Insulin secretion induced by allogeneic lymphocytes.
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Animals↗
Biomedical subjects
J C Basabe
Publications and source records attributed to J C Basabe.
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.
Age Factors↗
Insulin secretion stimulated by allogeneic lymphocytes in an inbred strain of mice.
Effects of intraperitoneal injection of allogeneic lymphocytes on insulin secretion were studied in incubated pancreas slices from BALB/c mice. Injection of allogeneic lymphocytes from C57BL/6J (H2b) mice increased insulin secretion, both in basal and 11-mM glucose-stimulated conditions. This effect was only present when at least 5 X 10(6) or 1 X 10(6) cells were injected (in basal and stimulated conditions, respectively). Glucose-induced insulin secretion (3.3-27.5 mM) was significantly increased in pancreata from mice injected with allogeneic lymphocytes. No effect was observed when glucose was not included in the incubation medium. Intraperitoneal injection of Dextran 70 produced no change in glucose-elicited insulin secretion. There were no differences in glucagon and somatostatin (SRIF) secretion obtained from pancreas of mice injected with allogeneic or syngeneic lymphocytes. Injection of allogeneic cells increases insulin secretion (basal and both phases of 11 mM glucose-stimulated secretion). Puromycin significantly inhibited the second phase of insulin secretion. These results suggest that: Injection of allogeneic lymphocytes raises both basal and glucose-stimulated insulin secretion. This effect seems to be connected with the major histocompatibility complex, and to be related to the number of allogeneic cells injected. Injection of allogeneic lymphocytes seems to sensitize the beta cell response to glucose stimulus. Neither glucagon nor SRIF secretion are altered by alloantigen injection. The stimulatory effect of allogeneic lymphocytes is related, at least in part, to insulin synthesis.
Animals↗
Insulin secretion induced by allogeneic lymphocytes in genetically diabetic mice C57BL/KsJ mdb.
Insulin and somatostatin (SRIF) secretion induced by alloantigen were studied in genetically diabetic mice from the C57BL/KsJ mdb-mdb strain. Diabetic (db) mice injected with allogeneic lymphocytes (A.L.) did not show any increase in their second phase of 27.5 mM glucose stimulated secretion and slightly increased their first phase. Normal A.L. injected mice showed a significant increase in both phases of 27.5 mM glucose stimulated secretion. SRIF secretion of A.L. injected normal and diabetic mice did not significantly differ from that obtained when injected with syngeneic lymphocytes. Lymphocytes from db mice injected into allogeneic mice caused an insulin secretion similar to that produced by allogeneic lymphocytes from non-diabetic mice. Lymphocytes from db mice injected into normal syngeneic mice caused an insulin secretion which was not significantly different from the one caused by syngeneic lymphocytes from non-diabetic mice, and is smaller than the secretion caused by A.L. injection. In summary, db mice showed an impaired hormone response to alloantigenic stimulus, while their lymphocytes maintained their alloantigenic action.
Animals↗
Increased insulin secretion induced by sodium salicylate: effect of puromycin.
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Animals↗
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.
Animals↗
Liver cells binding with high insulin doses at 37 C.
Insulin binding and receptor mediated insulin degradation were studied in isolated rat hepatocytes under physiological conditions (37 C, 100% oxygen, Krebs improved Ringer III with glutamate, pyruvate and fumarate, 150 mg% glucose, 1% bovine albumin). 10(6) rat hepatocytes/tube were incubated with various doses of insulin. Steady state binding with low insulin doses (0.05, 0.5 and 66 ng/tube) was reached in 15 minutes, that state being kept for the rest of the experimental time (75 min). Receptor mediated degradation (Kap) at 15 minutes was 0.0479 min-1, including doses of 5 000 and 50 000 ng/tube. Direct correlation was found between degradation and low doses of insulin, being the slope value equal to Kap. Intracellular accumulation of insulin was found at pharmacological concentrations of insulin (5 000 and 50 000 ng/tube) from the first 15 minutes. That accumulation was dose and time dependent. At 75 minutes, with a 0.2 microM insulin concentration, at least 53% of insulin was estimated as insulin accumulated in the cell, since it was not filtrable with acid medium on Sephadex G 50 superfine. When Triton or dodecyl sulphate were used to solubilize the cells, insulin recovery was complete after binding. Intracellular accumulation, however, was not demonstrated at the first two minutes. Binding studies with 16.67 microM insulin in the presence of degradation inhibitors, such as 2 mM N-ethylmaleimide and 5 mM tetracaine hydrochloride, demonstrated that intracellular accumulation of the hormone occurs when degradation is blocked. On the contrary, after trypsin digestion of receptors, degradation was not observed, while increases in binding were abolished, resembling non-specific binding. Under the experimental conditions reported here, neither intracellular accumulation of insulin nor extracellular release of insulin degradation products can be demonstrated at 2 minutes; insulin accumulation is dose dependent, and it is suggested by the fact that the velocity of insulin internalization exceeds its velocity of degradation.
Animals↗
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)
Adipose Tissue↗
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↗
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↗
Salicylate intoxication and glucose-induced insulin secretion in the rat.
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Animals↗
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↗
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↗
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↗
Effect of pancreatic adrenergic tone modifications prior to glucose-induced insulin secretion.
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Adrenergic alpha-Antagonists↗
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↗
Studies on the dynamics and mechanism of glibenclamide-induced insulin secretion.
Sustained, 60-minute perfusion of glibenclamide (0.5, 1.5 and 10 mug/ml) elicits a one-phase insulin release profile, formed by a rapid secretion peak followed by a second peak with lower insulin levels than the former. Basal insulin secretion values are observed during the period comprised between 13 and 60 minutes of perfusion. Concurrent stimulation with glucose (100, 150, 200 and 300 mg%) plus glibenclamide (1 mug/ml) causes a marked rise in both phases of insulin secretion. The addition of glibenclamide does not modify the biphasic secretion pattern caused by maximal glucose concentration (400 mg%). The maximal values of both phases of secretion in the dose-response curve elicited by different glucose concentrations shift to the left when glibenclamide is added to the perfusate. The increase in insulin secretion caused by glibenclamide is not inhibited by puromycin. Both theophylline and phentolamine modify and increase the glibenclamide-induced insulin release pattern. Propranolol and imidazole inhibit glibenclamide-induced insulin release. Our results suggest that: 1. Glibenclamide increases beta cell sensitivity to glucose stimulation. 2. Glibenclamide and glucose induce secretion of insulin originating in the same compartment. 3. Modification of alpha and beta adrenergic receptors may modify glibodulate the beta cell response to glibenclamide.
Adrenergic alpha-Antagonists↗
Evidence for a hypophyseal factor that stimulates insulin secretion by the pancreas (insulotrophine?)
The continuous infusion of glucose (1 mg/kg/min) via the carotid artery in anesthetized dogs produced a biphasic pattern of insulin secretion. The first peak reached a maximum 3 min after glucose infusion and decreased to basal level at 7 min. As long as glucose infusion persisted a slow and maintained increase in insulin level in the pancreatico-duodenal vein was observed. The same amount of glucose infused in to the carotid arteries of hypophysectomized dogs, failed to induce any change in plasma insulin level. Plasma sample obtained from the jugular vein of dogs receiving glucose via the carotid arteries were infused into a second dog via the pancreatico-duodenal artery. One minute after the onset of infusion a rise in insulin was observed in the pancreatico-duodenal vein. The stimulating effect was not due to the high blood glucose level present in the jugular vein of dogs undergoing the cephalic glucose infusion. Infusion through the pancreatico-duodenal artery of a glucose solution at a concentration equal to the highest blood glucose level observed in the jugular vein did not evoke insulin secretion. Plasma samples obtained from the jugular vein of dogs receiving saline via the carotid arteries did not evoke insulin secretion when receiving into the pancreatico-duodenal arteries of dogs. Pancreatic infusion of plasma obtained from the jugular vein of hypophysectomized dogs infused glucose through the carotid arteries did not evoke any pancreatic response. These findings are demonstrative of the presence of a hypophyseal humoral insulin stimulating factor in the jugular vein of dogs receiving a cephalic glucose load.
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