Studies on the mode of action of somatostatin on insulin secretion.

The effect of somatostatin on insulin release by incubated slices of rat pancreas was studied. Somatostatin inhibited insulin release induced by arginine/glucose (A/G), glucagon, glibenclamide, pentoxifyllin, 3',5'-adenosine monophosphate (cAMP), phentolamine, and KCl. When A/G was used as a stimulus, the quantial inhibitory effect of somatostatin was not neutralized by progressively increasing glucose concentrations. The alpha adrenergic blocking agent phentolamine, the phosphodiesterase inhibitors theophylline (10 mM) or pentoxifyllin (10 mM), and KCl partially reversed the inhibitory effect of somatostatin on A/G stimulation. The maximal reversal of somatostatin inhibition was obtained when the slices of pancreas were stimulated with A/G in the presence of the calcium ioniphore A23187 plus ATP. These results suggest that the inhibitory effect of somatostatin on insulin secretion could result from calcium translocation in pancreatic beta cells.

A temporal study of somatostatin secretion and its inhibitory effect in genetically diabetic mice (C57BL/KsJ-db/db).

Somatostatin (SRIF) acts as a physiological regulator of insulin and glucagon secretion. This study explored whether alterations in SRIF secretion and activity in perifused pancreas from spontaneously diabetic mice (C57BL/KsJ-db/db) could be correlated with hypersecretion of insulin by the beta-cells. SRIF release upon stimulation with 27.5 mM glucose was biphasic in controls, whereas a first phase peak was absent in 30-to 90-day-old diabetic mice (db/db). Twelve- to 28-day-old db/db mice showed two distinct patterns compared to controls: biphasic hypersecretion in the 12- to 20-day-old group compared to normal secretion in the 22- to 28-day-old group. Basal SRIF secretion showed a tendency to be elevated above controls in some of the db/db age groups, but the difference was not statistically significant. Insulin release from control pancreases was biphasic, whereas in db/db mice, basal hypersecretion, absence of the first peak, and second phase hypersecretion were observed. beta-Cell sensitivity to the inhibitory effect of SRIF was diminished in db/db mice aged 12-90 days. Diazoxide, on the contrary, inhibited insulin secretion from the pancreas of diabetic and normal mice to the same extent. The results suggest that altered secretion of and response to SRIF in C57BL/KsJ-db/db mice might provide an explanation for the anomalies in insulin secretion in the first stages of this type of diabetic syndrome.

Patient with an Xp21 contiguous gene deletion syndrome in association with agenesis of the corpus callosum.

The so-called Xp21 contiguous deletion syndrome or complex glycerol kinase deficiency (GKD) usually presents with classical Duchenne muscular dystrophy (DMD) or a milder dystrophic myopathy, adrenal hypoplasia, and GKD. A number of syndromic and nonsyndromic cases of agenesis of the corpus callosum (ACC) also map to that location. To date, none of the cases of complex GKD have been associated with ACC. Here, we report on a patient with a complex phenotype as a result of the Xp21 contiguous deletion syndrome in association with ACC. Biochemical, cytogenetic, and molecular analyses were performed to detect and establish the size of the genomic deletion. It is at least 3 million base pairs in length; however, exact limits could not be determined in the present study. Nevertheless, we suggest the presence of a primary gene involved in the embryogenesis of the corpus callosum between Xp21.1 and Xp22.11.

ATP inhibits insulin-degrading enzyme activity.

We studied the ability of ATP to inhibit in vitro the degrading activity of insulin-degrading enzyme. The enzyme was purified from rat skeletal muscle by successive chromatographic steps. The last purification step showed two bands at 110 and 60 kDa in polyacrylamide gel. The enzyme was characterized by its insulin degradation activity, the substrate competition of unlabeled to labeled insulin, the profile of enzyme inhibitors, and the recognition by a specific antibody. One to 5 mM ATP induced a dose-dependent inhibition of insulin degradation (determined by trichloroacetic acid precipitation and insulin antibody binding). Inhibition by 3 mM adenosine 5'-diphosphate, adenosine 5'-monophosphate, guanosine 5'-triphosphate, pyrophosphate, beta-gamma-methyleneadenosine 5'-triphosphate, adenosine 5'-O-(3 thiotriphosphate), and dibutiryl cyclic adenosine 5'-monophosphate was 74%, 4%, 38%, 46%, 65%, 36%, and 0%, respectively, of that produced by 3 mM ATP. Kinetic analysis of ATP inhibition suggested an allosteric effect as the plot of 1/v (insulin degradation) versus ATP concentration was not linear and the Hill coefficient was more than 1 (1.51 and 2.44). The binding constant for allosteric inhibition was KiT = 1.5 x 10(-7) M showing a decrease of enzyme affinity induced by ATP. We conclude that ATP has an inhibitory effect on the insulin degradation activity of the enzyme.

[Diabetic nephropathy in children: study using the amount of urinary albumin]. / Nefropatía diabética en niños: estudio mediante el dosaje de albúmina urinaria.

It is well known that 30 to 50% of patients with Type I Diabetes develop nephropathy and that chronic renal failure, it's final pathway, is the main cause of death. Assessment of urinary albumin becomes an essential tool for identifying the population at risk of developing nephropathy, to study its physiopathologic mechanisms and to evaluate the response to therapeutic trials. The present article is a preliminary report on the study of urinary albumin excretion rate (AER) in a pediatric population with Type I Diabetes and its relation to teh duration of the disease. A RIA technique with double antibody was developed for albumin assessment, with a displacement range of 1 to 300 ng/tube. Urine samples were collected during short periods with water load as suggested by Mogensen. Thirty nine children (30 patients and 9 controls) free of renal disease and with normal blood pressure were studied. Patients were divided according to duration of the disease in: Group I: less than 5 years, Group II: 5 to 10 years and Group III: more than 10 years. Results (mean +/- SD) in micrograms/min/1.73 m2 were: Control Group (n = 9) 4.30 +/- 2.53, GI: (n = 12) 10.44 +/- 9.47, GII (n = 10) 8.03 +/- 7.27 and GIII (n = 8) 8.56 +/- 4.26. The mean value of the Control Group (+3 SD) 12 micrograms/min/1.73 m2, was considered as the upper normal limit. Thus, 16.6%, 40%, and 12.5% of children in Groups I, II and III had microalbuminuria.(ABSTRACT TRUNCATED AT 250 WORDS)

[Insulin/glucagon relationship in spontaneous diabetic remission]. / La relación insulina/glucagon en la remisión diabética espontánea.

We define diabetic remission as the disappearance of clinical symptoms with normalization of blood glucose for a period over 15 days after withdrawal of insulin therapy. We studied 21 insulin-dependent diabetic children in remission (10 boys and 11 girls) and 29 normal children matched in age and sex as controls. Two tests were performed, intravenous glucose (IVGT) and glucose post-tolbutamide (PTGT). Two remission groups were studied with IVGT. Glucose, insulin, somatotropin and glucagon were determined in one and glucose and C-peptide in the other. Insulin secretion after IVGT was very low in the remission group, not surpassing basal value when stimulated. Only two girls showed normal or high insulin values during the study, and one of them showed the common hypoinsulinism of the remission group in a second study. The kinetics of glucagon and somatotropin secretion in the remission group were normal with low values of glucagon. When the integrated area (0-120 min) of hormone secretion (insulin, somatotropin and glucagon) was determined, the remission group had lower insulin and glucagon values (p less than 0.05) and identical growth hormone as the normal group. The insulin/glucagon ratio in normals and in remission were similar. During IVGT the remission group studied for C-peptide showed lower C-peptide values than normal group, resembling insulin behavior. In both groups, the glucose disappearance rate ("K" value) was higher in normals than in remissions (p less than 0.001). During the PTGT the normal group showed a peak of insulin secretion after tolbutamide and glucose stimulation. In the remission group, glucose was higher and insulin secretion lower than in the normal group, without a peak of insulin, and growth hormone and glucagon secretion were also lower.(ABSTRACT TRUNCATED AT 250 WORDS)

[Autoimmune hypoglycemia syndrome with specific anti-human insulin antibodies]. / Síndrome de hipoglucemia autoinmune con anticuerpos específicos anti-insulina humana.

A 33 year old woman with episodes of severe hypoglycemia is presented. The studies showed anti-insulin antibodies and variable C-peptide levels. Circulating insulin measured after acid-ethanol extraction, was of 1,600 uU/ml and shown to be human insulin after characterization by HLPC. Specific anti-human insulin antibodies were of high affinity (Ka1: 6.20 x 10(10) M-1; Ka2: 2.42 x 10(9) M-1). A small cross-reactive porcine and bovine antibody subpopulation was also detected (IgG, light k type chain). Plasmapheresis was undertaken when symptoms were spontaneously declining and turned antibody title negative. Prolonged follow-up showed no relapse of this syndrome.

Anti-idiotype guinea pig antibodies as response to insulin immunization.

The study was done using 39 guinea pigs grouped as followed; 18 were injected with 0.5 mg of porcine insulin emulsified in complete Freund's adjuvant; 12 were injected with saline and 9 were used as control of cardiac bleeding during the assay. Intraperitoneal glucose tolerance tests (IGTT) were carried out on days 0, 11, 32 and 38. Seven of the thirteen guinea pigs immunized with insulin which survived after the study, showed glucose intolerance on day 32 at 90 and 120 min (p < 0.01 and p < 0.001) and on day 38 at 120 min (p < 0.05). Anti-idiotypic IgG partially purified from a sera pool from these animals inhibited 125-Insulin binding to rat hepatocytes, immunoprecipitated 125I-rat insulin receptors and recognized the alpha-subunit of insulin receptor in immunoblotting. We conclude that insulin anti-idiotypes in guinea pigs offer a simple way to produce antibodies against insulin receptor binding site. The methodology for anti-idiotype identification can be applied to patients with insulin resistance.

Secretion and effect of somatostatin in early stages of the diabetic syndrome in C57BL/KsJ-mdb mice.

In a previous study in C57BL/KsJ (mdb) mice aged 12 to 90 days, we observed alterations in the secretion of insulin and somatostatin and in the inhibitory effect of the latter upon insulin secretion. This study explores whether hormonal alterations are to be found in the very early stages of the diabetic syndrome, i.e. between ages 4 and 12 days. The results demonstrate two distinct phases in the development of the syndrome: up to age 6 days, the perifused slices of pancreata of control animals present biphasic glucose-induced patterns of insulin and somatostatin secretion, whereas the diabetic animals show a diminished first peak of insulin secretion, but a similar pattern of somatostatin secretion, to that of the control animals; between ages 7 and 12 days, the pancreata of diabetic mice exhibit insulin hypersecretion in basal conditions, and an absence of the first secretion peak and insulin hypersecretion in the second phase in response to glucose stimulation. The glucose-induced pattern of somatostatin secretion presents hormonal hypersecretion in both phases. B-cell sensitivity to the inhibitory effect of somatostatin is diminished in mdb mice of the above-mentioned groups, an alteration which becomes more evident as diabetes evolves. The results show that, in very early stages of the evolution of the diabetic syndrome in C57BL/KsJ (mdb) mice, there are already alterations in insulin and somatostatin secretion patterns and in the inhibitory effect of the latter on insulin secretion.

Internalization and release of insulin from hepatocytes.

Degradation of internalized insulin was studied after binding at 25 degrees C and 37 degrees C to isolated hepatocytes. The cells were washed to avoid extracellular insulin contamination. Degradation of both, intracellular and extracellular 125I-insulin, was measured with TCA and insulin antibody. In these conditions binding at 25 degrees C and 37 degrees C was equal but both intra and extracellular degradation were greater at 37 degrees C than at 25 degrees C. At both temperatures, intracellular degradation was greater than extracellular degradation with accumulation of degraded and non-degraded intracellular insulin. To study in what state hepatocytes release internalized insulin into the medium, 125I-insulin association was performed at an intermediate temperature (30 degrees C). Extracellular insulin contamination (whether associated or not) was avoided by three methods: 1) washing; 2) treatment with insulin degrading enzyme(s) and washing; 3) treatment with insulin degrading enzyme(s) then with trypsin and washing. Kinetics of radioactivity released from the cells was identical in the three conditions and the radioactivity was released throughout the experiments. Complete degradation of the released insulin was observed by gel filtration when the previous binding was 0.4 ng insulin/10(6) cells. When the dose of associated insulin increased (25 ng/10(6) cells) 3.5% of non-degraded insulin was liberated and when the dose was 14,300 ng/10(6) cells, the insulin released was 44.3%. In one experiment during the first 30 min, the insulin released was 52.88% and in the last 45 min 39.59%. To study the biologic behavior of the insulin released from cells, a group of mice were injected with this insulin (8.4 mU/mouse) and blood glucose was measured. The released insulin behaved as intact insulin as far as blood glucose responses were concerned. We may conclude that liver cells have the ability to internalize insulin and release biologically active insulin after accumulation.

Degradation of soluble amyloid beta-peptides 1-40, 1-42, and the Dutch variant 1-40Q by insulin degrading enzyme from Alzheimer disease and control brains.

Insulin degrading enzyme (IDE) is a metalloprotease that has been involved in amyloid beta peptide (A(beta)) degradation in the brain. We analyzed the ability of human brain soluble fraction to degrade A(beta) analogs 1-40, 1-42 and the Dutch variant 1-40Q at physiological concentrations (1 nM). The rate of synthetic 125I-A(beta) degradation was similar among the A(beta) analogs, as demonstrated by trichloroacetic acid precipitation and SDS-PAGE. A 110 kDa protein, corresponding to the molecular mass of IDE, was affinity labeled with either 125I-insulin, 125I-Abeta 1-40 or 125I-A(beta) 1-42 and both A(beta) degradation and cross-linking were specifically inhibited by an excess of each peptide. Sensitivity to inhibitors was consistent with the reported inhibitor profile of IDE. Taken together, these results suggested that the degradation of A(beta) analogs was due to IDE or a closely related protease. The apparent Km, as determined using partially purified IDE from rat liver, were 2.2 +/- 0.4, 2.0 +/- 0.1 and 2.3 +/- 0.3 microM for A(beta) 1-40, A(beta) 1-42 and A(beta) 1-40Q, respectively. Comparison of IDE activity from seven AD brain cytosolic fractions and six age-matched controls revealed a significant decrease in A(beta) degrading activity in the first group, supporting the hypothesis that a reduced IDE activity may contribute to A(beta) accumulation in the brain.

Insulin binding and degradation in short time incubation at 37 C.

Studies on insulin-receptor binding in a short time incubations at 37 C have shown that neither internalization nor receptor-mediated insulin degradation are demonstrable during the first minutes. In the present study insulin receptor binding at 37 C in short time incubation periods was studied in mouse-hepatocytes, simultaneously determinating the proportion of degradation due to the cell activity. Degradation in the incubation buffer after cell separation was abolished during the experiment (900 sec) by a careful wash of the cells. 7.5 cells/ml were incubated with a tracer concentration (14.17 pM) of 125I-insulin and a pharmacological concentration (16.6 microM) of native insulin plus tracer. In the case of tracer insulin, 50% binding was reached in 55 sec and steady state in 160 sec. Once reached, steady state persisted along the experimental time. Binding follows a second order kinetics with k+1: 5 649 X 10(6) M-1 sec-1. In the presence of pharmacological insulin there is competitive inhibition of the tracer which reduces to zero the percent of binding. Binding increases along the time taking positive values, and the slope of binding versus time intersects the abscissa at 102 sec (r: 0.864). As long as binding of the tracer takes place, no degradation occurs until 635 sec, when a degradation slope abruptly appears (r: 0.722). Dissociation studies were followed previous incubation at 37 C during 200 sec with tracer and pharmacological doses. Specific dissociation follows a monoexponential kinetics with k-1: 3 067 X 10(-3) sec-1 and t 1/2: 226 sec. Eighty percent of bound insulin is dissociated with no changes in the slope (r: 0.820), thus suggesting that insulin-receptor binding in the present experimental conditions is basically a reversible process. No degradation was observed during dissociation, which demonstrates that insulin-receptor binding does not degrade insulin if internalization is not performed. At steady state, competitive inhibition curves showed two components: high and low affinity. Doses of 1.66 microM produce a 98% inhibition in the binding of 125I-insulin. The high affinity slope shows two components in the physiological range of insulin concentrations. The first one of very high affinity has a dissociation constant Ko: 7 075 X 10(-10), and a binding capacity of 1.5 X 10(-10). This study demonstrates that, with physiological concentrations of insulin, internalization is the only mechanism of insulin degradation in mouse-hepatocytes.

Insulin processing. Its correlation with glucose conversion to CO2.

Insulin-receptor binding, insulin degradation and biologic response (14C-glucose conversion into 14CO2) were studied in adipocytes of control (CG), fasted (FG-88 hr) and hyperinsulinic rats (HG-exogenous hyperinsulinism). The number of cells normalized to 3.5 X 10(5) cells/tube in all three groups. Insulin binding and degradation were studied at 5, 15, 30, 60 and 120 minutes of incubation with 3.5 X 10(-11) M, 6.66 X 10(-11) M, 1.0 X 10(-9) M, 6.66 X 10(-9) and 6.66 X 10(-6) M insulin. The net increments of 14CO2 taken into account (delta U-14C-glucose converted into 14CO2) ranged from the basal value to 10(6) microU in each case (30, 60 and 120 minutes). Quantitative analysis of results was performed with the Terris and Steiner degradation equation (formula; see text) (IR). Differences in insulin binding, comparing the three groups, lacked statistical significance, though FG data were systematically plotted above those of CG, occurring the opposite with HG. Degradation studies showed HG to have values statistically higher than the controls, while FG values were lower. HG also showed higher amounts of 14CO2, with basal levels more elevated than CG, while FG showed the inverse behavior. 14CO2 increased in the three groups along the 120-minutes incubation period (30, 60 and 120 minutes). Receptor-mediated degradation at 30 minutes, when binding is in steady state, showed a Kap value very close to that found by linear regression for the 2 and 10 microU doses (Kap min-1 CG: 0.1654, FG: 0.0824, HG: 0.5045; slope values for the 2 and 10 microU doses CG: 0.2181, FG: 0.0824, HG: 0.3718). The degradation velocity, considered as function of IR, was constant in each group at 30, 60 and 120 minutes. Since Kap values in the FG and HG indicate differences in their degradation velocities, this constant can be considered as indicative of the metabolic situations under study. At the same time, the biologic response (14C-glucose conversion into 14CO2) depends as well on the metabolic conditions. Glucose consumption and Kap value were then compared. All the groups showed linear correlation between the binding dependent velocity of degradation (Kap) and the net conversion of U-14C-glucose into 14CO2 at 30, 60 and 120 minutes, with ordinate close to zero (30 min: 0.1539; 60 min: -0.3812; 120 min: 0.1311). The slope increased along the incubation period, indicating that 14CO2 accumulation is time dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

A comparative study of two insulin secretion inhibitors: somatostatin and diazoxide.

We studied the effect of the ionophere A 23187 and of phosphodiesterase inhibitors and activators (Theophylline, Pentoxiphylline and Imidazol) on insulin secretion and on the pool of free tubulin in rat pancreas in the presence of somatostatin and diazoxide. The results suggest that: 1. The inhibitory effect of somatostatin on insulin secretion does not seem to be related mainly to an inhibition of cAMP production. The decrease in calcium translocation induced by somatostatin could inhibit the cAMP participation in the mechanism of hormonal secretion. 2. Somatostatin seems to inhibit the movement of calcium towards the cytoplasm from outside and from within the cell. Diazoxide seems to inhibit only the entrance of calcium from outside the cell but does not seem to inhibit the entrance determined by theophylline and pentoxiphylline from intracellular compartments. 3. Arginine glucose stimulation in the presence of A 23187-induced calcium translocation is able to determine insulin secretion although cAMP degradation is increased by imidazol. 4. Somatostatin and diazoxide inhibit pancreatic tubulin polimerization; however, the effect seem to be indirect and related to the inhibition of calcium translocation determined by both substances.

Insulin binding in mouse liver cells isolated with chelating agents.

Mouse liver cells were isolated with Ca2+ and K+ chelating agents. Cell concentrations in all experiments ranged from 2.5 X 10(5) to 1.44 X 10(6) cells/tube. The kinetics of insulin-receptor binding was studied at 2 C and 20 C. Binding of 1.67 X 10(-11) M 125I-insulin reached equilibrium at 2 C at 180 min; Ka at 50% binding was 0.736 X 10(7) M-1 sec-1. At 20 C equilibrium occurred at 30 min; Ka at 50% binding was 7.519 X 10(7) M-1 sec-1. Non-specific binding was measured by adding 16.6 microM native insulin. Kinetics studies of association point to a pure bimolecular reaction since the constant remains unaltered at different times. In studies of bound complex dissociation, insulin release from the receptor involves first order kinetics, 50% of the bound insulin becoming released during the experimental period. Dissociation was studied at 20 C only, either by dilution or addition of 16.6 microM native insulin. Both methods yielded the same result, showing the dissociation kinetics to be a first order reaction with a half-life of 101 min and Kd: 2.5 X 10(-4) sec-1. Competitive inhibition of native insulin (1.67 X 10(-10), 3.33 X 10(-10), 1.67 X 10(-9), 3.33 X 10(-9), 1.67 X 10(-8), 3.33 X 10(-8), 1.67 X 10(-7), 3.33 X 10(-7) M) against 1.67 X 10(-11) M 125I-insulin was studied in equilibrium. Heterogeneity among active binding sites was found: one population of high affinity and low capacity (2 C: K = 4.64 X 10(7) L/M, Ro = 213 X 10(-11) M; 20 C: K = 2.90 X 10(8) L/M Ro = 28.5 X 10(-11) M) and one of low affinity and high capacity (2 C: K = 6.81 X 10(7) L/M Ro: 836 X 10(-11) M; 20 C: K = 2.63 X 10(6) L/M, Ro: 1080 X 10(-11) M). The results show the use of chelating agents in the separation of liver cells to be of value in physicochemical studies of insulin-receptor interaction.

Increase of ionic strength for charcoal separation of B/F fractions in radioimmunoassays.

The increase in protein adsorption by charcoal as ionic strength increases (salting-out adsorption), was used to separate the bound and free fractions of glucagon, insulin, hGH, hLH and hPRL in the radioimmunoassay. The hormones were labelled with 125I and to express the immunocomplex, gamma-globulin was labelled with 125I. The charcoal used to produce the separation was suspended in magnesium sulfate 3 M (charcoal-SO4Mg). The optimum amount of charcoal and the final concentration of magnesium sulfate determined for each hormone were: glucagon (charcoal 5 mg/tube, 0.125 M); insulin (charcoal 5 mg/tube, 0.131 M); hGH (charcoal 40 mg/tube, 0. 447 M); hLH (charcoal 40 mg/tube, 0.447 M) and hPRL (charcoal 60 mg/tube, 0.321 M). The serum concentration was 1/20 for all hormones, excepting glucagon, where 1/10 was used. The stability of the immunocomplex was studied and it was shown that, under suitable conditions, increased ionic strength does not cause the dissociation of the bound fraction.