B-cell responses to intravenous glucose and glucagon in non-diabetic twins of patients with Type 1 (insulin-dependent) diabetes mellitus

Diabetologia, Nov 1989

D. A. Heaton, N. R. Lazarus, D. A. Pyke, R. D. G. Leslie

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B-cell responses to intravenous glucose and glucagon in non-diabetic twins of patients with Type 1 (insulin-dependent) diabetes mellitus

Diabetologia B-cell responses to intravenous glucose and glucagon in non-diabetic twins of patients with Type 1 (insulin-dependent) diabetes mellitus D. A. H e a t o n 0 N. R. Lazarus 0 D . A . Pyke a n d R. D. G. Leslie 0 0 Departments of Medicine and Diabetes, Kings College Hospital , London , UK Summary. The B-cells of patients with recently diagnosed Type 1 (insulin-dependent) diabetes may have no response to glucose when the response to glucagon is present but attenuated. This observation suggests that the recognition of glucose is more severely affected than that for non-glucose stimulants. To determine whether a similar selective decrease in glucose response was present before the onset of diabetes we studied two groups of non-diabetic identical twins of patients with recently diagnosed Type 1 diabetes: one group with complement-fixing islet cell antibodies who were at high risk of developing diabetes (four of the five have already developed diabetes) and a group without such antibodies at low risk of developing diabetes. In addition, a group of patients with chronic pancreatitis were studied to control for non-specific damage to the B-cell. Responses to i.v. glucose and i.v. glucagon were compared. Patients with chronic pancreatitis has similar responses to both glucose and glucagon and the responses did not differ from control subjects. The B-cells of the immune positive group showed evidence of pathology because the insulin and C-peptide responses to both stimuli were reduced when compared to either their control subjects or the immune negative twin group. However, the B-cell response to both glucose and glucagon in the immune positive twins was similar. Because the B-cell response to glucose was not less than that to glucagon, a selective destruction of the glucose recognition system cannot be a characteristic of all twins throughout the period before they develop Type 1 diabetes. B-cell responses; i; v; glucose; i; v; glucagon; nondiabetic twins - 9 Springer-Verlag1989 Type 1 (insulin-dependent) diabetes is due to specific destruction o f insulin secreting B-cells o f the pancreas. T h e disease process leading to Type 1 diabetes is slow, occurring o v e r m a n y m o n t h s a n d associated with a g r a d u a l deterioration in glucose t o l e r a n c e a n d a decline in the insulin r e s p o n s e to i.v. glucose [ 1, 2 ]. T h r e e recently d i a g n o s e d patients with Type I diabetes h a v e b e e n r e p o r t e d as h a v i n g no B-cell r e s p o n s e to glucose but an a t t e n u a t e d r e s p o n s e to g l u c a g o n [3]. It is not k n o w n at which stage o f the disease the B-cells first s h o w this p a t t e r n o f r e s p o n s e t h o u g h it has b e e n described in three n o n - d i a b e t i c relatives o f Type 1 diabetic patients [ 2 ]. I n o r d e r to d e t e r m i n e w h e t h e r the B-cells o f n o n - d i a b e t i c individuals genetically susceptible to Type I diabetes c o u l d s h o w a d e c r e a s e d res p o n s e to glucose c o m p a r e d to g l u c a g o n t h r o u g h o u t the p e r i o d b e f o r e t h e y d e v e l o p diabetes, we studied two g r o u p s o f n o n - d i a b e t i c identical twins o f Type 1 diabetic patients; one g r o u p with c o m p l e m e n t - f i x i n g islet cell antibodies w h o were at high risk o f d e v e l o p i n g Type 1 diabetes a n d a n o t h e r g r o u p without islet cell antibodies with a low risk o f d e v e l o p i n g the disease [ 4, 5 ]. I n addition, we studied the responses in two control groups, n o r m a l non-diabetic subjects a n d patients with chronic pancreatitis, in o r d e r to test if a n y changes in the twins' B-cell responses were disease specific. Subjects and methods We studied non-diabetic identical twins wi~fin five years of the diagnosis of Type 1 diabetes in their diabetic twin and a group of patients with chronic pancreatitis as a disease control group. All individuals had had an oral glucose test in order to exclude those with diabetes [ 6 ]. In the twin groups monozygosity was established as previously described [ 4 ]. The presence of islet cell antibodies was established by testing serum with indirect immunofluorescence on a fresh group 0 cryofixed human pancreas for the presence of both conventional and complement-fixing islet cell antibodies [ 5 ]. The titre of each sample was determined by serial doubling dilution. The same pancreas, reagents and incubation conditions gave end-point titres of 32 with the putative islet cell antibody standard (equivalent to 80Juvenile Diabetes Foundation units) being assessed for the Immunology and Diabetes workshop. The twins were divided into two groups according to their risk of developing Type 1 diabetes, as defined by the presence of islet cell antibodies [ 4-6 ]; group 1, five twins who had complement-fixing islet cell antibodies, four of whom have subsequently developed Type 1 diabetes (3, 3, 4 and 16 months after these studies), the fifth now has impaired glucose tolerance; group 2, five twins without islet cell antibodies in whom the risk of developing Type 1 diabetes is small, none have developed diabetes to date [ 7 ]. The patients with chronic pancreatitis were diagnosed clinically and the diagnosis was confirmed by endoscopic retrograde colangiopancreatography [ 8 ]; these patients were studied when clinically well and without symptoms. Each group was compared with unrelated healthy control subjects sought from the local community who were not members of the hospital or medical school community. The five twins in group 1 were compared with six control subjects of similar age (mean 19.0+ 5.1 vs 19.7 +4.8 years), sex (three males in each group) and body mass index (19.0 ___2.1 vs 20.5 + 1.4 kg/m2). The five twins in group 2 were compared with a further six control subjects who were also similar for age (20.3 _ 4.4 vs 19.4 ___3.2 years), sex (four males in each group) and body mass index (20.0+2.4 vs 20.4+ 1.5 kg/m2). The group of seven patients with chronic pancreatitis were compared with a group of seven control subjects who were similar in age (50.8+ 11.1 vs 49.4+ 10.9 years;), sex (five males in each group) and body mass index (23.2+ 3.1 vs 24.3 + 3.7 kg/m2). Each subject had i.v. glucose and i.v. glucagon given on separate occasions no more than two months apart. Subjects or their parents gave informed consent and the study was approved by the King's College Hospital ethical committee. Following an overnight fast, subjects were studied supine at least 15 min after a venous cannula was inserted into the antecubital vein under local anaesthesia. Basal blood samples were taken at - 10 and 0 min after which either glucose (0.5 g/kg dissolved in a 20% dextrose solution) or glucagon (1 mg dissolved in 1.1 ml of distilled water) were injected i.v. over a period of 2 min. Blood samples were then taken at 3, 5, 7.5,10,15, 20, 25 and 30 min, timed from the beginning of the i.v. injection, for the measurement of whole blood glucose, serum insulin and C-peptide. Whole blood glucose was measured by a glucose oxidase method (Yellow Springs Analyser, Ohio, Yellow Springs, USA). Serum insulin and C-peptide were measured by modifications of double antibody radioimmunoassay methods [ 8, 9 ]. All samples were batched so that they were run in the same assays at the same time. i.v. glucose clearance rates were calculated by a method of least squares, using the natural log of the glucose concentration from 15 to 30 rain. Total responses were calculated as areas under the curve and above the basal value from time 0 to 30 min following both glucose and glucagon. Statistical analysis Results are expressed as the mean + SD. The significant variables approximated to a normal distribution in that 66% of the values fell within one SD of their mean. Changes were compared using both a two-tailed Students t-test for paired and unpaired observations and a Wilcoxon's rank sum test; changes were considered significant at p < 0.05. Results Fasting concentrations Both groups of twins, the patients with pancreatitis and their respective control subjects had similar fasting concentrations of glucose, insulin and C-peptide before the glucose and glucagon tests (Tables 1, 2 and 3); except that the C-peptide levels both in the immune negative twins and in control subjects for the pancreatitis group were significantly lower before glucose than glucagon ( p < 0.01, respectively). Responses to i. v. glucose and i. v. glucagon Immune positive group o f twins and their control subjects. The B-cell responses of these twins to both glucose and glucagon were similar and reduced when compared to their control subjects (Table 1); though the difference for the total C-peptide response to glucose did not reach statistical significance. The insulin area response from 0 to 10 min (first phase, p<0.01), from 10 to 30 min (second phase, p < 0.05), and the total response Insulin pmol/L 500" 7oo-i~ 800 600" v (p<0.01) were all significantly reduced in twins compared with control subjects (Fig. 1). The B-cells of the control subjects responded similarly to both stimuli. The B-cells of one patient (M.W.) in the immune positive group three months before he became diabetic showed little response to glucose (total insulin and Cpeptide area responses to i.v. glucose were 0 and 183.3 pmol. 1-1.30 m i n - 1, respectively) as compared with total responses to glucagon (4041 and 6333.3 pmol- 1-1- 30 min-1, respectively); despite similar fasting glucose levels (5.3 and 4.7 mmol/1) and similar insulin and C-peptide basal levels. This patient's total insulin response to glucagon was only 54% of the mean control response (4041 vs 7490pmol. 1-1. 30 m i n - 1). Immune negative group o f twins and their control sub jects. The immune negative twins and their control subjects showed a similar response to the two stimuli (Table 2). Pancreatitis patients vs control subjects: a p < 0.01 Pancreatitis group and their control subjects. The pancre atitis patients and their control subjects showed a similar response to the two stimuli (Tabl[e 3), except that the total C-peptide response to glucagon was significantly lower in the patients with pancreatitis. Discussion Auto-immune endocrine diseases,, of which Type 1 diabetes is believed to be an example, are associated with specific stimulation, inhibition or destruction of a target cell [ 10 ]. The cells involved in these diseases are endocrine cells and it is not surprising that the immune process can target either the signal recognition systems, as in thyrotoxicosis, or the hormone secretion system, as in Hashimoto's thyroiditis. The differential response to glucose and non-glucose secretogogues in patients with Type I diabetes raised the possibility that the destructive process leading to Type 1 diabetes preferentially targets the glucose recognition system of the B-cells. The dose of glucose and glucagon used in this present study gave comparable B-cell responses in three separate groups of normal control subjects and in a group of patients with chronic pancreatitis. The nondiabetic twins we studied without islet cell antibodies were genetically susceptible to Type 1 diabetes but unlikely to develop the disease. These twins had a normal and comparable B-cell response to both glucose and glucagon suggesting that a differential response is not an inherited trait associated with Type I diabetes. In contrast, the B-cells of the immune positive group of twins, four of whom have already ,developed diabetes, had a diminished response to both glucose and glucagon. However, as a group, there was no evidence for a differential response to the two stimuli in the immune positive twins. It therefore appears that the glucose recognition system is not preferentially damaged throughout the pre-diabetic period. It remains possible that a differential response to glucose and non-glucose stimulants could occur at some stage of the pre-diabetic period which a cross-sectional study such as ours would not detect. In support of this argument the B-cells of one of our patients (M.W.) did show a zero response to i.v. glucose and an attenuated response (54% o f normal) to i.v. glucagon. This patient conforms to the three diabetic and three non-diabetic patients previously described who showed zero or markedly reduced insulin secretion to i.v. glucose with substantially higher, though still subnormal, insulin secretion to i.v. glucagon [ 2, 3 ]. The mechanisms which result in such a differential response to glucose and non-glucose stimulants in some pre-diabetic patients are not known. In summary, this study does not support our hypothesis that the initial lesion to the Bcell in Type i diabetes preferentially damages the glucose recognition system. Acknowledgements.This study was supported by the Wellcome Trust, Medical Research Council, Novo A/S, Eli Lilly, Kings College Hospital Joint Research Committee, and the British Diabetic Association. Tests for islet cell antibodies were kindly performed by Dr. G.F. Bottazzo (Middlesex Hospital Medical School). We are grateful to Mrs. J. John and Mrs. A.Corcoran for technical assistance and to Drs. J. Keating and R. Williams for letting us study their patients. 1. Eisenbarth GS ( 1986 ) Type 1 diabetes mellitus: a chronic autoimmune disease . N Engl J Med 304 : 1360 - 1368 2. Srikanta S , Ganda OP , Rabizadeh A , Soeldner JS , Eisenbarth GS ( 1985 ) First-degree relatives of patients with Type 1 diabetes mellitus: islet cell antibodies and abnormal insulin secretion . N Engl J Med 313 : 461 - 464 3. Ganda OP , Srikanta S , Brink SJ , Morris MA , Gleason RE , SoeldnerJS, Eisenbarth GS ( 1984 ) Differential sensitivity to beta cell secretagogues in "early" Type 1 diabetes mellitus . Diabetes 33 : 516 - 521 4. BarnettAH, EffC, Leslie RDG , PykeDA ( 1981 ) Diabetes in identical twins . Diabetologia 20 : 87 - 93 5. Tam AC , Thomas JM , Dean BM , Ingram D , Schwarz G , Bottazzo GF , Gale EAM ( 1988 ) Predicting insulin-dependentdiabetes . Lancet I: 845 - 850 6. National Diabetes Data Group ( 1979 ) Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance . Diabetes 28 : 1039 - 1057 7. SarlesH, Sahel J , Staub JL , BourryJ, Laugier R ( 1979 ) Chronic pancreatitis in the exocrine pancreas . In: Howat HT , Sarles H , (eds). WB Saunders , London, pp 402 8. Morgan DR , Lazarow A ( 1963 ) Immunoassayof insulin:two antibody system . Diabetes 20 : 1 - 9 9. Heding LG ( 1975 ) Radioimmunological determination of human C-peptide in serum . Diabetologia 11 : 541 - 548 10. Drexhage HA , van de Gaag RD , Wulffraat NM ( 1987 ) The multiplicity of stimulating and blocking autoantibodies in relation to thyroxid and extrathyroidal tissues . In: Doniach D , Bottazzo GF (eds) Endocrine and other organ-orientated autoimmune disorders , vol 1 . Bailliere Titndall ,London, pp 125 -140 Received: 6 April 1989 ; and in revised form: 21 July 1989 Dr . R.D.G. Leslie Chafing Cross and Westminster Medical School 17 , Horseferry Road London SWIP 2AR UK


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D. A. Heaton, N. R. Lazarus, D. A. Pyke, R. D. G. Leslie. B-cell responses to intravenous glucose and glucagon in non-diabetic twins of patients with Type 1 (insulin-dependent) diabetes mellitus, Diabetologia, 1989, 814-817, DOI: 10.1007/BF00264913