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Failure of high-dose insulin treatment to increase β-cell insulin content in diabetic non obese diabetic (NOD) mice
Diabetes Research and Clinical Practice 37 (1997) 9 – 14
Failure of high-dose insulin treatment to increase b-cell insulin content in diabetic non obese diabetic (NOD) mice Iben Bache *, Klavs H. Jørgensen, Karsten Buschard Bartholin Instituttet, Kommunehospitalet, DK-1399 Copenhagen K, Denmark Received 25 November 1996; received in revised form 26 May 1997; accepted 6 June 1997
Abstract High-dose insulin treatment in the first period after clinical onset of insulin-dependent diabetes mellitus (IDDM) has been found to reduce diabetic manifestations in humans. The aim of the present study was to examine whether high-dose insulin treatment of newly diagnosed diabetic non obese diabetic (NOD) mice would increase b-cell insulin content after termination of treatment in this experimental IDDM animal model. Newly diagnosed diabetic female NOD mice were randomized into three groups composed of a low-dose insulin treated group (n= 10) injected subcutaneously with 15 IU/kg per day of NPH for 14 days followed by 5 days without insulin, a high-dose insulin treated group (n=8) injected subcutaneously with 150 IU/kg per day of Actrapid for 14 days followed by 5 days without insulin and an untreated group sacrificed 3 days after diagnosis (n= 11). A reference group of age matched non-diabetic untreated female NOD mice (n= 11) was included in the study and sacrificed at the same time as the untreated diabetic mice. No significant difference in the amount of insulin extracted from the total pancreas was found by comparison of the three diabetic groups, consisting of the newly diagnosed untreated mice, the low-dose insulin treated mice and the high-dose insulin treated mice, respectively. The level was about 100-fold less than in the non-diabetic group. Blood glucose values in the two treated diabetic groups were at a high level (median\ 18 mM) throughout the study. We conclude that no increase in b-cell insulin content could be demonstrated in newly diagnosed diabetic NOD mice after early high-dose insulin treatment, at least not in the presence of high blood glucose values. © 1997 Elsevier Science Ireland Ltd. Keywords: NOD-mice; IDDM; Insulin; b-cells
* Corresponding author. Tel.: +45 33383831; fax: +45 33938566. 0168-8227/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 8 - 8 2 2 7 ( 9 7 ) 0 0 0 5 6 - 9
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I. Bache et al. / Diabetes Research and Clinical Practice 37 (1997) 9–14
1. Introduction
2. Materials and methods
Besides conventional insulin treatment of diabetics, aiming at normalizing blood glucose levels, insulin treatment has been used with substantial success for prevention of diabetes in BB Wistar rats [1], female non obese diabetic (NOD) mice [2] and prediabetic humans [3]. An alternative way of insulin treatment has also been applied in a group of newly diagnosed insulin-dependent diabetic patients, receiving insulin by continuous infusion for a 2 week period with maintenance of blood glucose levels between 3.3 and 4.4 mmol/l [4]. During the 2 weeks the experimental group received four times as much insulin as the conventionally insulin-treated control group in the same period. Thereafter both groups were treated conventionally with insulin. After 1 year both b-cell function (as judged from mealstimulated C-peptide secretion) and metabolic control (as judged from glycohemoglobin values) were significantly improved in the experimental group compared with the control group. To our knowledge no information regarding the status of patients has been published since. The effect observed in the above mentioned study was suggested to be a result of a more sustained suppression of the activity of remaining b-cells, thereby prolonging their survival and restoring their ability of functioning. However, we have made the hypothesis, that the effect, alternatively or complementarily, may have been due to neogenesis or replication of b-cells as a consequence of the provoked high insulin concentration in pancreas, since studies have shown IGF-I like activity of insulin [5] and insulin stimulation of islet cell replication in neonatal rat pancreatic monolayer cultures [6]. The aim of the present study was to test our hypothesis by examining whether or not treatment of newly diagnosed diabetic NOD mice with substantially increased insulin doses, compared with a reference treatment, would increase b-cell insulin content after termination of treatment. The aim should be seen in light of the finding that content of immunoreactive insulin in pancreatic extracts correlate positively with b-cell function [7].
2.1. Animals Female NOD mice, 47 (17 weeks old) were purchased from Bomholtgaard, Laboratory Animal Breeding and Research Centre, Ry, Denmark. Of the 47 NOD mice, 36 were newly diagnosed at Bomholtgaard as diabetic, based on clinical appearance, polyuria and glucosuria. On arrival, the diabetic mice were re-examined and all included in the study since they fulfilled the following criteria: blood glucose values ]13 mmol/l (Glucometer Elite™, Blood Glucose Meter, Bayer Diagnostics) and urine glucose values ] 0.1% (TesTape, Lilly, Indianapolis). The 11 non-diabetic NOD mice were tested at Bartholin Instituttet and found to have blood glucose 5 12 mmol/l and no glucosuria. Since their state was found unchanged after 3 days they were all included as non-diabetic control animals. All mice were kept under conventional conditions at Bartholin Instituttet with free access to food and water. They were observed and weighed daily.
2.2. Study design The diabetic mice were randomized into three groups: A, B and C. The non-diabetic mice were referred to as Group D. Group A. Each mouse was injected subcutaneously with 9 ml of 40 IU/ml Protaphan (NPH) MC pork insulin suspension (Novo Nordisk, Bagsværd, Denmark) at 09:00 h, and with 30 ml of saline as placebo at 11:00 and 13:00 h, from the day of arrival and throughout a 14 day period. The daily dose of insulin corresponded to approximately 15 IU/kg. All mice had free access to a 10% glucose solution (besides normal water and food) from 09:00 to 15:00 h daily. Days, 5, after the end of treatment all mice were sacrificed. Of the 13 treated mice, one died and two were killed because of fatal clinical appearance. Group B. Each mouse was injected subcutaneously with 31 ml of 40 IU/ml of Actrapid MC pork insulin solution (Novo Nordisk) three times daily at 09:00, 11:00 and 13:00 h for 14 days
I. Bache et al. / Diabetes Research and Clinical Practice 37 (1997) 9–14
starting from the day of arrival. The total daily dose of insulin corresponded to approximately 150 IU/kg. All mice had free access to 10% glucose water solution (besides normal water and food) from 09:00–15:00 h daily. Days, 5, after the end of treatment, the animals were sacrificed. Of the 12 treated mice, two died and two were killed because of fatal clinical appearance. Group C. These 11 diabetic mice were untreated. They were sacrificed 3 days after arrival. Group D. The 11 non-diabetic mice were sacrificed 3 days after arrival. The treatments of groups A and B were initiated within 4 days after onset of diabetes. In addition to the initial blood glucose measurements blood glucose was also measured by glucometer at day 8 just before the morning insulin injection and at day 15 in the morning.
2.3. Samples The mice were sacrificed by CO2-intoxication. Blood was taken immediately for blood glucose determination, the value was measured in duplicate by the glucose oxidase method. Serum was prepared and stored frozen. The entire pancreas was excised within 5 min after sacrifice, frozen, weighed and stored in a tube at −80°C.
11
quent collections. Of a mixture, 600 ml, of 100 vol 60% (v/v) ethanol and 1 vol 1.4 mol/l H2SO4 was added to the pancreas residue. Homogenization, centrifugation, transfer of supernatant to the syringe, pressing down and collection of the eluate were performed as before. The latter procedure was repeated twice. Finally the cartridges were washed three times with 500 ml of 60% (v/v) ethanol and eluates collected. The pH of the pool of eluates was then adjusted by addition of 1.5 mol/l NH3 ( 220 ml) to a value between 7 and 8 controlled by use of indicator paper. The pool was then evaporated to dryness in a vacuum centrifuge and the residue dissolved in 5 ml of a solution containing 9 g/l NaCl, 10 g/l albumin (Bovine, RIA-grade, Sigma, St. Louis, MO) 3.72 g/l Na2EDTA, 5.77 g/l Na2HPO4,2H2O and 1.05 g/l NaH2PO4,H2O; pH 7.4. A small precipitate was removed by centrifugation and decantation. The final extract was stored at − 20°C. For control of insulin recovery, extractions of normal mouse pancreas were performed in one experiment with added 125I-human-insulin, possessing the same molecular charge as mouse I insulin in acid solution, and in another experiment with 125 I-B-Lys-Arg-A single chain insulin [8], possessing the same molecular charge as mouse II insulin in acid solution. The distribution of radioactivity between fractions was followed by a monitor.
2.4. Extraction of pancreas 2.5. Radioimmunochemical analyses The frozen pancreas (weight×mg) was added (0.9 × +136) ml of 100% ethanol and 100 ml of 1.4 mol/l H2SO4. The mixture was homogenized (Polytron PT 10–35, Kinematica Littau, Switzerland) at room temperature. The tube was centrifuged at 10 000×g and at 15 – 20°C. The supernatant was transferred to a 2 ml syringe placed on top of a C18 Cartridge (Sep-Pak Light, Waters, Milford, Massachusetts) and a CM Cation exchange Cartridge (Sep-Pak Accell™ Plus CM, Waters) in series, the whole system being previously washed with 2 ml of 100% ethanol, followed by 2 ml of water and 2 ml of 60% (v/v) ethanol. The supernatant was pressed slowly through the cartridges (down to the surface of the C18 cartridge) and the eluate collected in a 10 ml tube, which was also used for the subse-
The contents of insulin in the final pancreatic extracts were determined by radioimmunoassay [9]. Rat insulin supplied by Novo Nordisk (composed of Rat I insulin=Mouse I insulin and Rat II insulin=Mouse II insulin) was used as standard. The results were calculated as mean values and considered acceptable, if the underlying determinations deviated less than 25% and could be based on measurements above the detection limit, within the linear part of the standard curve and originating from at least two dilutions in the same assay. Insulin antibodies in serum from diabetic mice were detected by a method described earlier [10] but using 125I-human insulin. The percent of bound radioactivity was calculated and corrected
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I. Bache et al. / Diabetes Research and Clinical Practice 37 (1997) 9–14
Table 1 Group characteristics and results at time of sacrifice Group
A (10)
B (8)
C (11)
D (11)
Diabetic
+
+
+
−
Insulin treatment
15 IU/kg/daya
150 IU/kg/dayb
None
None
Blood glucose (mmol/l) Mean Range
31 15–40
32 26– 45
35 25 – 54
9 7 – 12
Insulin content in pancreas (ng) Mean 55 Range 7–120
88 4– 437
120 16 – 365
20 200 1100 – 39 300
% Insulin binding in serum Corrected mean Corrected range
32 11– 53
5 −6 – 13
— —
24 −4–49
Figures in brackets represent the number of mice in each group. a Protaphan insulin. b Actrapid insulin.
for the co-precipitation of 125I-insulin with non-diabetic mouse serum (group D).
2.6. Statistical analyses The difference in proportions of mice not completing the study in groups A and B was tested with Fishers two-tailed exact test. Differences in initial body weight and body weight change were tested using Student’s t-test. Other parameters were analysed for significant differences using Kruskal–Wallis and Mann – Whitney non-parametric tests. Differences with P 50.05 were considered statistically significant.
3. Results The proportions of mice not completing the entire study in groups A and B were not significantly different (P =0.67). On the day of arrival the body weights of the three groups of diabetic mice (25 g) were not significantly different. During the 14 days of insulin treatment the body weight change in group A ranged between − 4.0 and 4.4 g and in group B between − 4.4 and 1.2 g, and did not differ significantly.
Median blood glucose values (measured by glucometer) at day 8 were 23 and \ 28 mmol/l (non significant) for group A and B respectively. At day 15 the values were \ 28 and 18 mmol/l (PB 0.05), respectively. Blood glucose values (measured by the glucose oxidase method) at time of sacrifice of the three diabetic NOD mice groups, A, B and C, and the non-diabetic group, D, are seen in Table 1. There were no significant differences between the blood glucose values in treated (A and B) compared with newly diagnosed untreated (C) mice and no difference between the two treatments (A and B). The differences to the non-diabetic mice (D) were highly significant (PB 0.001). The insulin content of pancreas was defined as the total amount of insulin in the final extract of pancreas. The recovery of radioactivity in the final pancreatic extract was 95% with added 125I-insulin and 90% with added 125I-B-Lys-Arg-A single chain insulin. Comparison of results from assays performed on the same extract at 2 different days showed no time-dependent changes. The average insulin contents of pancreas are shown in Table 1. The insulin contents were not significantly different between the three diabetic groups (C, A and B) consisting of the newly diagnosed untreated
I. Bache et al. / Diabetes Research and Clinical Practice 37 (1997) 9–14
mice, the low-dose insulin treated mice and the high-dose insulin treated mice, respectively. The non-diabetic control mice (group D) were found to have an average insulin content more than 100-fold greater than each of the three diabetic mice groups (P B 0.001). Insulin antibodies as estimated by percent insulin binding were detected in the two treated groups of diabetic mice (A and B) at a significantly higher level (P B0.005) compared with the newly diagnosed untreated diabetic mice (group C) as seen in Table 1. Between the two groups of treated mice no significant difference was found. The corrected percents of insulin binding (mean 5%) for the untreated diabetic mice (group C) were not significantly different from the corresponding values (mean defined as 0%) for the non-diabetic mice (group D).
4. Discussion The present study has shown that the insulin content in the pancreas of diabetic mice after treatment with high-dose insulin was no greater than that of low-dose insulin treated diabetic mice and that of newly diagnosed untreated diabetic mice. This content was found to be in the order of 1% of that present in the pancreas of non diabetic control mice sacrificed at the same time as the untreated diabetic mice. Accordingly, the serum glucose levels were equally high at the end of the study in the two treated groups and at the same level as for newly diagnosed untreated diabetic mice. These results do not support the hypothesis that high-dose insulin treatment in the first period after clinical onset of IDDM increase b-cell insulin content, at least not in the presence of high blood glucose. In this study strict blood glucose control was not obtained in contrast with the human study [4]. However, this is not important in the light of the purpose of the study: to test whether high-dose insulin treatment, per se, would increase b-cell insulin content after termination of treatment. Protaphan (NPH) was used for low-dose insulin treatment in order to cover the demand of insulin during the day. The low-dose was chosen at the
13
same level, in terms of IU/kg, as used for substitution treatment of diabetic BB rats with protracted-acting insulin [1] because of no available information about substitution therapy of diabetic NOD. In case of high-dose insulin treatment the risk of fatal hypoglycaemia was considered to be too high, if Protaphan had been used. Therefore, the faster acting Actrapid was chosen instead. For further safety the mice were given free access to glucose water during the hours of treatment. In order to avoid introduction of a new differential experimental factor glucose water was also offered to the Protaphan treated group within the same hours. Before completion of the present study some spontaneous and provoked deaths occurred in both insulin treated groups. Presumably these events do not invalidate comparisons of results because no significant difference was found between the groups, regarding death rates. The remaining mice were sacrificed 5 days after the last day of treatment, allowing a period of time for the b-cells to recover from the functional depression induced by exogeneous insulin administration [11]. The period was limited to 5 days in order to minimize the possibility of the disease mechanism to resume. The extraction method as described was developed with the purpose of obtaining high recovery of insulin combined with removal of proteolytic enzymes. It was rendered likely that the recovery of mouse insulin was more than 90% and we found no evidence of proteolytic degradation of insulin in the final aqueous extract. A raised level of insulin antibodies was detected in the insulin treated compared with the untreated diabetic mice probably due to injections of unhomologous insulin. Despite a ten-fold difference in amount of injected insulin the percent insulin binding level was not significantly different between the two groups of insulin treated mice, indicating that the intended difference in exposure to free insulin was not masked by binding to antibodies. Previous studies of humans have shown improved b-cell function after high-dose insulin treatment the first 2 or 4 weeks after IDDM diagnosis. In the studies of both Madsbad [12]
I. Bache et al. / Diabetes Research and Clinical Practice 37 (1997) 9–14
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and Perlman [13] the effect did not last beyond 3 months. In contrast, Shah et al. [4] showed a higher plasma level of meal stimulated C-peptide and lower glycohemoglobin values still after 1 year compared with the conventionally treated group. The effect was suggested by the authors to be a result of a more sustained suppression of the activity of remaining b-cells, thereby prolonging their survival and restoring their ability of functioning. Based on considerations of a positive effect of high-dose insulin on b-cell neogenesis and/or replication, rather than b-cell conservation, we have examined, whether the insulin content of the endocrine pancreas was affected in diabetic NOD mice after termination of high-dose insulin treatment. Obviously it was not the case. On the other hand, the result of the present study does not rule out the conception of strict glycaemic control as a cause of improvement of the functional state of existent b-cells in IDDM.
Acknowledgements The authors wish to thank Aage Vølund (Novo Research Institute) for statistical advice and Novo Nordisk A/S for supply of 125I-insulins, anti-insulin serum for radioimmunoassay and insulin preparations for injection.
References [1] C.F. Gotfredsen, K. Buschard, E.K. Frandsen, Reduction of diabetes incidence of BB Wistar rats by early prophylactic insulin treatment of diabetes-prone animals, Diabetologia 28 (1985) 933–935. [2] M.A. Atkinson, N.K. Maclaren, R. Luchetta, Insulitis and diabetes in NOD mice reduced by prophylactic insulin therapy, Diabetes 39 (1990) 933–937.
.
[3] R.J. Keller, G.S. Eisenbarth, R.A. Jackson, Insulin prophylaxis in individuals at high risk of type 1 diabetes, Lancet 341 (1993) 927 – 928. [4] S.C. Shah, J.I. Malone, N.E. Simpson, A randomized trial of intensive insulin therapy in newly diagnosed insulin-dependent diabetes mellitus, N. Engl. J. Med. 320 (1989) 550 – 554. [5] G.L. King, C.R. Kahn, M.M. Rechler, S.P. Nissley, Direct demonstration of separate receptors for growth and metabolic activities of insulin and multiplicationstimulating activity (an insulinlike growth factor) using antibodies to the insulin receptor, J. Clin. Invest. 66 (1980) 130 – 140. [6] A. Rabinovitch, C. Quigley, T. Russell, Y. Patel, D.H. Mintz, Insulin and multiplication stimulating activity (an insulin-like growth factor) stimulate islet b-cell replication in neonatal rat pancreatic monolayer cultures, Diabetes 31 (1982) 160 – 164. [7] D.K. McCulloch, D.J. Koerker, S.E. Kahn, S. BonnerWeir, J.P. Palmer, Correlations of in vivo b-cell function tests with b-cell mass and pancreatic insulin content in streptozotocin-administered baboons, Diabetes 40 (1991) 673 – 679. [8] L. Thim, M.T. Hansen, K. Norris, et al., Secretion and processing of insulin precursors in yeast, Proc. Natl. Acad. Sci. USA 83 (1986) 6766 – 6770. [9] L.G. Heding, Determination of total serum insulin (IRI) in insulin-treated diabetic patients, Diabetologia 8 (1972) 260 – 266. [10] J. Schlichtkrull, J. Brange, Aa.H. Christiansen, et al., Monocomponent insulin and its clinical implications, Horm. Metab. Res. (Suppl. Ser.) 5 (1974) 134 – 143. [11] Y.T. Kruszynska, L. Villa-Komaroff, P.A. Halban, Islet b-cell dysfunction and the time course of recovery following chronic overinsulinisation of normal rats, Diabetologia 31 (1988) 621 – 626. [12] S. Madsbad, T. Krarup, O.K. Faber, C. Binder, L. Regeur, The transient effect of strict glycaemic control on B cell function in newly diagnosed Type 1 (insulindependent) diabetic patients, Diabetologia 22 (1982) 16 – 20. [13] K. Perlman, R.M. Ehrlich, R.M. Filler, A.M. Albisser, Sustained normoglycemia in newly diagnosed type 1 diabetic subjects. Short-term effects and 1-year follow-up, Diabetes 33 (1984) 995 – 1001.
Failure of high-dose insulin treatment to increase b-cell insulin content in diabetic non obese diabetic (NOD) mice Iben Bache *, Klavs H. Jørgensen, Karsten Buschard Bartholin Instituttet, Kommunehospitalet, DK-1399 Copenhagen K, Denmark Received 25 November 1996; received in revised form 26 May 1997; accepted 6 June 1997
Abstract High-dose insulin treatment in the first period after clinical onset of insulin-dependent diabetes mellitus (IDDM) has been found to reduce diabetic manifestations in humans. The aim of the present study was to examine whether high-dose insulin treatment of newly diagnosed diabetic non obese diabetic (NOD) mice would increase b-cell insulin content after termination of treatment in this experimental IDDM animal model. Newly diagnosed diabetic female NOD mice were randomized into three groups composed of a low-dose insulin treated group (n= 10) injected subcutaneously with 15 IU/kg per day of NPH for 14 days followed by 5 days without insulin, a high-dose insulin treated group (n=8) injected subcutaneously with 150 IU/kg per day of Actrapid for 14 days followed by 5 days without insulin and an untreated group sacrificed 3 days after diagnosis (n= 11). A reference group of age matched non-diabetic untreated female NOD mice (n= 11) was included in the study and sacrificed at the same time as the untreated diabetic mice. No significant difference in the amount of insulin extracted from the total pancreas was found by comparison of the three diabetic groups, consisting of the newly diagnosed untreated mice, the low-dose insulin treated mice and the high-dose insulin treated mice, respectively. The level was about 100-fold less than in the non-diabetic group. Blood glucose values in the two treated diabetic groups were at a high level (median\ 18 mM) throughout the study. We conclude that no increase in b-cell insulin content could be demonstrated in newly diagnosed diabetic NOD mice after early high-dose insulin treatment, at least not in the presence of high blood glucose values. © 1997 Elsevier Science Ireland Ltd. Keywords: NOD-mice; IDDM; Insulin; b-cells
* Corresponding author. Tel.: +45 33383831; fax: +45 33938566. 0168-8227/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 1 6 8 - 8 2 2 7 ( 9 7 ) 0 0 0 5 6 - 9
10
I. Bache et al. / Diabetes Research and Clinical Practice 37 (1997) 9–14
1. Introduction
2. Materials and methods
Besides conventional insulin treatment of diabetics, aiming at normalizing blood glucose levels, insulin treatment has been used with substantial success for prevention of diabetes in BB Wistar rats [1], female non obese diabetic (NOD) mice [2] and prediabetic humans [3]. An alternative way of insulin treatment has also been applied in a group of newly diagnosed insulin-dependent diabetic patients, receiving insulin by continuous infusion for a 2 week period with maintenance of blood glucose levels between 3.3 and 4.4 mmol/l [4]. During the 2 weeks the experimental group received four times as much insulin as the conventionally insulin-treated control group in the same period. Thereafter both groups were treated conventionally with insulin. After 1 year both b-cell function (as judged from mealstimulated C-peptide secretion) and metabolic control (as judged from glycohemoglobin values) were significantly improved in the experimental group compared with the control group. To our knowledge no information regarding the status of patients has been published since. The effect observed in the above mentioned study was suggested to be a result of a more sustained suppression of the activity of remaining b-cells, thereby prolonging their survival and restoring their ability of functioning. However, we have made the hypothesis, that the effect, alternatively or complementarily, may have been due to neogenesis or replication of b-cells as a consequence of the provoked high insulin concentration in pancreas, since studies have shown IGF-I like activity of insulin [5] and insulin stimulation of islet cell replication in neonatal rat pancreatic monolayer cultures [6]. The aim of the present study was to test our hypothesis by examining whether or not treatment of newly diagnosed diabetic NOD mice with substantially increased insulin doses, compared with a reference treatment, would increase b-cell insulin content after termination of treatment. The aim should be seen in light of the finding that content of immunoreactive insulin in pancreatic extracts correlate positively with b-cell function [7].
2.1. Animals Female NOD mice, 47 (17 weeks old) were purchased from Bomholtgaard, Laboratory Animal Breeding and Research Centre, Ry, Denmark. Of the 47 NOD mice, 36 were newly diagnosed at Bomholtgaard as diabetic, based on clinical appearance, polyuria and glucosuria. On arrival, the diabetic mice were re-examined and all included in the study since they fulfilled the following criteria: blood glucose values ]13 mmol/l (Glucometer Elite™, Blood Glucose Meter, Bayer Diagnostics) and urine glucose values ] 0.1% (TesTape, Lilly, Indianapolis). The 11 non-diabetic NOD mice were tested at Bartholin Instituttet and found to have blood glucose 5 12 mmol/l and no glucosuria. Since their state was found unchanged after 3 days they were all included as non-diabetic control animals. All mice were kept under conventional conditions at Bartholin Instituttet with free access to food and water. They were observed and weighed daily.
2.2. Study design The diabetic mice were randomized into three groups: A, B and C. The non-diabetic mice were referred to as Group D. Group A. Each mouse was injected subcutaneously with 9 ml of 40 IU/ml Protaphan (NPH) MC pork insulin suspension (Novo Nordisk, Bagsværd, Denmark) at 09:00 h, and with 30 ml of saline as placebo at 11:00 and 13:00 h, from the day of arrival and throughout a 14 day period. The daily dose of insulin corresponded to approximately 15 IU/kg. All mice had free access to a 10% glucose solution (besides normal water and food) from 09:00 to 15:00 h daily. Days, 5, after the end of treatment all mice were sacrificed. Of the 13 treated mice, one died and two were killed because of fatal clinical appearance. Group B. Each mouse was injected subcutaneously with 31 ml of 40 IU/ml of Actrapid MC pork insulin solution (Novo Nordisk) three times daily at 09:00, 11:00 and 13:00 h for 14 days
I. Bache et al. / Diabetes Research and Clinical Practice 37 (1997) 9–14
starting from the day of arrival. The total daily dose of insulin corresponded to approximately 150 IU/kg. All mice had free access to 10% glucose water solution (besides normal water and food) from 09:00–15:00 h daily. Days, 5, after the end of treatment, the animals were sacrificed. Of the 12 treated mice, two died and two were killed because of fatal clinical appearance. Group C. These 11 diabetic mice were untreated. They were sacrificed 3 days after arrival. Group D. The 11 non-diabetic mice were sacrificed 3 days after arrival. The treatments of groups A and B were initiated within 4 days after onset of diabetes. In addition to the initial blood glucose measurements blood glucose was also measured by glucometer at day 8 just before the morning insulin injection and at day 15 in the morning.
2.3. Samples The mice were sacrificed by CO2-intoxication. Blood was taken immediately for blood glucose determination, the value was measured in duplicate by the glucose oxidase method. Serum was prepared and stored frozen. The entire pancreas was excised within 5 min after sacrifice, frozen, weighed and stored in a tube at −80°C.
11
quent collections. Of a mixture, 600 ml, of 100 vol 60% (v/v) ethanol and 1 vol 1.4 mol/l H2SO4 was added to the pancreas residue. Homogenization, centrifugation, transfer of supernatant to the syringe, pressing down and collection of the eluate were performed as before. The latter procedure was repeated twice. Finally the cartridges were washed three times with 500 ml of 60% (v/v) ethanol and eluates collected. The pH of the pool of eluates was then adjusted by addition of 1.5 mol/l NH3 ( 220 ml) to a value between 7 and 8 controlled by use of indicator paper. The pool was then evaporated to dryness in a vacuum centrifuge and the residue dissolved in 5 ml of a solution containing 9 g/l NaCl, 10 g/l albumin (Bovine, RIA-grade, Sigma, St. Louis, MO) 3.72 g/l Na2EDTA, 5.77 g/l Na2HPO4,2H2O and 1.05 g/l NaH2PO4,H2O; pH 7.4. A small precipitate was removed by centrifugation and decantation. The final extract was stored at − 20°C. For control of insulin recovery, extractions of normal mouse pancreas were performed in one experiment with added 125I-human-insulin, possessing the same molecular charge as mouse I insulin in acid solution, and in another experiment with 125 I-B-Lys-Arg-A single chain insulin [8], possessing the same molecular charge as mouse II insulin in acid solution. The distribution of radioactivity between fractions was followed by a monitor.
2.4. Extraction of pancreas 2.5. Radioimmunochemical analyses The frozen pancreas (weight×mg) was added (0.9 × +136) ml of 100% ethanol and 100 ml of 1.4 mol/l H2SO4. The mixture was homogenized (Polytron PT 10–35, Kinematica Littau, Switzerland) at room temperature. The tube was centrifuged at 10 000×g and at 15 – 20°C. The supernatant was transferred to a 2 ml syringe placed on top of a C18 Cartridge (Sep-Pak Light, Waters, Milford, Massachusetts) and a CM Cation exchange Cartridge (Sep-Pak Accell™ Plus CM, Waters) in series, the whole system being previously washed with 2 ml of 100% ethanol, followed by 2 ml of water and 2 ml of 60% (v/v) ethanol. The supernatant was pressed slowly through the cartridges (down to the surface of the C18 cartridge) and the eluate collected in a 10 ml tube, which was also used for the subse-
The contents of insulin in the final pancreatic extracts were determined by radioimmunoassay [9]. Rat insulin supplied by Novo Nordisk (composed of Rat I insulin=Mouse I insulin and Rat II insulin=Mouse II insulin) was used as standard. The results were calculated as mean values and considered acceptable, if the underlying determinations deviated less than 25% and could be based on measurements above the detection limit, within the linear part of the standard curve and originating from at least two dilutions in the same assay. Insulin antibodies in serum from diabetic mice were detected by a method described earlier [10] but using 125I-human insulin. The percent of bound radioactivity was calculated and corrected
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I. Bache et al. / Diabetes Research and Clinical Practice 37 (1997) 9–14
Table 1 Group characteristics and results at time of sacrifice Group
A (10)
B (8)
C (11)
D (11)
Diabetic
+
+
+
−
Insulin treatment
15 IU/kg/daya
150 IU/kg/dayb
None
None
Blood glucose (mmol/l) Mean Range
31 15–40
32 26– 45
35 25 – 54
9 7 – 12
Insulin content in pancreas (ng) Mean 55 Range 7–120
88 4– 437
120 16 – 365
20 200 1100 – 39 300
% Insulin binding in serum Corrected mean Corrected range
32 11– 53
5 −6 – 13
— —
24 −4–49
Figures in brackets represent the number of mice in each group. a Protaphan insulin. b Actrapid insulin.
for the co-precipitation of 125I-insulin with non-diabetic mouse serum (group D).
2.6. Statistical analyses The difference in proportions of mice not completing the study in groups A and B was tested with Fishers two-tailed exact test. Differences in initial body weight and body weight change were tested using Student’s t-test. Other parameters were analysed for significant differences using Kruskal–Wallis and Mann – Whitney non-parametric tests. Differences with P 50.05 were considered statistically significant.
3. Results The proportions of mice not completing the entire study in groups A and B were not significantly different (P =0.67). On the day of arrival the body weights of the three groups of diabetic mice (25 g) were not significantly different. During the 14 days of insulin treatment the body weight change in group A ranged between − 4.0 and 4.4 g and in group B between − 4.4 and 1.2 g, and did not differ significantly.
Median blood glucose values (measured by glucometer) at day 8 were 23 and \ 28 mmol/l (non significant) for group A and B respectively. At day 15 the values were \ 28 and 18 mmol/l (PB 0.05), respectively. Blood glucose values (measured by the glucose oxidase method) at time of sacrifice of the three diabetic NOD mice groups, A, B and C, and the non-diabetic group, D, are seen in Table 1. There were no significant differences between the blood glucose values in treated (A and B) compared with newly diagnosed untreated (C) mice and no difference between the two treatments (A and B). The differences to the non-diabetic mice (D) were highly significant (PB 0.001). The insulin content of pancreas was defined as the total amount of insulin in the final extract of pancreas. The recovery of radioactivity in the final pancreatic extract was 95% with added 125I-insulin and 90% with added 125I-B-Lys-Arg-A single chain insulin. Comparison of results from assays performed on the same extract at 2 different days showed no time-dependent changes. The average insulin contents of pancreas are shown in Table 1. The insulin contents were not significantly different between the three diabetic groups (C, A and B) consisting of the newly diagnosed untreated
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mice, the low-dose insulin treated mice and the high-dose insulin treated mice, respectively. The non-diabetic control mice (group D) were found to have an average insulin content more than 100-fold greater than each of the three diabetic mice groups (P B 0.001). Insulin antibodies as estimated by percent insulin binding were detected in the two treated groups of diabetic mice (A and B) at a significantly higher level (P B0.005) compared with the newly diagnosed untreated diabetic mice (group C) as seen in Table 1. Between the two groups of treated mice no significant difference was found. The corrected percents of insulin binding (mean 5%) for the untreated diabetic mice (group C) were not significantly different from the corresponding values (mean defined as 0%) for the non-diabetic mice (group D).
4. Discussion The present study has shown that the insulin content in the pancreas of diabetic mice after treatment with high-dose insulin was no greater than that of low-dose insulin treated diabetic mice and that of newly diagnosed untreated diabetic mice. This content was found to be in the order of 1% of that present in the pancreas of non diabetic control mice sacrificed at the same time as the untreated diabetic mice. Accordingly, the serum glucose levels were equally high at the end of the study in the two treated groups and at the same level as for newly diagnosed untreated diabetic mice. These results do not support the hypothesis that high-dose insulin treatment in the first period after clinical onset of IDDM increase b-cell insulin content, at least not in the presence of high blood glucose. In this study strict blood glucose control was not obtained in contrast with the human study [4]. However, this is not important in the light of the purpose of the study: to test whether high-dose insulin treatment, per se, would increase b-cell insulin content after termination of treatment. Protaphan (NPH) was used for low-dose insulin treatment in order to cover the demand of insulin during the day. The low-dose was chosen at the
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same level, in terms of IU/kg, as used for substitution treatment of diabetic BB rats with protracted-acting insulin [1] because of no available information about substitution therapy of diabetic NOD. In case of high-dose insulin treatment the risk of fatal hypoglycaemia was considered to be too high, if Protaphan had been used. Therefore, the faster acting Actrapid was chosen instead. For further safety the mice were given free access to glucose water during the hours of treatment. In order to avoid introduction of a new differential experimental factor glucose water was also offered to the Protaphan treated group within the same hours. Before completion of the present study some spontaneous and provoked deaths occurred in both insulin treated groups. Presumably these events do not invalidate comparisons of results because no significant difference was found between the groups, regarding death rates. The remaining mice were sacrificed 5 days after the last day of treatment, allowing a period of time for the b-cells to recover from the functional depression induced by exogeneous insulin administration [11]. The period was limited to 5 days in order to minimize the possibility of the disease mechanism to resume. The extraction method as described was developed with the purpose of obtaining high recovery of insulin combined with removal of proteolytic enzymes. It was rendered likely that the recovery of mouse insulin was more than 90% and we found no evidence of proteolytic degradation of insulin in the final aqueous extract. A raised level of insulin antibodies was detected in the insulin treated compared with the untreated diabetic mice probably due to injections of unhomologous insulin. Despite a ten-fold difference in amount of injected insulin the percent insulin binding level was not significantly different between the two groups of insulin treated mice, indicating that the intended difference in exposure to free insulin was not masked by binding to antibodies. Previous studies of humans have shown improved b-cell function after high-dose insulin treatment the first 2 or 4 weeks after IDDM diagnosis. In the studies of both Madsbad [12]
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and Perlman [13] the effect did not last beyond 3 months. In contrast, Shah et al. [4] showed a higher plasma level of meal stimulated C-peptide and lower glycohemoglobin values still after 1 year compared with the conventionally treated group. The effect was suggested by the authors to be a result of a more sustained suppression of the activity of remaining b-cells, thereby prolonging their survival and restoring their ability of functioning. Based on considerations of a positive effect of high-dose insulin on b-cell neogenesis and/or replication, rather than b-cell conservation, we have examined, whether the insulin content of the endocrine pancreas was affected in diabetic NOD mice after termination of high-dose insulin treatment. Obviously it was not the case. On the other hand, the result of the present study does not rule out the conception of strict glycaemic control as a cause of improvement of the functional state of existent b-cells in IDDM.
Acknowledgements The authors wish to thank Aage Vølund (Novo Research Institute) for statistical advice and Novo Nordisk A/S for supply of 125I-insulins, anti-insulin serum for radioimmunoassay and insulin preparations for injection.
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