more specific defect characterized by poor responsiveness to certain stimuli. Failure of the gut hormonal insulin-secretagogues to stimulate insulin secretion cannot explain the early delay in insulin secretion since an inadequate insulinsecretory response is even more readily evident when hyperglycemia is induced by intravenous administration of glucose 6*ithan by oral administration of glucose. The poor response to I.V. glucose observed in the present study is in accord with the observations of Perley and Kipnisa and Seltzer et al.? It appears that the glycemic stimulus per se is less than normally effective in maturity-onset diabetes.6,7 In view of the similar rates of disappearance of injected insulin in the five cases studied here we can attribute differences in plasma insulin to differences in secretory rate rather than to differences in metabolic turnover rate. In the present study, the diabetic group showed a significant elevation in fasting blood glucose in spite of significantly higher plasma insulin concentrations. In earlier studies1v2v4too, this association has been observed repeatedly. The elevated plasma insulin concentrations undoubtedly simply reflect the response to a persistent hyperglycemic stimulus for some hours but the persistent hyperglycemia might reflect resistance to or diminished effectiveness of the fasting hyperinsulinemia. So, too, the maintenance of blood glucose concentrations in the diabetics following tolbutamide injection might support the hypothesis of insulin resistance; however, a strong case cannot be made for this suggestion since the initial fall in blood sugar (within the first 10 minutes) was at least as
VARSANO-AHARON
414
INTRAVENOUS GLUCOSE TOLERANCE TEST
ET
AL.
INTRAVENOUS TOLBUTAMIDE TEST
GLUCOSE mg/lOOml
i i
9........._... * .....~,.
“‘\ 2.. “. . . . .. .. . ....*
I ‘. . . .
i
INSULIN /+J$l
1
e-”
. .. . . .. . . ...__
~~
,....
.
1
-0
O-0 TIME
IN MINUTES
Fig. 3.-Plasma insulin and glucose concentrations during intravenous glucose tolerance test and intravenous tolbutamide test in uatient with diabetes secondary I to chronic pancreatitis.
R
TIME
Fig. 4.-Disappearance of exogenous onset diabetic and pancreatic diabetic subtracting fasting endogenous insulin total insulin concentration (x) in each
IN MINUTES
beef insulin from plasma of normal, maturitysubjects. Dotted straight line was obtained by concentration (shown in stippled area) from specimen.
marked in the diabetics as in the control group although the later response was less impressive in the diabetics in spite of the significantly higher plasma insulin concentrations from 1O-90 minutes. It is also relevant to note that a very low level of plasma insulin was adequate
EARLY
INSULIN
RESPONSES
415
to maintain plasma glucose below 130 mg./lOO ml. in the patient with pancreatic diabetes. Whereas the plasma insulin response to IV. glucose was much greater than to I.V. tolbutamide in the control group, quite the reverse was true of the maturity-onset diabetics. Indeed, as noted, the integrated insulin-secretory response to tolbutamide was greater in the diabetics than in the controls but the most striking difference observed was between the responses to tolbutamide and glucose in the diabetic subjects. This difference was most marked in the first five minutes. A rapid release of what must have been preformed insulin15 was triggered by both glucose and tolbutamide in the control group but only by tolbutamide in the diabetics. It appears therefore that the delayed insulinsecretory response to a glycemic stimulus (glucose IV. or P.O. ) seen in maturityonset diabetics who appear to be capable of synthesizing significant quantities of insulin, might be due to a reduced ability to release insulin when a high concentration of glucose is presented to the islet ,Q cell rather than to a lack of available insulin; the availability of mobilizable insulin may be evidenced on the exhibition to tolbutamide. These findings were not observed by Perley and Kipni9 whose diabetic patients showed slightly subnormal responses to I.V. tolbutamide in the 5-15 minutes after injection as well as subnormal responses to I.V. glucose. However Boden et al.16 observed normal insulin-secretory responses to IV. tolbutamide associated with depressed responses to IV. glucose in prediabetic subjects (children of two diabetic parents) who had normal oral glucose tolerance tests and normal cortisone-primed glucose tolerance tests. The mechanism by which changes in glucose concentration regulate insulin secretion is unknown. Presumably the islet p cell is stimulated by some glucose metabolite beyond the stage of glucose-6-phosphate in the glycolytic pathway since 2-deoxy-D-glucose inhibits the insulin-secretory response to glucose17 and since the 6-phosphate derivative of 2-deoxy-D-glucose is known to inhibit competitively the further metabolism of glucose-6-phosphate.ls It is known also that mannoheptulose blocks the insulin-secretory response to glucosel” but not to tolbutamideZO in rabbit pancreas in vitro. In studies carried out in this laboratory,“l it has been demonstrated in man that although 2-deoxy-D-glucose blocks insulin-secretory responses to glucose and glucagon, the responses to tolbutamide and arginine are not impaired. Since sulfonylureas stimulate insulin secretion in vitro in the absence of glucose2” it seems reasonable to conclude that tolbutamide and glucose stimulate insulin secretion by mechanisms that differ in some way and that the effect of tolbutamide is not dependent on the presence of glucose. The present study is consistent with these conclusions. Tolbutamide was clearly an effective insulin-secretagogue in a group of maturity-onset diabetics who responded poorly to glucose intravenously. The possibility is therefore suggested that an early defect in ,Q cell function in maturity-onset diabetes is expressed specifically in a failure of insulin release to be triggered promptly by glucose. That these responses may not be unique to genetic diabetes is suggested by two recent studies in diabetes associated with pancreatic disease. Joffe et al.‘3 found that in patients with chronic pancreatitis due to chronic alcoholism and oral glucose intolerance associated with hypoinsulinism, intravenous tolbutamide
416
VARSANO-AHARON
ET AL.
given 30 minutes after the oral glucose administration caused a prompt doubling of the plasma insulin concentration, which however was less of a rise than in normal controls. Handwerger et al.?* studied patients with cystic fibrosis and moderate to severe oral glucose intolerance. In two such patients given intravenous tolbutamide and two of three given intravenous glucagon two hours after the oral glucose there was a significant rise in plasma insulin beyond that achieved by glucose alone. The authors could not find evidence for significant loss of /3 cells on pathological examination in six patients with the disease and attributed the physiopathologic abnormalities to architectural disorganization of the islets by the fibrotic process. In our patient with pancreatic diabetes there was no significant insulin response to glucose or tolbutamide. In a large group of juvenile diabetics studied by Drash et al.,25 there was no insulin response to glucose, glucagon, tolbutamide or arginine. Similar results except for the absence of testing with glucagon were reported by Parker et al.?” The differences in insulin secretory responses to intravenous tolbutamide and glucose in our patients with maturity-onset diabetes appear much more striking than the differences described above in the patients with pancreatic disease given oral glucose followed by intravenous tolbutamide and suggest an almost complete inability of the /3 cell to respond rapidly to intravenous glucose but normal ability to respond to tolbutamide. However, the distribution between the two kinds of diabetes may be more of degree than of kind and dependent more on the stage than on the nature of the disease. If so, /3 cell dysfunction in diabetics, genetic or acquired through pancreatic damage, is characterized in its initial stages, not so much by an inability to synthesize and/or store insulin but by a unique failure to release it promptly in response to the physiologic stimulus of hyperglycemia. REFERENCES 1. Yalow, R. S., and Berson, S. A.: Immunoassay of endogenous plasma insulin in man. J. Clin. Invest. 39:1157, 1960. 2. -, and -: Plasma insulin concentrations in non-diabetic and early diabetic subjects. Diabetes 9:254, 1960. 3. Rabinowitz, D., and Zierler, K. L.: Forearm metabolism in obesity and its response to intraarterial insulin. Characterization of insulin resistance and evidence for adaptive hyperinsulinism. J. Clin. Invest. 41: 2173, 1962. 4. Karam, J. H., Grodsky. G. M., and Forsbam, P. H.: Excessive insulin response to glucose in obese subjects as measured by immunochemical assay. Diabetes 12: 197, 1963. 5. Yalow, R. S., Glick, S. M., Roth, J., and Berson, S. A.: Plasma insulin and growth hormone levels in obesity and diabetes. Ann. N. Y. Acad. Sci. 131:357, 1965. 6. Perley, M., and Kipnis, D. M.: Plasma
insulin responses to glucose and tolbutamide of normal weight and obese diabetic and nondiabetic subjects. Diabetes 15: 867. 1966. 7. Seltzer, H. S., Allen, E. W., Herron. A. L., Jr., and Brennan, M. T.: Insulin secretion in response to glycemic stimulus: Relation of delayed initial release to carbohydrate intolerance in mild diabetes mellitus. J. Clin. Invest. 46:323, 1967. 8. Berger, S., and Vongaraya, N.: Insulin response to ingested protein in diabetes. Diabetes 15:303, 1966. 9. Graber, A. L., Wood, F. C., Jr., and Williams, R. H.: Serum immunoreactive insulin response during prolonged glucose infusions in nondiabetic and diabetic humans. Diabetes 16:145, 1967. 10. Reaven, G., and Miller, R.: Study of the relationship between glucose and insulin responses to an oral glucose load in man. Diabetes 17:560, 1968. 11. Danowski, T. S., Morgan, C. R.,
EARLY
INSULIN
RESPONSES
Sunder, J. H., Moses, C., and Sabeh, G.: Immunoassayable insulin and glucose tolerance in nonobese and obese subjects. Exp. Med. Surg. 25:122, 1967. 12. Buchanan, K. D., and McKiddie, M. T.: Factors determining the plasma insulin response to oral glucose in diabetes mellitus. Diabetes 16:466, 1967. 13. Simpson, R. G., Benedetti, A., Grodsky, G. M., Karam, J. H., and Forsham, P. H.: Early phase of insulin release. Diabetes 17:684, 1968. 14. Hoffman, W. S.: A rapid photoelectric method for the determination of glucose in blood and urine. J. Biol. Chem. 120:51, 1937. 15. Curry, D. L., Bennett, L. L., and Grodsky, G. M.: Dynamics of insulin secretion by the perfused rat pancreas. Endocrinology 83:.572, 1968. 16. Boden, G., Soeldner, J. S., Gleason, R. E., and Marble, A.: Elevated serum human growth hormone and decreased serum insulin in prediabetic males after intravenous tolbutamide and glucose. J. Clin. Invest. 47:729, 1968. 17. R-Candela, J. L., Castrillon, A. M., Martin-Hernandez, D., and Castilla-Cortazar, T.: Effect of glucose and citrate on insulin secretion in vitro by the pancreas pretreated with 2-deoxyglucose. Med. Exp. (BaseI) 11:47, 1964. 18. Wick, A. N., Drury, D. R., Nakada, H. I., and Wolfe, J. B.: Localization of the primary metabolic block produced by 2deoxy glucose. J. Biol. Chem. 224:963, 1957. 19. Coore, H. G., Randle, P. J., Simon,
417 E., Kraicer, P., and Shelesnyak, M. C.: Block of insulin secretion from the pancreas by D-mannoheptulose. Nature 197: 1264, 1963. 20. Coore, H. G., and Randle, P. J.: Regulation of insulin secretion studied with pieces of rabbit pancreas incubated in vitro. Biochem. J. 93:66, 1964. 21. Goldsmith, S. J., Yalow, R. S., and Berson, S. A. : Effect of 2-deoxy-d-glucose on insulin-secretory responses to intravenous glucose, glucagon, tolbutamide and arginine in man. Diabetes (in press). 22. Mehnert, H., Schaefer, G., Kaliampetosis, G., Stuhlfauth, K., and Engelhardt, W.: Insulin secretion of the pancreas in extracorporeal perfusion. II. Perfusions of the pancreas with periston, glucose, carbutamide and biguanides. Klin. Wschr. 40: 1146, 1962. 23. Joffe, B. I., Banks, S., Jackson, W. P. U., Keller, P., O’Reilly, I. G., and Vinik, A. I.: Insulin reserve in patients with chronic pancreatitis. Lancet 2:890, 1968. 24. Handwerger, S., Roth, J., Gorden, P., di Sant’Agnese, P., Carpenter, D. F., and Peter, G.: Glucose intolerance in cystic fibrosis. N. Eng. J. Med. 281:451, 1969. 25. Drash, A., Field, J. B., Garces, L. Y., Kenny, F. M., Mintz, D., and Vazquez, A. M.: Endogenous insulin and growth hormone response in children with newly diagnosed diabetes mellitus. Pediat. Res. 2:94, 1968. 26. Parker, M. L., Pildes, R. S., Chao, K-L., Cornblath, M., and Kipnis, D. M.: Juvenile diabetes mellitus, a deficiency in insulin. Diabetes 17:27, 1968.
to Glucose
in Maturity-onset
Diabetes
By NORA VARSANO-AHARON, ELSA ECHEMENDIA, ROSALYN S. YALOW AND SOLOMON A. BERSON
Intravenous glucose and tolbutamide tolerance tests were performed in six nonobese, nondiabetic control subjects, in six untreated nonobese maturity-onset diabetic patients without pancreatic diabetes and in one insulin-non-requiring patient with diabetes secondary to chronic panereatitis. Following injection of glucose the control group responded with a sharp rise in plasma insulin from a fasting level of 8 ? 2 ~&J/ml. to a peak value of 216 ? 29 &U/ml. at 2 minutes whereas the maturity-onset diabetics showed a very slight increase in plasma insulin from a fasting level of 19 f 4 &J/ml. to 24 f 10 au/ml. at 2 minutes and a peak value of 35 -C 9 &U/ml. at 30 minutes. In contrast initial insulin secretory responses of both groups to intravenous tolbutamide were essentially the same with peak values of 69 & 1 J&J/ ml. and 72 + 20 &U/ml. being obtained at 2 minutes from fasting levels of 6 ? 1 pUlm1. and 13 + 5 au/ml. in the control and diabetic groups, respectively. The
response of the maturity-onset diabetics was biphasic and there appeared to be a sustained insulin release for over 30 minutes. The subject with pancreatic diabetes had virtually no response to either glucose or tolbutamide. The rate of disappearance of crystalline beef insulin from the plasma of two subjects with maturity-onset diabetes, two control subjects and the subject with pancreatic diabetes was about the same for all, with half-times ranging from 9-11 minutes after the initial distribution phase. The differences in plasma insulin concentration therefore reflect differences in secretory rate rather than differences in metabolic turnover. The possibility is suggested that the early lesion in p cell function in the maturity-onset diabetics studied here is expressed specifically in a failure of insulin release to be triggered promptly by glucose rather than in a generally defective mechanism for synthesizing and storing insulin. (Metabolism 19: No. 6, June, 409-417, 1970)
N
ONDIABETIC ADULT HUMAN SUBJECTS respond to an orally administered glucose feeding with a brisk rise in the concentration of plasma insulin, which reaches a peak value within 30-60 minutes and then declines within the next two hours to or below fasting control levels.lz” In contrast, subjects with mild maturity-onset diabetes, who are not insulin requiring, characteristicahy exhibit a delayed initial rise in plasma insulin; however, in response to persistent hyperglycemia, plasma insulin may continue to increase for 2-3 From the Radioisotope Service, Veterans Administration Hospital, Bronx, N. Y., and the Department of Medicine, Mount Sinai School of Medicine, New York, N. Y. Received for publication November 17, 1969. NORA VARSANO-AHARON, M.D., ELSA ECHEMENDIA, M.D., ROSALYN S. YALOW, PH.D., AND SOLOMON A. BERSON, M.D.: Radioisotope Service, Veterans Administration Hospital, Bronx, N. Y., and Department of Medicine, Moant Sinai School of Medicine, New York, N. Y. METABOLISM, VOL. 19, No. 6 (JUNE), 1970
409
410
VARSANO-AHARON ET AL.
hours, reaching levels higher than the peak levels shown in non-diabetic controls.l,g Although obese, nondiabetic subjects may show elevated fasting plasma insulin concentrations” and increased insulin-secretory responses to glucose loading,4 the late high plasma insulin levels seen in maturity-onset diabetes during the course of an oral glucose tolerance test are exhibited by lean as well as by obese patients.“-l” The failure of diabetic subjects to respond to hyperglycemia with a rapid outpouring of insulin is more evident on intravenous than on oral administration of glucose. When an intravenous dose of glucose is given as a single rapid injection, the nondiabetic subject displays a peak insulin response in a few minutes whereas in the diabetic, the plasma insulin concentrations rise only slowly to a relatively low plateau .6*7 That the islet /3 cell of the diabetic subject has greater insulin-secretory reserve than is revealed by the response to intravenously administered glucose is suggested, first by the high plasma insulin levels frequently achieved 2-3 hours after oral administration of glucose,‘,” and second by the observation of Simpson et al. I:{ that a greater initial insulin response can be obtained with glucagon than with glucose in maturity-onset diabetics. The present study was initiated to compare the very early plasma insulin responses to intravenously administered glucose and sodium tolbutamide in normal subjects and in patients with maturity-onset diabetes. MATERIALS AND METHODS Intravenous glucose tolerance (IVGTT) and tolbutamide tests were performed in six untreated, nonobese maturity-onset, mild diabetics, six nonobese, nondiabetic controls (hospital personnel or patients with minor illness) and one subject with pancreatic diabetes secondary to chronic pancreatitis who did not require insulin. All subjects were within 100 + 10 per cent of the mean ideal weight. After an overnight fast, subjects were kept recumbent and venous blood samples were drawn a few minutes before and 2, 5. 10, 20, 30, 40, 50. 60, 90, 120 minutes after rapid injection of 25 Gm. glucose in 50 ml. water, or 1 Gm. sodium tolbutamide in 10 ml. water. Both glucose and tolbutamide were given within 30 seconds and timing was started from the onset of injection. Blood samples taken into heparinized syringes were immediately centrifuged under refrigerated conditions: plasma was separated and stored frozen at - 15’ C until assay. The IVGTT preceded the tolbutamide test by two or three days. The IVGTT was repeated in two of the diabetics several days following the tolbutamide test. Plasma glucose concentrations were determined by the method of Hoffmanr-’ in the Technicon autoanalyser. Plasma insulin concentrations were determined by radioimmunoassay1 using crystalline human insulin (Novo) as standard. The rate of disappearance of insulin from plasma was studied in two subjects with maturity-onset diabetes, two control subjects and in one subject with pancreatic diabetes. Following injection of crystalline beef insulin 0.1 U/Kg. body weight, plasma insulin using the injected material as a concentrations were measured by radioimmunoassay’ standard. RESULTS The results of the mean plasma intravenous glucose are given in Fig. glucose in the control group was 95 mean of 129 k 10 mg./lOO ml. in
glucose and insulin concentrations after 1 and Table 1. The mean fasting plasma i- 4 mg. (SEM)/lOO ml., compared to a the maturity-onset diabetic group. Corre-
EARLY
INSULIN
411
RESPONSES
AVERAGE CONTROL
OF 6 LEAN SUBJECTS
AVERAGE OF 6 LEAN MATURITY -ONSET DIABETICS
‘%L~~ 220
Fig l.-Plasma insulin and glucose concentrations during intravenous glucose tolerance tests in nondiabetic and in maturity-onset diabetic subjects.
TIME IN MINUTES
Table 1 .-Plasma
Time (min.)
0 2 5 10 20 30 40 SO 60 90 120
Glucose and Insulin Concentrations During Intravenous Glucose Tolerance Tests (25 Gm.)
Controls Plasma Glucose ‘p&g ‘$$I
95 280 247 220 179 163 137 117 116 100 92
+ 4 f 18 k 14 r 19 f 14 k 11 f 11 f 11 -I- 12 -c 6 ?I 4 ~-
(6)
Plas;s,I;3yh Mean + SkM
8r 216 76 42 31 36 27 22 24 14 14
f +-c k f k f f f -c
2 29 13 7 4 7 4 6 6 5 5
Diabetics Plasma Glucose (ggl$&j
129 318 270 245 245 237 203 187 188 146 127
4 -t 2 -t r k 2 k f zz +-
10 74 35 28 30 1.5 15 15 14 20 17
(6) Plasma Insulin (N/ml. 1 Mean 2 SEM
19 24 32 32 26 3.5 26 24 33 32 28
t 2 _’ i t k -c zk + k k
4 10 9 9 4 9 9 9 8 8 7
sponding plasma insulin concentrations were 8 + 2 pU/ml. and 19 rt 4 pU/ml., respectively. Following injection of glucose the control group responded with a sharp rise in plasma insulin to a peak value of 216 4 29 pU/ml. at 2 minutes (the earliest sample drawn) which was followed by a precipitous drop to 76 + 13 ,&J/ml. at 5 minutes and to 42 + 7 pU/ml. at 10 minutes after the injection. By the end of 20 minutes plasma insulin had declined further to 31 +- 4 @/ml. and after a small, but statistically insignificant, secondary rise at 30 minutes, continued to fall as the glucose concentration returned to near control levels. In contrast to the controls the maturity-onset diabetics showed only a small initial insulin-secretory response to intravenous glucose. At 2 minutes the circulating insulin level had risen only 5 $J/ml. and continued to increase only slightly. The maximal increase was reached at 30 minutes which coincided with the small second peak exhibited by the normal control group. Thereafter, from
VARSANO-AHARON
412
ET AL.
60 to 120 minutes insulin concentrations changed little, remaining above control levels and plasma glucose concentrations fell slowly. The insulin-secretory response to intravenous tolbutamide and the mean plasma glucose concentrations in control and diabetic groups are shown in Fig. 2 and Table 2. The responses in both groups were almost instantaneous; the peak plasma insulin levels were obtained in the first samples drawn at 2 minutes. The mean increments in control and diabetic groups were 63 and 59 $J/m.l., respectively. The response of the normals was characterized by a single pulse with rapid, smooth decline of the circulating insulin to the preinjection levels. In contrast, the response of the maturity-onset diabetics was biphasic. After the initial pulse, there appeared to be a sustained release of insulin, continuing for about 30 minutes, which was then followed by a slow decline in plasma insulin concentration.
Fig. Z.-Plasma insulin and glucose concentrations during intravenous tolbutamide tests in nondiabetic and in maturity-onset diabetic subjects.
Table 2.-Plasma
Time (min.)
0 2 5 10 20 30 40 50 60 90
Glucose and Insulin Concentrations Tolbutamide Test (1 Gm.)
Controls (6) Plasma Insulin Plasma Glucose (mg./loo ml.) NJ/d) Mean t SEM Mean -c SEM
98 f
8
6cl
98 98 83 70 66 65 70 79 82
8 5 4 3 3 4 3 4 4
69 k 43 k 31 * 19 -c 12 k 10 k 9k2 8&2 7+-7
-c +-c t 5 f +?I +-
1 3 3 2 3 2
During Intravenous
Diabetics 6) Plasma Glucose Plasma Insulin (mg./100 ml.) wJ/rnl.) Mean i SEM Mean t SEM
130 118 100 110 102 98 92 93 90 91
I? 18 r 4 k 16 r 16 k 14 c 16 k 13 -e 13 r13 r 8
13 72 42 38 39 36 22 19 18 16
t 5 t 20 t 12 t 7 rf- 9 t 6 ? 5 2 4 k 5 i- 7
413
EARLYINSULINRESPONSES
The integrated increment in plasma insulin concentration over the period O-90 minutes, averaged 40 per cent greater in diabetics than in the control group ( 1139 PU-min./ml. and 8 15 PU-min./ml., respectively). The mean maximum fall in plasma glucose was 31 per cent and occurred at 60 minutes in the maturity-onset diabetics compared to 34 per cent at 40 minutes in the control group. The subject with pancreatic diabetes (Fig. 3) had a fasting plasma insulin concentration of only 1.5 pU/ml. and showed virtually no insulin-secretory response to the IVGTT or to tolbutamide stimulation; the plasma glucose curve remained flat after tolbutamide. There were no significant differences in the rates of disappearance of injected insulin in the control group, the maturity-onset diabetics and the subject with pancreatic diabetes (Fig. 4). DISCUSSION That the initial insulin-secretory response to a glucose load is defective in insulin-non-requiring, maturity-onset diabetics has been recognized from the earliest studies on immunoreactive plasma insulin. Iv2 In itself the characteristic delay in insulin secretion might signify nothing but a diminished capacity to synthesize or release insulin as a consequence of a reduction either in the number of /3 cells or of the function of each of a normal number of p cells. However, the ability to achieve high plasma insulin levels late during the course of an oral GTT associated with an obvious defect in the early secretion of insulinlJ raises the possibility that the secretory capacity of the islet ,Q cell may not be generally depressed in maturity-onset diabetes but rather that there may be a more specific defect characterized by poor responsiveness to certain stimuli. Failure of the gut hormonal insulin-secretagogues to stimulate insulin secretion cannot explain the early delay in insulin secretion since an inadequate insulinsecretory response is even more readily evident when hyperglycemia is induced by intravenous administration of glucose 6*ithan by oral administration of glucose. The poor response to I.V. glucose observed in the present study is in accord with the observations of Perley and Kipnisa and Seltzer et al.? It appears that the glycemic stimulus per se is less than normally effective in maturity-onset diabetes.6,7 In view of the similar rates of disappearance of injected insulin in the five cases studied here we can attribute differences in plasma insulin to differences in secretory rate rather than to differences in metabolic turnover rate. In the present study, the diabetic group showed a significant elevation in fasting blood glucose in spite of significantly higher plasma insulin concentrations. In earlier studies1v2v4too, this association has been observed repeatedly. The elevated plasma insulin concentrations undoubtedly simply reflect the response to a persistent hyperglycemic stimulus for some hours but the persistent hyperglycemia might reflect resistance to or diminished effectiveness of the fasting hyperinsulinemia. So, too, the maintenance of blood glucose concentrations in the diabetics following tolbutamide injection might support the hypothesis of insulin resistance; however, a strong case cannot be made for this suggestion since the initial fall in blood sugar (within the first 10 minutes) was at least as
VARSANO-AHARON
414
INTRAVENOUS GLUCOSE TOLERANCE TEST
ET
AL.
INTRAVENOUS TOLBUTAMIDE TEST
GLUCOSE mg/lOOml
i i
9........._... * .....~,.
“‘\ 2.. “. . . . .. .. . ....*
I ‘. . . .
i
INSULIN /+J$l
1
e-”
. .. . . .. . . ...__
~~
,....
.
1
-0
O-0 TIME
IN MINUTES
Fig. 3.-Plasma insulin and glucose concentrations during intravenous glucose tolerance test and intravenous tolbutamide test in uatient with diabetes secondary I to chronic pancreatitis.
R
TIME
Fig. 4.-Disappearance of exogenous onset diabetic and pancreatic diabetic subtracting fasting endogenous insulin total insulin concentration (x) in each
IN MINUTES
beef insulin from plasma of normal, maturitysubjects. Dotted straight line was obtained by concentration (shown in stippled area) from specimen.
marked in the diabetics as in the control group although the later response was less impressive in the diabetics in spite of the significantly higher plasma insulin concentrations from 1O-90 minutes. It is also relevant to note that a very low level of plasma insulin was adequate
EARLY
INSULIN
RESPONSES
415
to maintain plasma glucose below 130 mg./lOO ml. in the patient with pancreatic diabetes. Whereas the plasma insulin response to IV. glucose was much greater than to I.V. tolbutamide in the control group, quite the reverse was true of the maturity-onset diabetics. Indeed, as noted, the integrated insulin-secretory response to tolbutamide was greater in the diabetics than in the controls but the most striking difference observed was between the responses to tolbutamide and glucose in the diabetic subjects. This difference was most marked in the first five minutes. A rapid release of what must have been preformed insulin15 was triggered by both glucose and tolbutamide in the control group but only by tolbutamide in the diabetics. It appears therefore that the delayed insulinsecretory response to a glycemic stimulus (glucose IV. or P.O. ) seen in maturityonset diabetics who appear to be capable of synthesizing significant quantities of insulin, might be due to a reduced ability to release insulin when a high concentration of glucose is presented to the islet ,Q cell rather than to a lack of available insulin; the availability of mobilizable insulin may be evidenced on the exhibition to tolbutamide. These findings were not observed by Perley and Kipni9 whose diabetic patients showed slightly subnormal responses to I.V. tolbutamide in the 5-15 minutes after injection as well as subnormal responses to I.V. glucose. However Boden et al.16 observed normal insulin-secretory responses to IV. tolbutamide associated with depressed responses to IV. glucose in prediabetic subjects (children of two diabetic parents) who had normal oral glucose tolerance tests and normal cortisone-primed glucose tolerance tests. The mechanism by which changes in glucose concentration regulate insulin secretion is unknown. Presumably the islet p cell is stimulated by some glucose metabolite beyond the stage of glucose-6-phosphate in the glycolytic pathway since 2-deoxy-D-glucose inhibits the insulin-secretory response to glucose17 and since the 6-phosphate derivative of 2-deoxy-D-glucose is known to inhibit competitively the further metabolism of glucose-6-phosphate.ls It is known also that mannoheptulose blocks the insulin-secretory response to glucosel” but not to tolbutamideZO in rabbit pancreas in vitro. In studies carried out in this laboratory,“l it has been demonstrated in man that although 2-deoxy-D-glucose blocks insulin-secretory responses to glucose and glucagon, the responses to tolbutamide and arginine are not impaired. Since sulfonylureas stimulate insulin secretion in vitro in the absence of glucose2” it seems reasonable to conclude that tolbutamide and glucose stimulate insulin secretion by mechanisms that differ in some way and that the effect of tolbutamide is not dependent on the presence of glucose. The present study is consistent with these conclusions. Tolbutamide was clearly an effective insulin-secretagogue in a group of maturity-onset diabetics who responded poorly to glucose intravenously. The possibility is therefore suggested that an early defect in ,Q cell function in maturity-onset diabetes is expressed specifically in a failure of insulin release to be triggered promptly by glucose. That these responses may not be unique to genetic diabetes is suggested by two recent studies in diabetes associated with pancreatic disease. Joffe et al.‘3 found that in patients with chronic pancreatitis due to chronic alcoholism and oral glucose intolerance associated with hypoinsulinism, intravenous tolbutamide
416
VARSANO-AHARON
ET AL.
given 30 minutes after the oral glucose administration caused a prompt doubling of the plasma insulin concentration, which however was less of a rise than in normal controls. Handwerger et al.?* studied patients with cystic fibrosis and moderate to severe oral glucose intolerance. In two such patients given intravenous tolbutamide and two of three given intravenous glucagon two hours after the oral glucose there was a significant rise in plasma insulin beyond that achieved by glucose alone. The authors could not find evidence for significant loss of /3 cells on pathological examination in six patients with the disease and attributed the physiopathologic abnormalities to architectural disorganization of the islets by the fibrotic process. In our patient with pancreatic diabetes there was no significant insulin response to glucose or tolbutamide. In a large group of juvenile diabetics studied by Drash et al.,25 there was no insulin response to glucose, glucagon, tolbutamide or arginine. Similar results except for the absence of testing with glucagon were reported by Parker et al.?” The differences in insulin secretory responses to intravenous tolbutamide and glucose in our patients with maturity-onset diabetes appear much more striking than the differences described above in the patients with pancreatic disease given oral glucose followed by intravenous tolbutamide and suggest an almost complete inability of the /3 cell to respond rapidly to intravenous glucose but normal ability to respond to tolbutamide. However, the distribution between the two kinds of diabetes may be more of degree than of kind and dependent more on the stage than on the nature of the disease. If so, /3 cell dysfunction in diabetics, genetic or acquired through pancreatic damage, is characterized in its initial stages, not so much by an inability to synthesize and/or store insulin but by a unique failure to release it promptly in response to the physiologic stimulus of hyperglycemia. REFERENCES 1. Yalow, R. S., and Berson, S. A.: Immunoassay of endogenous plasma insulin in man. J. Clin. Invest. 39:1157, 1960. 2. -, and -: Plasma insulin concentrations in non-diabetic and early diabetic subjects. Diabetes 9:254, 1960. 3. Rabinowitz, D., and Zierler, K. L.: Forearm metabolism in obesity and its response to intraarterial insulin. Characterization of insulin resistance and evidence for adaptive hyperinsulinism. J. Clin. Invest. 41: 2173, 1962. 4. Karam, J. H., Grodsky. G. M., and Forsbam, P. H.: Excessive insulin response to glucose in obese subjects as measured by immunochemical assay. Diabetes 12: 197, 1963. 5. Yalow, R. S., Glick, S. M., Roth, J., and Berson, S. A.: Plasma insulin and growth hormone levels in obesity and diabetes. Ann. N. Y. Acad. Sci. 131:357, 1965. 6. Perley, M., and Kipnis, D. M.: Plasma
insulin responses to glucose and tolbutamide of normal weight and obese diabetic and nondiabetic subjects. Diabetes 15: 867. 1966. 7. Seltzer, H. S., Allen, E. W., Herron. A. L., Jr., and Brennan, M. T.: Insulin secretion in response to glycemic stimulus: Relation of delayed initial release to carbohydrate intolerance in mild diabetes mellitus. J. Clin. Invest. 46:323, 1967. 8. Berger, S., and Vongaraya, N.: Insulin response to ingested protein in diabetes. Diabetes 15:303, 1966. 9. Graber, A. L., Wood, F. C., Jr., and Williams, R. H.: Serum immunoreactive insulin response during prolonged glucose infusions in nondiabetic and diabetic humans. Diabetes 16:145, 1967. 10. Reaven, G., and Miller, R.: Study of the relationship between glucose and insulin responses to an oral glucose load in man. Diabetes 17:560, 1968. 11. Danowski, T. S., Morgan, C. R.,
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RESPONSES
Sunder, J. H., Moses, C., and Sabeh, G.: Immunoassayable insulin and glucose tolerance in nonobese and obese subjects. Exp. Med. Surg. 25:122, 1967. 12. Buchanan, K. D., and McKiddie, M. T.: Factors determining the plasma insulin response to oral glucose in diabetes mellitus. Diabetes 16:466, 1967. 13. Simpson, R. G., Benedetti, A., Grodsky, G. M., Karam, J. H., and Forsham, P. H.: Early phase of insulin release. Diabetes 17:684, 1968. 14. Hoffman, W. S.: A rapid photoelectric method for the determination of glucose in blood and urine. J. Biol. Chem. 120:51, 1937. 15. Curry, D. L., Bennett, L. L., and Grodsky, G. M.: Dynamics of insulin secretion by the perfused rat pancreas. Endocrinology 83:.572, 1968. 16. Boden, G., Soeldner, J. S., Gleason, R. E., and Marble, A.: Elevated serum human growth hormone and decreased serum insulin in prediabetic males after intravenous tolbutamide and glucose. J. Clin. Invest. 47:729, 1968. 17. R-Candela, J. L., Castrillon, A. M., Martin-Hernandez, D., and Castilla-Cortazar, T.: Effect of glucose and citrate on insulin secretion in vitro by the pancreas pretreated with 2-deoxyglucose. Med. Exp. (BaseI) 11:47, 1964. 18. Wick, A. N., Drury, D. R., Nakada, H. I., and Wolfe, J. B.: Localization of the primary metabolic block produced by 2deoxy glucose. J. Biol. Chem. 224:963, 1957. 19. Coore, H. G., Randle, P. J., Simon,
417 E., Kraicer, P., and Shelesnyak, M. C.: Block of insulin secretion from the pancreas by D-mannoheptulose. Nature 197: 1264, 1963. 20. Coore, H. G., and Randle, P. J.: Regulation of insulin secretion studied with pieces of rabbit pancreas incubated in vitro. Biochem. J. 93:66, 1964. 21. Goldsmith, S. J., Yalow, R. S., and Berson, S. A. : Effect of 2-deoxy-d-glucose on insulin-secretory responses to intravenous glucose, glucagon, tolbutamide and arginine in man. Diabetes (in press). 22. Mehnert, H., Schaefer, G., Kaliampetosis, G., Stuhlfauth, K., and Engelhardt, W.: Insulin secretion of the pancreas in extracorporeal perfusion. II. Perfusions of the pancreas with periston, glucose, carbutamide and biguanides. Klin. Wschr. 40: 1146, 1962. 23. Joffe, B. I., Banks, S., Jackson, W. P. U., Keller, P., O’Reilly, I. G., and Vinik, A. I.: Insulin reserve in patients with chronic pancreatitis. Lancet 2:890, 1968. 24. Handwerger, S., Roth, J., Gorden, P., di Sant’Agnese, P., Carpenter, D. F., and Peter, G.: Glucose intolerance in cystic fibrosis. N. Eng. J. Med. 281:451, 1969. 25. Drash, A., Field, J. B., Garces, L. Y., Kenny, F. M., Mintz, D., and Vazquez, A. M.: Endogenous insulin and growth hormone response in children with newly diagnosed diabetes mellitus. Pediat. Res. 2:94, 1968. 26. Parker, M. L., Pildes, R. S., Chao, K-L., Cornblath, M., and Kipnis, D. M.: Juvenile diabetes mellitus, a deficiency in insulin. Diabetes 17:27, 1968.