Parallel Dysfunctions of Pancreatic A, B and PP Cells in Insulin

Enaocrinol. Japon. 1980, S. R. No.1, 127-133

Parallel Dysfunctions of Pancreatic A, B and PP Cells in Insulin Dependent Diabetes KYOHEI NONAKA, HIROYUKITOYOSHIMA, TOSHIAKIHANAFUSA AND SEIICHIROTARUI The Second Department of Internal Medicine, Osaka University Medical School, Fukushima-ku Osaka 553, Japan

Synopsis

To test the possibility that insulitis might play an etiological role in the pathogenesis of insulin dependent diabetes, functions of 3 kinds of islet constituting cells (A, B and PP cells) were estimated by quantifying secretory responses of glucagon-, C- peptide-and pancreatic polypeptide-producing cells to hyperglycemia and hypoglycemia. In insulin dependent diabetes, all 3 hormonal respo:ses were severely impaired to the same extent. On the other hand, 3 islet cell functions were uniformly but less severly impaired in insulin independent diabetics without a diabetic family history. These results suggest that A, B and PP cells of islet of Langerhans are evenly destroyed in parallel fashion at least in insulin dependent diabetes and in some insulin independent diabetes, suggesting insulitis as a possible cause of these types of diabetes.

Recently insulitis has been recognized to play a certain role in the etiology of Materials and Methods insulin dependent diabetes mellitus. Insulitis occurs as the result of a direct attack on B- 9 insulin dependent and 20 insulin independent diabetics participated in the present study. Of 20 in- cells by viruses such as coxsackie B4(Coleman sulin independent diabetics, 10 were without a family et al., 1973, Yoon et al., 1979) and ence- history of diabetes and 10 with it. Insulin dependent phalomyocarditis virus (Craighead and Mc- diabetes was defined as diabetes of juvenile onset, Lane, 1968) or as the result of a subsequent ketosis prone or uncontrollable without using in- autoimmune mechanism (Irvine, 1977). The sulin. 0.5 g per kg of body weight of glucose was given intravenously to induce hyperglycemia. C- islet cell damage caused by these agents peptide immunoreactivity (CPR) in response to hy- is not limited to any particular cell type perglycemia was regarded as a measure of islet B of 4 islet constituting cells (Bottazzo et al., cell function. After 30 min of glucose administration, 0.2-0.4 U per kg of body weight of purified 1974). Therefore, glucagon producing (A), regular insulin, monocomponent actrapid insulin, insulin producing (B) and pancreatic poly- was injected intravenously to induce hypoglycemia. peptide producing (PP) cells are possibly Immunoreactive glucagon (IRG) and pancreatic all damaged to the same extent. We tried polypeptide (HPP) in response to hypoglycemia were assayed as measures of islet A and PP cell func- to see if these changes could be observed tions. The radioimmunoassay of IRG was de- by quantifying these cell functions in insulin scribed elsewhere (Nonaka and Foa 1969). HPP dependent and insulin independent diabetics was measured by the double antibody method using of our outpatient clinic. highly purified human pancreatic polypeptide for standard and bovine pancreatic polypeptide for iodi- nation, both of which were generous gifts from Dr. R. E. Chance (Lilly Research Laboratories). CPR Endocrinol. Japon. 128 NONAKA et al. December 1980

was assayed by using a kit from Daiichi Isotope Laboratory. Plasma glucose was measured by the glucose oxidase method with Beckman's Glucose Analyzer.

Results

1. Changes of plasma glucose to glucose and insulin administration In normal controls shown in the shadow of the upper panel of Fig. 1, the mean plasma

glucose concentration increased from the basal level of 96•}3 (mean•}S.E.) mg/dl at 0 min to a peak of 368•}7 mg/d/ at 2 min after glucose administration, then decreased

gradually. After insulin injection, the plasma glucose level continued to decline and reached a nadir of 30•}2 mg/dl at 60 min, then increased gradually toward normo-

glycemia. In diabetics, the mean plasma glucose of insulin dependent and independent diabetics increased from the basal level of 141•}9 mg/dl to 391•}17 mg/dl at 2 min in response to intravenous glucose, then decreased much more slowly than in controls

until 30 min. After insulin injection, plasma Fig. 1. Top panel: Mcan•}S.E. of plasma glucose (PG) in response to hyper- and hypoglycemic glucose of diabetics declined as fast as in stimuli in insulin dependent (•œ-•œ) and inde- controls to a nadir of 43•}3 mg/dl at 120 pendent (•›- - -•›) diabetics. Hatchei.shadow rep- min. Due to the fact that initial levels of resent mean•}S.D. of normals. Middle panel: Mean•}S.E. of CPR. Lines and shadow are the plasma glucose in diabetics vary markedly same as in the top panel. Large circles represent from one case to another, the time needed significant changes over basal level. Bottom panel: to reach hypoglycemia differs also from one Mean•}S.E. of IRG. Lines, shadow and large patient to another. All diabetics in the pre- circles are the same as in the middle panel. As- sent study showed hypoglycemic signs and terisk indicate significant differences between two symptoms at various times from 90 to 150 groups of diabetes (*p<0.05, **p<0.025, ***p< 0.01). min, with average glasma glucose of 35•} 2 mg/dl. In insulin independent diabetics the

2. Plasma CPR responses (middle panel plasma CPR gradually increased from the of Fig. 1) basal level of 1.6•}0.1 ng/ml to a blunt

Plasma CPR in normals increased from peak of 2.4•}0.2 ng/m/ at 30 min, then the basal level of 1.4•}0.2 ng/m/ to a peak decreased slowly. In insulin dependent of 4.2•}0.3 ng/m/ at 2 min, than decreased diabetics, the basal CPR level was as low

gradually, remaining elevated for next 60 as 0.6•}0.2 ng/ml, which is significantly min. After 90 min it fell to less than the basal lower than the normal level, and was not level and decreased gradually in response increased significantly by the glucose load. to hypoglycemia until the end of the test. As shown in Fig. 1, CPR levels in two types S. R. No.1 PARALLEL DYSFUNCTIONS OF ISLET HORMONES IN IDDM 129

of 133•}28 pg/ml at 120 min. On the other hand, in insulin dependent diabetics, IRG response to hyperglycemia was poor and there was hardly any response to hypo-

glycemia.

4. HPP responses (Fig. 2) In normal controls (In Fig. 2, represented

by the shaded area at the bottom) plasma HPP tended to decline in response to hyper-

glycemia from a basal level of 54•}17 pg/ml to a minimum of 34•}5 pg/m/ at 10 min after the injection of glucose (Fig. 2) In response to hypoglycemia, plasma HPP rose steeply from a level of 57•}27 mg/ml at 60 min, when the nadir of plasma glucose was achieved, to a peak level of 1320•}200 pg/ ml at 75 min, and then was gradually reduced toward the end of the test. As shown in the bottom panel of Fig. 2, HPP increments of two types of diabetes behaved quite differently in response to hypoglycemia. Namely, a significantly lower response to hypoglycemia was observed in Fig. 2. Top panel: Changes of plasma glucose insulin dependent diabetics than in insulin (mean•}S.E.) to glucose and insulin. Lines and shadow represent the same as Fig. 1. Lower panal: independent diabetics at 105 and 120 min Increment of plasma HPP level (mean•}S.E.) over (p<0.05), in spite of the fact that the dura- basal value. tion of hypoglycemia was rather longer in the insulin dependent group.

of diabetes differed markedly and signifi To quantify a substantial HPP response cantly at any time except at 150 min. to hypoglycemia, we introduced an "HPP- area" index in individual subject. The "HPP-

3. Plasma IRG responses (bottom panel of area" was defined as the area under the Fig. 1) curve of plasma HPP from the blood sam-

In normal controls, as shown in the pling time when plasma glucose first fell shadow at the bottom of the Fig. 1, the to less than 40 mg/dl to the time 30 min mean basal plasma IRG was 55•}11 pg/ml. later. This index was regarded as the It was not changed by the glucose load. substantial response of HPP to hypoglycemia. After insulin administration, however, it The results obtained with this procedure increased to 93•}30 pg/ml at 60 min and are given in Fig. 3. The "HPP-area" in dia- reached a peak of 177•}39 pg/ml at 75 min, betics (15370•}3270 pg•min/ml) was signi- remaining at that level until the end of the ficantly lower than that in normals (28490 test. The plasma IRG of insulin indepen- ±4630pg・min/ml, p<0.05). Among dia- dent diabetics decreased from the basal value betes, the "Hpp-area" in insulin independent of 76•}12 pg/ml to significantly lower diabetics (21630•}4720 pg•Emin/ml) was not levels at 20, 50, 60 and 75 min after glucose significantly different from that in normals, injection, then increased gradually to a peak and the HPP-area in insulin dependent Endocrinol. Japan. 130 NONAKA et al. December 1980 response over basal level (4CPR) to hyperglycemia as a measure of B cell function and maximal IRG respone over basal level (△IRG) to hypoglycemia such as that of A cell function. As shown in the right panel of Fig. 4, CPR response in insulin dependent diabetes was reduced to about one tenth that of normals and that in insulin independent diabetes to one third. In the latter type of diabetes, there was no difference in the response of CPR regarding the presense or absence of diabetic heredity in their first degree relatives. IRG response in insulin dependent diabetes also decreased significantly to one third that of normals . On the other hand, IRG response in insulin independent diabetes did not. However, when we divide the latter type of diabetes into two groups, the one without diabetic heredity responded significantly poorly to insulin induced hypoglycemia and the other group with heredity responded as much as normal controls. This may indicate there was no A cell abnormality in the latter Fig. 3. "HPP-area" in normals and diabetics. "HPP- area" is an index of the substantial response of group. If insulitis plays a role in insulin HPP to hypoglycemia. See text for details. Each dependent diabetes, there must be various column with bar represents mean•}S.E . N : nor- grades of impairment of all islet constituting mals, D: diabetics, IID: insulin indendent dia- cells from normal function to severly da- betics, IDD: insulin dependent diabetics , AN maged stage. To investigate this possibility, (-): diabetics without autonomic neuropathy, the correlation btween pancreatic A cell AN (+): diabetics with autonomic neuropathy , n: numbers of subjects, *p<0.05, **p<0.025, function, maximal IRG increment (y), and ***p<0 .005, NS: not significant. B cell function, maximal CPR increment (X), was examined (Fig. 5). Indeed, there was a positive correlation between the two diabetics (7860•}3020) was significantly parameters: y=47x+52, r=0.63 (p<0.001). smaller than either that in normals or in Accordingly, A and B cell functions seemed insulin independent diabetics (p<0.005, to be impaired in parallel fashion. p<0.025, respectively). Although the "HPP- area" in diabetics with autonomic neuro- 6. Parallel dysfunction of A, B and PP pathy tended to be smaller than that in cells in diabetes mellitus diabetics without autonomic neuropathy, To further ascertain the parallelism of the difference was not significant. the impairment of islet A, B and PP (F) cells, 3 cell functions were depicted in 5, Correlation between A and B cell insulin dependent diabetes, insulin indepen- functions dent diabetes and in normal controls. As To quantify individual pancreatic A and shown in Fig. 6, 3 islet cell functions were B cell function, we adopted maximal CPR damaged in parallel fashion. The result did S. R. No.1 PARALLEL DYSFUNCTIONS OF ISLET HORMONES IN IDDM 131

Fig. 4. Left panel: Maximal IRG increment in hypoglycemia. Each column with bar represents mean•}S.E. Figures in parenthesis indicate the numbers of subjects. Heredity (-) or (+) show insulin independent diabetics without or with diabetic family history. Right panel: Maximal CPR increment in hyperglycemia. Others indicate the same as in left panel.

not contradict the aforementioned hypothesis animals. Encephalomyocarditis virus (Craig- that insulitis may play a pathological role head et al., 1968) and coxsackie B4 virus in insulin dependent diabetes. (Coleman et al., 1973) are well documented as the causative agents for insulitis. Re- cently coxsackie B4 virus was shown to Discussion cause insulitis in man (Yoon et al., 1979). Another factor other than viruses inducing Insulitis-mononuclear cell infiltration insulitis is autoimmune mechanism. Dia- in the islet of Langerhans-has been found betics with autoimmune polyendocrine in juvenile acute onset type diabetes (von deficiency often possess an islet cell antibody Myenburg 1940 ; Gepts et al., 1965). It is which is considered to be a marker for known to be observed in the pancreas of islet cell destruction (Bottazzo et al., 1974). virus induced acute diabetes in experimental Whatever the mechanisms of insulitis may Fig. 5. The correlation between maximal IRG response to hypoglycemia and maximal CPR response to hyperglycemia in diabetic and normal subjects.•œ:insulin dependent diabetics,•›: insulin independent diabetics, •¢: Normal controls

Fig. 6. Parallel dysfunctions of A, B and PP (F) cells in diabetes. Each column represents maximal secretory capacity of A, B and PP (F) cells, mean•}S.E. IDD : insulin dependent diabetics, IID: insulin independent diabetics. S. R. No.1 PARALLEL DYSFUNCTIONS OF ISLET HORMONES IN IDDM 133 be, one result is the destruction of islets. There are some reports that the number References of islets is largely decreased in insulin de- Bottazzo, G. F., A. Florin-Christensen and D. Do- pendent diabetes (Doniach, 1974 ; Saito et niach (1974). Lancet 2, 1279. al., 1978). These findings may indicate Coleman, T. J., D. R. Gamble and K. W. Taylor that islets of Langerhans are destroyed (1973). Br. Med. J. 3, 25. acutely by viral attack or by being dam- Craighead, J. E. and M. F. McLane (1968). Science aged chronically by an autoimmune me- 162, 913. Doniach, I. Immunity and autoimmunity in diabetes chanism in insulin dependent diabetes. mellitus. Excerpta Medica, Amsterdam. p. 175 Therefore if insulitis play a role in the (1974). pathogenesis of insulin dependent diabetes, Gepts, W.(1965). Diabetes 14, 619. dysfunctions of islet cell should be detected Irvine, W. J.(1977). Lancet 1, 638. Meyenburg, H. V.(1940). Schweiz. Med. Wochenschr.. by estimating islet cell secretory function. 70, 554. The results of the present studies indeed Nonaka, K. and P. P. Foa (1969). Proc. Soc. Exp.. disclosed the parallel dysfunction of islet Biol. Med. 130, 330. constituting cells in insulin dependent dia- Saito, K., T. Takahashi, N. Yaginuma and N. Iwama (1978). Tohoku J. Exp. Med. 125, 185. betes and insulin independent diabetes Yoon, J. W., M. Austin, T. Onodera and A. without diabetic family history (Fig. 5 and Notkins (1979). N. Engl. J. Med. 300, 1173. 6). In insulin independent diabetes, those with a family history of diabetes had a near normal pancreatic A cell function, suggesting rather limited specific B cell impairment. An unknown hereditary factor may be involved in this abnormality.