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.

Indication and results of insulin therapy for gestational diabetes mellitus.

The aim of this study was to determine whether amniotic fluid insulin concentration (AFI) is a better parameter than mean maternal blood glucose values (MBG) for deciding about insulin therapy in patients with gestational diabetes. MBG's were calculated on the base of 9 blood glucose levels during a 24 hour period after one week of diet therapy. In a prospective trial between 1987 and 1989 in Karlsburg, 123 gestational diabetic patients were randomized into two groups. Treatment was either based on the concentration of AFI or MBG levels. In a second series in Berlin, 103 patients were offered amniocentesis. 81 patients agreed and 22 refused. Treatment was then analogous to that in Karlsburg. In both groups of the randomized population, strict metabolic control was achieved. There was no difference regarding pregnancy complications. Earlier labor induction and higher cesarean section rates were seen in the non-invasive group (p < 0.05). The incidence of diabetic fetopathy and neonatal hypoglycemia was significantly lower in the invasive group (p < 0.01), even though the metabolic control parameters did not differ between the two groups. The results in Berlin correspond to these findings. In conclusion, AFI enables the recognition of any hyperinsulinism reaction to the maternal metabolic situation. We recommend the additional measurement of the AFI concentration between 28 and 36 weeks as the direct fetal parameter for deciding about insulin treatment.

Validation of red cell sodium-lithium countertransport measurement--influence of different loading conditions.

Increased sodium-lithium countertransport in erythrocytes from patients with long-standing type I (insulin-dependent) diabetes mellitus has been considered as an early marker of nephropathy. Since the activity and kinetics of the sodium-lithium countertransport may critically depend on loading conditions, this study was aimed at determining sodium-lithium countertransport activity, Michaelis constant Km and maximum velocity Vmax in erythrocytes loaded in two different Li+ solutions. Sodium-lithium countertransport activity was determined in erythrocytes in 8 healthy control subjects after loading with 150 mmol/l LiCl compared with those loaded with 150 mmol/l LiHCO3. Sodium-lithium countertransport activity was similar for both loading procedures, although the erythrocyte lithium content did significantly differ (mean +/- SEM, 7.0 +/- 0.5 for LiCl and 8.9 +/- 0.5 mmol/l of cells for 150 mmol/l LiHCO3). There were no significant changes in the Km and Vmax. Increase of osmolality in efflux media containing 200 and 250 mmol/l NaCl resulted in a negligible shrinking of the red blood cells, not exceeding 2.2%. The main advantage is the short loading time of 15 min for LiHCO3 compared with 3 hours for LiCl. Under these conditions saturating intracellular Li+ concentrations can be obtained much more rapidly than with LiCl loading, thereby minimising alterations of the cell membranes. LiHCO3 loading shortens the experimental time considerably and enables a greater number of samples to be screened from larger population cohorts.

Discordant results in the assay of cytoplasmic islet cell autoantibodies with ELISA and immunohistochemical techniques.

We evaluated 6 batches of a solid phase enzyme-linked immunosorbent assay (ELISA) Isletest-ICA kit commercially available for the determination of autoantibodies to pancreatic islet cells, and compared the results with those obtained by a standardized immunohistochemical method. Following the immunohistochemical determination of autoantibodies to pancreatic islet cells, sera from patients with insulin-dependent diabetes mellitus, both positive and negative for autoantibodies to pancreatic islet cells, were randomly selected and analysed by ELISA. Sera from healthy control subjects, as well as standards recommended by the International Diabetes Workshop (IDW) ICA (Autoantibodies to Pancreatic Islet Cells) Proficiency Program, were included. Of the sera testing positive for autoantibodies to pancreatic islet cells in the immunohistochemical assay, only 14 +/- 5% were found to give a positive reaction in the ELISA. Among the sera from healthy control subjects and pancreatic islet cell autoantibody-negative insulin-dependent diabetes mellitus patients, 25 +/- 7% and 1 +/- 1%, respectively, yielded false-positive readings for autoantibodies to pancreatic islet cells. These results clearly show that the ELISA test presently available does not reliably detect autoantibodies to pancreatic islet cells, even qualitatively. Thus, it cannot be used for screening subjects at risk of developing diabetes.

Autoantibodies to insulin and to proinsulin in type 1 diabetic patients and in at-risk probands differentiate only little between both antigens.

To answer the question whether insulin or proinsulin would be the true antigen for both insulin and proinsulin autoantibodies, displacement experiments of 125I-insulin and -proinsulin binding with both unlabeled antigens were performed in sera of four groups of antibody-positive probands: first-degree relatives of Type 1 diabetic patients, pre-Type 1 diabetic persons, recent-onset Type 1 diabetic patients, insulin-treated Type 1 diabetic patients. In subjects who were primarily screened to constitute these groups, prevalences of insulin and proinsulin autoantibodies were nearly identical. In antibody-positive sera, 125I-insulin and -proinsulin binding values in general were closely correlated to each other with regression coefficients near 1.0. In all groups of probands, mean values of 125I-insulin and -proinsulin binding did not significantly differ. With the exception of a few sera, insulin and proinsulin antibodies differentiated only little between both antigens. Epitopes of the insulin molecule are therefore preferred. Nevertheless, insulin and proinsulin autoantibodies are not completely identical nor are insulin autoantibodies merely a subgroup of proinsulin autoantibodies: In each group, in the mean, insulin antibodies as well as proinsulin antibodies reacted somewhat (but significantly) stronger with their respective antigen. In some cases a distinct (relative) specificity for either antigen of insulin and proinsulin autoantibodies were observed, the latter being still present after some months of insulin treatment. In conclusion, despite detectable differences in antigen specificity, insulin and proinsulin autoantibodies seem to be equally potent markers of Type 1 diabetes mellitus.

Erythrocyte sodium-lithium countertransport, adenosine triphosphatase activity and sodium-potassium fluxes in insulin-dependent diabetes.

Increased erythrocyte sodium-lithium countertransport activity has been implicated in the pathogenesis of diabetic nephropathy. However, its relationship to other cation membrane transport systems in incipient nephropathy is not yet clear. The present study was thus performed to: (1) explore associations between sodium-lithium countertransport and changes in the activity of other cation transport pathways and (2) to compare the sodium transport activities with clinical characteristics of insulin-dependent diabetic patients with and without evidence of incipient diabetic nephropathy. We measured erythrocyte sodium-lithium countertransport, passive sodium/potassium flux (at 1 degree C), adenine nucleotide content in intact erythrocytes and sodium/potassium-, magnesium- and calcium-dependent ATPase activity in erythrocyte membrane preparations from 34 insulin-dependent diabetic patients without microalbuminuria, 8 diabetic patients with microalbuminuria, and 8 age-matched healthy control subjects. Sodium-lithium countertransport was elevated in diabetic patients with normo- and microalbuminuria compared with control subjects [268 +/- 99 and 299(277-465), respectively, vs. 166 +/- 65 mumol/(1 cells x h)] and was positively correlated (r = 0.36, P < 0.05) with the albumin excretion rate. However, the activity of erythrocyte membrane ATPases was significantly decreased compared with control subjects. The ATP and ADP content was found to be significantly higher (P < 0.001) in erythrocytes from diabetic patients compared with control subjects (1,196 +/- 276 vs. 833 +/- 253 mumol/l cells and 353 +/- 97 vs. 255 +/- 64 mumol/l cells, respectively). The extent of erythrocyte potassium leakage correlated with hemoglobin A1c (r = 0.39, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

Monoclonal antibody-mediated cytotoxicity against rat beta cells detected in vitro does not cause beta-cell destruction in vivo.

Two monoclonal Beta-cell surface antibodies M10H6 und K14D10 were obtained by fusion of spleen cells of Balb/c mice with the myeloma cell line P(3)0. The monoclonal antibody M10H6 was induced by immunization with rat insulinoma cells finally boostered with disintegrated rat islets, whereas the K14D10 was generated after immunization with porcine proinsulin. Both monoclonals belong to the IgG2A isotype and were screened with insulin-producing rat insulinoma cells by an indirect immunofluorescence test as well as by a cellular enzyme linked immunosorbent assay. In addition to the cell surface binding on living Beta cells the monoclonals react with islets on cryostat sections of rat pancreas. The anti-islet cytotoxic potential of these monoclonals was measured by 51Chromium-release in the presence of complement or Fc-receptor bearing leucocytes using 51Chromium-labelled rat islet cells as target. Both antibody secreting hybridomas were propagated in syngeneic mice resulting in high levels of islet cell surface antibodies in ascites and sera from the recipient. High anti-islet cytotoxicity was mediated by ascites fluid, but no mouse developed hyperglycaemia. Furthermore, the repeated injections of the monoclonals into rats did not exert a diabetogenic action and failed to reduce the pancreatic insulin content although the attraction of the K14D10 to the pancreatic islets in vivo could be demonstrated. We conclude that islet cell surface antibody-mediated Beta-cell lysis in vitro may not be relevant to Beta-cell destruction in vivo.

Alteration of pancreatic B-cells in Wistar rats treated with non-diabetogenic doses of cyclosporin A.

We have investigated the effect of non-immunosuppressive cyclosporin A (CS-A) doses on glucose tolerance, pancreatic insulin content, insulin content of isolated islets and insulin secretion in vitro in response to glucose. Within 12 weeks animals treated permanently with a dose of 2.5 mg CS-A/kg b.wt. developed glucose intolerance (but not hyperglycaemia) accompanied by a decrease of pancreatic insulin content due to a decrease of islet insulin content, whereas the relative B-cell volume density was not changed. Isolated islets obtained from rats treated for 4 weeks had a diminished sensitivity for glucose, whereas islets obtained from animals treated for greater than 4 weeks showed a diminished half-maximum and maximum secretion rate. Rats treated for 12 weeks with 1.25 mg CS-A/kg b.wt. developed an impaired glucose tolerance after 8 weeks accompanied by a diminished pancreatic insulin content. Despite continued treatment the pancreatic insulin content was able to increase and the glucose tolerance to normalize, indicating an adaptation of pancreatic B-cells to CS-A. The results support the theory that a potential toxic effect of cyclosporin A can be diagnosed by functional tests (e.g. insulin secretion in response to a stepwise increase of glucose) before the irreversible (e.g. morphological) alterations occur.

The effect of serum concentrations and sources on DNA synthesis and insulin secretion of cultured islets.

We investigated the effect of different sera, as newborn calf serum, fetal calf serum, rat serum and human serum at various concentrations on biofunctions of cultured pancreatic islets. Islets isolated from newborn LEW. 1 W rats were maintained at 37 degrees C in TCM 199 containing 10 mmol/l glucose and either 1%, 10% or 50% newborn calf serum (NCS), fetal calf serum (FCS), serum obtained from adult syngeneic rats (RS) or different batches of human serum (HS). While exposure of islets to increasing concentrations of NCS significantly inhibited the islet DNA synthesis as well as the glucose stimulated insulin secretion after 2, 4 and 8 days of culture, HS at different concentrations failed to inhibit both, the islet DNA synthesis and the secretory response to glucose at each time point investigated. The action of FCS, RS and 2 further batches of HS on islet DNA synthesis was analysed by short-term culture. The supplementation of the medium with 50% FCS and RS resulted in a marked decrease of H-thymidine incorporation into islet DNA similar to that observed with 50% NCS. In contrast, exposure of islets to different batches of HS never resulted in an inhibition of DNA synthesis. Moreover, the 50% concentration of HS 465 caused a significant stimulation of thymidine incorporation.

Mathematical modelling of competitive labelled-ligand assay systems. Theoretical re-evaluation of optimum assay conditions and precision data for some experimentally established radioimmunoassay systems.

A mathematical theory of competitive labelled-ligand assays was developed with the intention of theoretically re-evaluating the optimal assay conditions and precision data of assay systems established by experiment. Our theory is based upon the assumptions of a simple bimolecular reaction mechanism, homogeneous reactants, as well as kinetically indistinguishable labelled and non-labelled ligands. The general case of two-step (non-equilibrium) assay was considered including the one-step (equilibrium) assay as a special case. The solution of the system of corresponding kinetic differential equations was used to mathematically construct standard curves. Furthermore, intraassay precision profiles and indices as well as detection limits were calculated considering solely the pipetting error, epsilon, as a source of experimental error. A procedure was outlined to mathematically determine the optimal incubation conditions for any assay system targeted to a given analyte concentration, P, at which the standard deviation of assay results is to be minimized. Estimates of both the content of binding sites and the equilibrium constant, K, of the specific binding agent are necessary, and these can be derived from Scatchard plots. For six RIA systems, of which three were one-step and three were two-step assays, experimental assay conditions and precision data were compared with theoretical predictions. Experimentally determined antibody binding site concentrations agreed fairly well with those independently evaluated by mathematical optimization. Mean precision indices, defined as constituting an average over the complete precision profile, were found to be within the theoretically predicted range, i.e. two- to threefold the pipetting error.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of human amniotic fluid in supporting long-term survival of pancreatic islets in tissue culture.

Pancreatic islets obtained from newborn Lewis rats were kept free-floating in TCM 199 either in the presence of 50% human amniotic fluid (HAF), 50% Hank's balanced salt solution (HBSS) or 10% fetal calf serum (FCS) for up to 14 days. The culture, in the presence of HAF, resulted in a good islet viability as demonstrated by islet number, islet insulin content and insulin release into the medium. Contradictory results were obtained when HBSS was used as the medium supplement. Moreover, the islets cultured in HAF-supplemented medium are characterized by a marked replicatory activity as reflected by the incorporation of labeled thymidine into islet DNA and gradual increase in DNA content with the progression of culture time. Even if compared to DNA synthesis of islets cultured under so called standard culture conditions as e.g. supplementation of 10% FCS to the medium, 50% HAF but not yet 10% HAF was found to stimulate the islet replication twofold already after 4 days of culture. It was also demonstrated that FCS has no additional effect on HAF-stimulated DNA synthesis. These findings support the view that HAF-enriched culture medium may have a use in supporting the long-term survival of well preserved endocrine pancreatic tissue.

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.

[Enzyme immunoassay for the determination of albumin in urine]. / Enzymimmunoassay zur Bestimmung von Albumin im Urin.

Two modifications of a double antibody enzyme immunoassay for the determination of urinary albumin content are described. The method is simple, rapid and precise and can be carried out in test tubes and on microtiter plates as well. In 1:10 diluted urine samples albumin concentrations of 1.25 to 20 mg/l (corresponding to the normal range) can be determined. For a control sample with 0.3 mg/l albumin the intra- and interassay coefficients of variation were 4.9% (n = 11) and 10.4% (n = 21), respectively, on microtiter plates.

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.

A sensitive sandwich enzyme immunoassay for measurement of insulin on microtiter plates.

A sensitive enzyme immunoassay for the measurement of insulin in human sera on microtiter plates was established. The assay is based on the sandwich technique with guinea pig anti-insulin IgG adsorbed at microtiter plate wells, human insulin as standard and the same anti-insulin IgG labeled with horseradish peroxidase. Standards used cover a range from 0 to 1200 pmol/l with a detection limit of 10 pmol/l. Coefficients of variation between 3-7% for intraassay precision and 5-11% for interassay precision were obtained over the concentration range of 80-1000 pmol/l. The correlation of EIA-data with those of a commercially available double antibody radioimmunoassay (r = 0.98) could be expressed by the equation: EIA = 0.97 RIA - 57 pmol/l. Normal fasting serum insulin concentrations in healthy subjects ranged from 11-165 pmol/l. In subjects with potentially diminished basal values concentrations of 10-79 pmol/l were determined. The insulin response in oral glucose tolerance tests of children was discussed, who had a constitutional tall stature or Turner's syndrome, respectively.

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.