Characterization of pseudo-islets formed from pancreatic islet cell suspensions of neonatal rats.

Well-preserved pancreatic islet cell suspensions were prepared from islets of Langerhans of neonatal rats by gentle trypsin treatment. Within a culture period of 4-6 days the islet cells reaggregate spontaneously and form pseudo-islets of different size and of a variable insulin content. While the ratio of insulin to glucagon in isolated islets of Langerhans is constant (18 +/- 1.9), the hormone ratio of the pseudo-islets is strongly variable and increased, indicating an excess of insulin. Glucose enhancement from 1.5 mmoles/l to 15 mmoles/l results in a significant stimulation of (pro)insulin biosynthesis whereas insulin secretion of the pseudo-islets is only slightly increased. At high glucose concentration (15 mmoles/l) insulin secretion of the pseudo-islets can be potentiated (by a factor of 4.5 +/- 0.46) by 3-isobutyl-l-methylxanthine (IBMX). Compared with the initial islet cell suspension, the cell aggregation during pseudo-islet formation did not result in an enhanced secretory response on glucose stimulation.

Modulation of the effect of humoral-mediated cytotoxicity on isolated rat pancreatic islets by glucose.

Rat islets of Langerhans exposed for 20 h at high glucose (20 mmol/l) to 50% or 20% experimentally raised rabbit anti-rat islet cell surface antiserum (ICSA-positive serum) plus complement exhibited an irreversible loss of glucose-stimulated insulin secretion. In contrast, islets treated with 50% ICSA-positive serum at low glucose (5.5 mmol/l) could overcome this alteration within a subsequent 48 h recovery period at 10 mmol/l glucose in the absence of ICSA, and islets affected at 5.5 mmol/l glucose by 20% ICSA-positive serum even retained the insulin secretory potential and responded on glucose challenge already immediately after the removal of ICSA. The islet insulin content was reduced by the effect of 50% as well as of 20% ICSA-positive serum and complement irrespective of whether the glucose level amounted to 5.5 or 20 mmol/l during serum influence. However, islets altered in a normoglycaemic environment at 5.5 mmol/l glucose by 20% ICSA-positive serum restored their insulin content up to the level of control islets, whereas those islets affected under hyperglycaemic conditions at 20 mmol/l glucose only partially recovered. Thus, beta-cell loss and/or impairment of the insulin secretory mechanisms result from the simultaneous action of humoral-mediated anti-islet cytotoxicity and elevated glucose level, and cause the diminished insulin secretory potential of the islets. These results support the hypothesis that decreasing the insulin secretory activity of beta cells may protect them from cytotoxic immunological attacks.

Enhanced synthesis, storage, and secretion of insulin in pancreatic islets derived from obese subjects.

Insulin biosynthesis, content, and secretion were investigated in islets derived from pancreas specimens of normal weight (100.4 +/- 1.1% of ideal body weight) and obese (137.2 +/- 5.9% of ideal weight) patients. The pancreatic islets from the obese subjects were characterized by a significantly enhanced glucose-induced insulin secretion and biosynthesis and by an insulin content that was nearly double when compared with islets from the nonobese subjects. The results support the hypothesis that an enhanced beta-cell reactivity significantly contributes to the insulin hyperresponse observed in the obese state.

Glucose tolerance behaviour before the onset of type I (insulin-dependent) diabetes in young people as a predictor of the further course of the disease: a retrospective analysis of 33 cases.

A study was made of glucose tolerance and insulin secretion in 33 persons who later developed insulin-dependent diabetes (aged 4-24 years) and observation continued further in the first years after manifestation. Patients who developed the typical labile type of diabetes were of normal weight and had either normal glucose tolerance tests before diagnosis or had impaired glucose tolerance (IGT) for a short interval of 2-16 months. Subjects with IGT over a significantly (p less than 0.01) longer period of 32.30 +/- 6.25 (normal body weight) or 94.71 +/- 20.62 (obese) months developed a milder form of diabetes with retarded insulin dependency in obese subjects. The severe and mild form of IDDM are distinct with respect to insulin requirement (0.75 +/- 0.03 or 0.28 +/- 0.04 U/kg b.w., P less than 0.01) and glucagon stimulated C-peptide (0.18 +/- 0.05 or 1.41 +/- 0.27, P less than 0.01) in the first 2.5-3.5 years after onset. The two forms were not different regarding HLA-DR antigens. Islet cell surface antibodies investigated in 15 probands at 27 occasions before diabetes onset had no prognostic value. The development of a mild form of IDDM may be expected in cases with pre-existing IGT for more than one year. The insulin secretion is of low predictive value under these conditions. The observation is of practical use and theoretical interest.

Insulin treatment improves islet function in type 2 diabetic Chinese hamsters.

To study whether normalization of hyperglycemia improves islet function in long-standing type 2 diabetes, hyperglycemic CHIG/Han subline of the genetic type 2 diabetic Chinese hamster (>15 mmol/l: n=23) were either treated with insulin implants (liberating 1 U/day) or vehicle for two weeks. Islets were isolated and incubated for 3 h in the presence of 10 mmol/l glucose with or without 0.1 mmol/l 3-isobutyl-1-methylxanthine (IBMX). Specimens were also taken for immunocytochemical analysis of insulin cells. Glucose-stimulated insulin secretion was reduced by 83% in the vehicle-treated diabetic hamsters compared to non-diabetic controls (p<0.001). This impairment was not improved by the two-week insulin treatment. IBMX potentiated glucose-stimulated insulin secretion; this effect was markedly reduced in vehicle-treated diabetics compared to controls (p<0.001). In fact, the linear relation between IBMX-potentiated and glucose-stimulated insulin secretion in controls was absent in islets from diabetic animals. The two week insulin treatment normalized this relation, although still the total insulin secretory response to IBMX and glucose was lower than in controls. Furthermore, the islet insulin content was significantly increased by the 2 week normalization of glucose and, finally, the severe degranulation and lowering of insulin staining in islet beta cells in diabetic animals were markedly improved by insulin treatment. The results suggest that two-weeks of normalization of glycemia in long-standing type 2 diabetes in non-obese Chinese hamster improves beta cell signaling induced by the cyclic AMP pathway in conjunction with improved islet insulin content and beta cell morphology.

Impaired beta-cell function in the Chinese hamster CHIG/Han subline.

Plasma glucose and insulin levels were measured in the genetically diabetic CHIG/Han and the diabetes-resistant CHIA/Han subline of the Chinese hamster. At 31 +/- 8 wk of age, the CHIG hamsters were grouped into nondiabetic, mildly and severely diabetic, according to their levels of glycemia. Hyperinsulinemia, occurring in nondiabetic and mildly diabetic CHIG hamsters, was attenuated in severely diabetic animals. Light microscopy and immunohistochemistry revealed initial beta-cell hyperplasia, followed by extensive degranulation and loss of immunoreactive insulin in islets of severely diabetic animals. Staining intensity of glucagon-immunoreactive cells was unchanged in nondiabetic and mildly diabetic animals, but was increased in islets from the severely diabetic hamsters. A static incubation system was used to examine the insulin response of pancreatic islets isolated from the diabetic and nondiabetic CHIG hamsters, and the diabetes-resistant CHIA subline. Compared with the nondiabetic CHIG hamsters, islets from mildly and severely diabetic animals displayed increased basal insulin release at 1.5 mmol/l and a deficient response at 10 mmol/l glucose which was associated with 61 and 77% decreases (p < 0.01 and p < 0.001) in the islet insulin content. The addition of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) enhanced glucose-stimulated insulin release from islets of nondiabetic and mildly diabetic CHIG hamsters, although the response elicited was lower than from CHIA islets. However, IBMX failed to significantly increase the glucose-stimulated insulin response of islets from severely diabetic hamsters. A negative correlation (r = -0.73, p < 0.001, n = 48) was found between islet insulin content and plasma glucose levels. The data suggest that the reduced secretory capacity represents an early islet beta-cell dysfunction, and the decrease in the insulin content contributes to the islet abnormalities in the diabetes-susceptible CHIG hamsters.

Characterization of beta-cell specific function of isolated pancreatic islets from hyperglycemic C57BL/KsJ-db/db mice.

Isolated pancreatic islets from male hyperglycemic C57BL/KsJ-db/db mice were used to study (pro)insulin biosynthesis, insulin release and insulin content. A diminution of the insulin content to about 36% and of the insulin release (20 mmol/l glucose) to nearly one half was observed, whereas [3H]leucine incorporation into (pro)insulin was increased. Insulin biosynthesis as well as insulin release showed no further increase, when the glucose concentration was increased to 20 or 30 mmol/l. Cultivation of such islets in TCM 199 (10 mmol/l glucose or additionally increased [Mg++] to 5.3 mmol/l) for 48 h resulted in a significant increase of the insulin content and a restoration of the glucose dependent insulin release.

The effect of mixing regular- and intermediate-acting insulins on their absorption and biological activity.

Many insulin-dependent diabetics are now treated with different regimens of short- and intermediate-acting insulins. The new L-insulin S.N.C. (Berlin-Chemie) is thought to be appropriate for a combination with regular insulin. We investigated whether or not absorption kinetics and biological activity changes when L-insulin S.N.C. is mixed with regular insulin S.N.C. Eight non-obese healthy male subjects were connected to the BIOSTATOR. After an overnight fast 0.1 U of insulin S.N.C. and 0.3 U per kg b.w. of L-insulin S.N.C. or Monotard MC insulin (NOVO) were administered either in a mixture or separately. Injecting L-insulin S.N.C. and insulin S.N.C. in a mixture there was some, but not a statistically significant, delay in absorption of insulin. A mixture of insulin S.N.C. and Monotard provoked a marked delay of absorption during the first two hours. To examine biological activity we used the BIOSTATOR (glucose controlled dextrose infusion system). Glucose infusion rate necessary to keep blood glucose steady state concentration was taken as a measure of insulin action. Whereas mixing of insulin S.N.C. and L-insulin S.N.C. only slightly changed the profile of biological activity, a mixture of insulin S.N.C. and Monotard has a clear-cut higher potency during the late phase when compared with separately injected insulins. Therefore, insulin S.N.C. and Monotard should not be mixed. A mixture of insulin S.N.C. and L-insulin S.N.C. is also not recommended but can be done in exceptional cases.

Spontaneous recovery of streptozotocin diabetes in mice.

With the aim of developing a model of experimental diabetes by which spontaneous recovery processes can be investigated, we used a lower (1) and a higher (2) dose of streptozotocin (SZ) to find out the lowest possible dose definitely inducing diabetes in mice through beta cell loss but preventing excessive damage to the endocrine pancreas which would exclude restoration processes. After application of SZ (1) to neonatal mice only male animals showed an overt diabetes in adult life. 70 percent of these mice had recovered 15 weeks after appearance of diabetes. Recovery was indicated by normalization of blood glucose, serum insulin, insulin secretion and biosynthesis of isolated pancreatic islets and a reenhancement of the pancreatic insulin content from lower than 10 to 30 percent of control values. After SZ (2) both sexes became hyperglycaemic, and the recovery rate was lower, but was increased by pregnancy in female mice. By means of this model it will be possible to investigate mechanisms and promoting factors of such restoration processes in more detail.

Characterization of insulin biosynthesis and secretion of pancreatic islets prepared from hyperglycaemic sand rats. II. Investigations of islets after different periods of cultivation.

Cultivation of pancreatic islets of sand rats in the presence of high glucose led to a very high insulin release and to strongly diminished insulin contents independent of the metabolic stage of the sand rats. Changes in the [3H]-leucine incorporation into (pro)insulin were not found in dependence of cultivation.

Prospective investigations of long-term normoglycaemic BB/OK-rats: serial determination of glucose tolerance, insulitis, B-cell volume density and pancreatic insulin content.

Pancreatic tissue was obtained by repeated surgical biopsies from BB/OK rats (n = 62), which maintained normoglycaemia up to 250 days. The tissue was used to determine pancreatic insulin content, islet volume density, pancreatic B-cell volume density and the presence of mononuclear cell infiltrations within and around pancreatic islets (insulitis). The BB/OK rats were also characterized by determination of glucose tolerance. In 50 d old BB/OK rats lymphatic infiltration of pancreatic islets are rare. The mean value of relative B-cell volume density amounted to 0.71 +/- 0.05% and the pancreatic insulin content was 21.31 +/- 1.29 pmol/mg wet weight. 70 d old BB/OK rats are characterized by an identical relative B-cell volume density and pancreatic insulin content, although in 70% of the animals an invasion of immunocytes could be observed. At an age of 90 and 120 d the BB/OK rats were characterized by an increased number of islets having insulitis accompanied by a decrease of pancreatic insulin content and B-cell volume density. The individual evaluation of the investigated BB/OK rats revealed in each animal the presence of insulitis accompanied by a decreased B-cell volume density and a diminished pancreatic insulin content. Animals older than 120 d do reenhance the pancreatic insulin content and B-cell volume density attended by an impairment of glucose tolerance. The results suggest that all BB/OK rats are characterized by a spontaneous pancreatic B-cell destruction, which was arrested and/or interrupted in the animals maintaining normoglycaemia up to 250 d.

Influence of polyethylene glycol (PEG) extraction on the C-peptide determination in sera from insulin-dependent diabetic patients with circulating insulin antibodies.

To investigate whether the unexpectedly high C-peptide levels in some insulin-dependent diabetic (IDDM) patients are due to co-determination of proinsulin bound to circulating insulin antibodies, 36 randomly selected sera from IDDM patients were assayed for C-peptide immunoreactivity (CPR) after polyethylene glycol (PEG) extraction, preceding incubation with proinsulin binding antibodies (LAB + PEG) or without pretreatment of the sera. Recovery of proinsulin was checked by addition of 1 nmol/l proinsulin to all sera. Recovery was found to be 101.5 +/- 4.0%. The mean values of concentrations were significantly lower (p less than 0.001) after treatment with PEG and IAB + PEG compared to the untreated sera. There was also a significant difference (p less than 0.05) between sera extracted with PEG alone or after IAB + PEG-treatment. However, no correlation (p greater than 0.1) was found to bound insulin (total minus free insulin) or to insulin binding capacity (IBC) of the sera. If an antiserum is not available with very low cross-reactivity with proinsulin to determine human C-peptide then sera should not be extracted with PEG alone but after additional incubation with a proinsulin binding antiserum. In spite of the extraction in some cases unexplicably high C-peptide levels may still be expected.

Intraindividual comparison of pharmacokinetics of insulin after intravenous, portal, subcutaneous and peritoneal administration.

To compare the kinetics of praehepatic and of posthepatic administered insulin, short term insulin deprived diabetic dogs were sequentially injected with 200 mU/kg of a monocomponent porcine insulin using either the intravenous, portal, subcutaneous or peritoneal route. After peritoneal insulin was applied, the peripheral plasma insulin levels increased immediately, their maxima were in the same range as after subcutaneous injection but the duration of elevation was shorter. There were portal-peripheral insulin-quotients greater than 1 after peritoneal and portal insulin administration but quotients less than 1 after subcutaneous and intravenous application. The time constant of insulin elimination was identical regardless of whether the praehepatic or the posthepatic route was used for application. The effectiveness of the administered insulin dose on blood glucose was found to be dependent on the posthepatic elevation of plasma insulin and its duration. The decrease in glycemia was initially identical in all tests but, on the whole, it was smaller after the two intravascular routes were used because of the shorter duration of elevated insulin levels. It is concluded that in an optimized management of insulin-dependent diabetes, the regime (doses and intervals or algorithms) must be adapted to the pharmacokinetic implications of the employed route of application.

Pregnancy-associated changes in the endocrine pancreas of normoglycaemic streptozotocin-treated Wistar rats.

The effect of pregnancy on pancreatic insulin content and relative B-cell volume has been studied in normoglycaemic Wistar rats treated with streptozotocin 14 days before mating. A single intravenous injection of streptozotocin (30 mg/kg body weight) caused a significant reduction of pancreatic insulin content and B-cell volume. The islet insulin content was 60% of control values. However, pregnancy-associated adaptation was preserved in these streptozotocin-treated animals. Plasma insulin levels, pancreatic insulin and B-cell volume were significantly enhanced compared with non-pregnant rats investigated on the same date. The incorporation of [3H]-thymidine into islets from pregnant rats (day 10.5) was higher than that in islets isolated from non-pregnant animals. After delivery insulin content and B-cell volume returned to pre-pregnant values. Also during a longer period after streptozotocin treatment (156 days), no measurable enhancement of B-cell volume and pancreatic insulin content was observed indicating the unresponsiveness of residual B cells to compensate spontaneously for the loss despite persisting normoglycaemia.