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Biomedical subjects

J C Cresto

Publications and source records attributed to J C Cresto.

At least 37 records · Page 2Linked to original sources
PubMed1983

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.

Aging↗
PubMed1982

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.

Animals↗
PubMed1981

Preparation of biologically active mono-125I-insulin of high specific activity.

Pork insulin was labeled by the chloramine T technique (phosphate buffer 0.25 M; pH 7.5; EDTA 0.001 M; chloramine T: 0.2625 mg/ml; sodium metabisulfite 2.4 mg/ml) in a reaction volume of 50 microliters, adding chloramine T every 30 seconds twice (2.1 micrograms in 1 minute) and halting the reaction with 5 microliters metabisulfite. Three fractions were separated in preparative starch gel: F1 (mono-125I-insulin contaminated with cold insulin), F2 (mono-125I-insulin free from cold insulin), and F3 (di-125I-insulin). Insulins with low and high specific activity (iodine/insulin ratios 0.1/1 and 1/1 respectively) were prepared for study purposes, and quality was assessed by means of dose-response curves with antibodies and with liver cells. Specific activity of F2 as obtained from dose-response curves utilizing Scatchard's plot was 323 and 382 mCi/mg. Specific activity of F1 varied according to the extent of contamination with cold insulin. A reduction in the F2 B/F ratio was observed upon iodination with iodine/insulin ratios of 1/1 or in the neighborhood. The mass and immunoreactivity of F3, as well as its B/F ratios were constant, regardless of specific activity. The behavior with antibodies was ratified upon observations on uptake by liver cells and glucose consumption by isolated fat cells. In conclusion, F2 labeled with 0.1/1 iodine/insulin ratios was separated from cold insulin in preparative starch gel, thus increasing its specific activity (360 mCi/mg approximately) without alteration of its immunologic or biologic properties.

Animals↗
PubMed1981

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.

Adsorption↗
PubMed1981

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.

Animals↗
PubMed1981

Isolation of liver cells with Ca2+ and K+ chelating agents. Biochemistry and cell morphology.

Cell morphology, glutamic pyruvic (GTP) and glutamic oxalacetic transaminases (GOT) concentrations, and the ability to produce glucose or urea from different substrates (pyruvate, alanine, fructose, lactate and glutamine) were studied in isolated mouse and rat liver cells in the presence of Ca2+ and K+ chelating agents (0.1 M sodium perchlorate and 0.027 M sodium citrate with 1 mg/ml bovine albumin; ionic strength: 0.198, pH: 7.4). The chelating agent is perfused through the portal vein of an in situ liver, at low pressure (8 ml/min) at 20 C for 15 min. Cell dispersion is obtained by cutting liver lobes and "massaging" the tissue with a plastic spatula. Wash and cell concentration may be obtained by sedimentation or centrifugation in Krebs III, glucose 150 mg %, improved with 0.16 M pyruvate, 0.1 M fumarate and 0.16 M glutamate. This procedure furnished 53.06 +/- 3.33 X 10(6) cells, which was highly significant (p less than 0.001) with respect to saline controls: 6.11 +/- 1.91 X 10(6). After staining with Papanicolaou, hematoxylin-eosin, and PAS, the cellular material obtained was classified optically into: normal isolated parenchymal liver cells, hepatocyte clumps, "burst" cells, normal blood or reticuloendothelial cells, cellular debris and non-cellular material. Cell morphology showed that a constant perfusion (8 ml/min) with a minimal mechanical treatment, 82.5% of the liver cells appears normal. Biochemical study showed that transaminases are indeed lost, but this loss is below the amount capable of effecting metabolic blockade (3/4 of transaminases remain in liver cells; GOT in cells: 692 +/- 218; GPT in cells. 264 +/- 94; GOT in supernatant: 152 +/- 29; GPT in supernatant: 79 +/- 12 mUI/10(6) cells, after recovering 60 min at 37 C) (means +/- SEM). Conversion of substrates (sodium pyruvate 10 mM, 20 mM D-L alanine, 10 mM fructose and 20 mM D-L sodium lactate) into glucose was statistically significant with respect to the baseline when the liver cells were isolated and recovered (rat liver cells, basal: 25.37 +/- 3.73; pyruvate: 54.04 +/- 7.98; DL-alanine: 62 +/- 10.07; fructose: 264.67 +/- 20.51; DL-lactate: 78.05 +/- 17.99 mmoles/10(6) cels, means +/- SEM). Urea production from 5 mM DL-glutamine was statistically highly significant to the basal with rat liver cell isolated and recovered (basal: 160.60 +/- 3.76; DL-glutamine: 608.47 +/- 16.15 mmoles/10(6) cells; means +/- SEM). The results obtained suggest that liver cells isolated with Ca2+ and K+ chelating agents used as described above are of value for biochemical studies.

Alanine Transaminase↗
PubMed1977

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.

Animals↗
PubMed1977

Half life of injected 125I-insulin in control and ob/ob mice.

The t-1/2 of moniodo 125I-insulin in ob/ob mice and their lean litter mates is 10 min. No difference was found between the two groups. Further, 48 hr of fasting did not alter the t-1/2 in ob/ob mice. In view of the markedly enlarged insulin pool in ob/ob mice, one must conclude that the mass of insulin degraded in unit time is increased. However, these findings indicate that the cause of hyperinsulinism in ob/ob mice is unrelated to rates of insulin degradation.

Animals↗
PubMedDate not supplied

Effect of salicylate poisoning on insulin secretion. Studies on its mechanism of action.

Sodium salicylate poisoning increased glucose-induced insulin secretion by slices of rat pancreas. The alpha adrenergic blocker phentolamine, but not salicylate poisoning, overcame the inhibitory effect of epinephrine on insulin secretion. Theophylline (5, 10 and 15 mM) significantly increased the insulin secretion induced by 11 mM glucose. The highest insulin response was obtained when theophylline was used at a 10 mM concentration. However, when pancreas slices from salicylate poisoned rats were used, a 5 mM concentration was sufficient to achieve maximal insulin response. Salicylate poisoning diminished the free tubulin pool, an action that was impaired by imidazole; however, imidazole did not modify the effect of the ionophore A23187. The results suggest that: a) sodium salicylate poisoning increases B-cell response to glucose; b) changes in alpha adrenergic activity are not related to the mechanism of action of salicylate; c) an increment in the cAMP concentration may mediate the stimulatory effect of salicylate poisoning on insulin secretion; d) the effect of salicylate on the microtubular system is indirect and probably mediated through an increment in pancreatic cAMP.

Animals↗
PubMedDate not supplied

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.

Animals↗