Establishment of serum based essentiall free of proteolytic activity for the culture of mouse pancreatic islets.

The present study demonstrates that a) serum based culture medium degrades 125I inhibits its proteolytic activity leading to the recovery of more insulin secreted by islets cultured in the presence of high glucose concentration alone or with glucagon; c) aprotinin also favoured the accumulation of secreted insulin by protecting the hormone from a residual degradative capacity of the hear treated serum.     

Pancreastatin (33–49) enhances the priming effect of glucose in the rat pancreas

Short-term exposure to glusoe increases insulin secretion during subsequent stimulation. We investigated the effect of the new regulatory peptide pancreastatin on this priming effect of glucose in the perfused rat pancreas. Pancreastatin (33–49) at a concentration of 10^–8 M inhibited insulin release when stimulated by glucose at a concentration of 16.7 mM. However, after a second pulse of 16.7 mM glucose, pancreastatin potentiated the priming effect of glucose on insulin secretion. The modulation of insulin secretion by pancreastatin results in a potentiation of the priming effect of glucose in the rat pancreas, suggesting a role for pancreastatin in the adaptation of the B cell to glucose-stimulated insulin secretion.     

Effect of insulin in vivo on the synthesis of free fatty acids (FFA) in chicken heart and skeletal muscle

Summary The effect of insulin on the synthesis of free fatty acids from glucose in the skeletal and heart muscles of chicken is examined. 10 min after glucose-(U-¹⁴C) administration, labeled free fatty acids (FFA) appeared in both skeletal and heart muscles. 0.75 IU of insulin kg^–1 b. wt significantly increased the labeled FFA at the 30, 60 and 120 min intervals, with a maximum at 60 min.     

Fehlende immunogene Wirkung exogenen Insulins bei Hunden nach langsamer Adaptation an Insulin

Summary Subcutaneous injection of exogenous insulin always results in a production of antibodies against insulin. But after a slowly increased dose of insulin (a daily end dose of 3.5–4.2 U/kg) insulin antibodies could not be found in the serum of 2 dogs. This failure of immunogenic action of insulin is discussed as an induction of immunotolerance by the modus of initial antigen application.     

Insulin acts on the hypothalamic glucose-facilitated neurons to induce hyperglycemia and hyperinsulinemia in the rat

Microinjection of insulin (0.04–0.12 IU/l) into the anterior hypothalamus or the lateral hypothalamus, but not the vertromedial hypothalamus of the rat brain, caused a dose-dependent rise in blood glucose and in serum insulin. The majority (71.5%) of the glucose-facilitated neurons recorded in the lateral hypothalamic area were excited by intracerebral injection of insulin. The data indicate that insulni acts on the hypothalamic glucose-facilitated neurons to induce hyperglycemia and hyperinsulinemia. It is unknown whether insulin normally reaches the hypothalamic area, or how it might do so.This work was supported by grants from the National Science Council (Taipei, Taiwan, Republic of China).     

The effect of insulin on the electrophoretic mobility of rat hepatocytes

Summary The addition of insulin (10 U) to a suspension of isolated hepatocytes in Krebs-Ringer bicarbonate solution. causes an increase in the negative electrophoretic mobility of the cells from –1.68 m sec^–1 V^–1 cm to 2.26 m sec^–1 V^–1 cm. This observation supports the findings by other workers that the binding of insulin to its receptor leads to a marked change in the membrane.Acknowledgment. We wish to thank the Medical Research Council for the provision of the microelectrophoresis apparatus and initial running costs of the project.     

Effect of gentamycin on insulin release and45Ca net uptake by isolated islets

Summary In isolated islets, gentamycin reduced both the⁴⁵Ca net uptake and insulin release induced by glucose, but failed to inhibit the insulin secretion provoked by the combination of Ba²⁺ and theophylline. This indicates that the inhibitory effect of gentamycin is due to a reduction of Ca²⁺ entry into B-cells instead of to a harmful effect upon the integrity of the effector system, responsible for the extrusion of the insulin containing granules.Supported by a Grant (No. 79/1872) from the São Paulo State Research Foundation (FAPESP), Brazil.     

Hyperthermia-induced apoptosis and the inhibition of DNA laddering by zinc supplementation and withdrawal of calcium and magnesium in suspension culture of tobacco cells

In the present paper we report examination of stereotypic hallmarks of apoptosis in heat-treated tobacco cells. Hyperthermia (44 °C, 4 h) caused apoptosis in 53.6% of cells when assayed 24 h after heat treatment. The induction of apoptosis by heat treatment was confirmed by flow cytometric assay. Cytological observations revealed condensation of the cytoplasm and nucleus, as well as nuclear collapse. DNA ladders were observed in DNA extracted from heat-treated cells, whereas DNA from control cells remained undegraded. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay revealed that 51.8% of the heat-treated cells (44 °C, 4 h) show positive reaction after a 24-h recovery. When cells were cultured in a medium supplemented with 0.4–5.0 mM ZnSO₄, internucleosomal DNA fragmentation induced by heat shock was completely negated. Strikingly, when cells were cultured in Ca²⁺ and/or Mg²⁺ free medium for 44 h followed by heat treatment, DNA laddering was not observed. The results suggest hyperthermia-induced apoptosis and a correlation between the regula tion of endonucleases and heat shock signal in apoptotic tobacco cells. Received 17 September 1998; received after revision 4 January 1999; accepted 4 January 1999     

Dynamic insulin secretion from perifused rat pancreatic islets

Insulin secretion from isolated pancreatic islets of 8- to 12-day-old rats was investigated in a dynamic in vitro (perifusion) system. The aims of the study were (i) to describe a carefully controlled in vitro method to study the mechanism of insulin secretion and to analyse the effects and dynamic interactions of bioactive compounds on isolated rat pancreatic islets, (ii) to validate the method by comparing fundamental data on the functions of the islets obtained with this method to those collected with other techniques; and (iii) to find novel features of the control of insulin secretion. The method was carefully designed to maintain the functional capacity of the explanted cells. A functional standardization system was elaborated consisting of (i) analysis of the changes in the basal hormone secretion of the cells; (ii) evaluating responses to a standard, specific stimuli (50 mM glucose for 3 min); (iii) determining the alteration of the momentary size of the hormone pool with responses to KCl; and (iv) direct determination of the total intracellular hormone content from the extract of the column. The technique provides accurate quantitative data on the dynamic responses to biologically active compounds that act directly on the pancreatic islets. The islets maintained their full responsiveness for up to 7 days, and responses as close as in 1-min intervals could be distinguished. A linear dose-response relationship was found on the glucose-induced insulin release in case of 3-min stimulation with 4 and 500 mM of glucose (lin-log graph). Utilizing this method, we showed that no desensitization to glucose-induced insulin release can be observed if the responsiveness of the cells is properly maintained and the parameters of the stimulation are carefully designed. Exposure of the explanted islets to 10 μM acetylcholine or 30 mM arginine (Arg) induced a transitory elevation of insulin release similar in shape to that experienced after glucose stimulation. Norepinephrine (NE), dopamine (DA) and somatostatin (SS) did not induce any detectable alteration on the basal insulin secretion of the islets. However, 100 nM SS given together with 50 mM glucose, 30 mM Arg or 10 μM acetylcholine significantly reduced the insulin-releasing effect of these substances (by 75.5, 71.5 and 72.5%, respectively). At the same time, SS did not alter the insulin response of the islets to 100 mM elevation of K⁺ concentration. SS also inhibited glucose-induced insulin release in a dose-dependent way (ED₅₀ = 22 nM). A similar dose-dependent inhibitory effect on glucose-induced insulin release was found with NE (ED₅₀ = 89 nM) and DA (ED₅₀ = 2.2 μM). γ-Aminobutyric acid (GABA) did not influence insulin release under similar circumstances. Received 16 January 1998; received after revision 6 May 1998; accepted 8 May 1998     

Insulin activates Gαil,2 protein in rat hepatoma (HTC) cell membranes

Insulin action is initiated by binding to its cognate receptor, which then triggers multiple cellular responses by activating different signaling pathways. There is evidence that insulin receptor signaling may involve G protein activation in different target cells. We have studied the activation of G proteins in rat hepatoma (HTC) cells. We found that insulin stimulated binding of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-³⁵S) to plasma membrane proteins of HTC cells, in a dose-dependent manner. This effect was completely blocked by pertussis toxin treatment of the membranes, suggesting the involvement of G proteins of the Gα _i/Gα _o family. The expression of these Gα proteins was checked by Western blotting. Next, we used blocking antibodies to sort out the specific Gα protein activated by insulin stimulation. Anti-Gα _il,2 antibodies completely prevented insulin-stimulated GTP binding, whereas anti-Gα _o,i3 did not modify this effect of insulin on GTP binding. Moreover, we found physical association of the insulin receptor with Gα _i1,2 by copurification studies. These results further support the involvement of a pertussis toxin-sensitive G protein in insulin receptor signaling and provides some evidence of specific association and activation of Gα _i1,2 protein by insulin. These findings suggest that Gα _i1,2 proteins might be involved in insulin action. Received 23 September 1998; received after revision 23 November 1998; accepted 25 November 1998     

Nitric oxide-related pancreatic endocrine responses to hyperglycaemia in the conscious calf

Mean plasma insulin concentration was reduced and mean plasma glucose concentration increased following the administration of N-nitro-L-arginine methyl ester (L-NAME; 100 mol kg^–1 i.a.) in conscious calves given continuous infusions of exogenous glucose (30–60 mol min^–1 kg^–1 i.v.). It is concluded that the rise in plasma insulin concentration which occurs in these animals in response to glucose is mediated, at least in part, by a nitric oxide-related factor (NOx).     

Endocrine and exocrine pancreatic function after camostate-induced growth of the organ

It is well known that oral administration of camostate induces hyperplasia and hypertrophy of the rat pancreas. It is not clear, however, whether pancreatic hormone and enzyme secretion are affected by camostate treatment.In rats, daily administration of 200 mg camostate/kg b. wt for 14 days significantly increased pancreatic weight and pancreatic content of DNA, protein, amylase, lipase, trypsin and chymotrypsin, as well as the amount of insulin, glucagon and somatostatin. In the intact animal, blood glucose levels and serum concentrations of insulin and glucagon in response to an oral glucose load were not impaired after camostate treatment. In the isolated perfused pancreas, however, insulin and glucagon secretions were reduced, whereas somatostatin release was not affected. The volume of pancreatic juice produced by the unstimulated isolated perfused organ, as well as protein and enzyme secretion, were increased after camostate treatment. Likewise, the isolated perfused pancreas from camostate-treated rats secreted a larger volume of pancreatic juice and more protein in response to cholecystokinin (CCK), while enzyme secretion was affected in a non-parallel manner: amylase release was markedly reduced, lipase release was unchanged, and release of trypsin and chymotrypsin was increased.     

Role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion

The role of protein kinase C and Ca²⁺ in glucose-induced sensitization/desensitization of insulin secretion was studied. A 22–24h exposure of mouse pancreatic islets to glucose (16.7 mmol/l) in TCM 199 culture medium, with 0.26 mmol/l or 1.26 mmol/l Ca²⁺, reduced total islet protein kinase C activity to approx. 85% and 60% of control values, respectively. At 0.26 mmol/l Ca²⁺ in TCM 199 medium, exposure to glucose (16.7 mmol/l) led to a potentiation of both phase 1 and phase 2 of glucose-induced insulin secretion, and caused a shift in the dose-response curve with 10 mmol/l and 16.7 mmol/l glucose exhibiting equipotent effects in stimulation of insulin secretion. In glucose-sensitized islets, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (0.16 μmol/l) did not further potentiate induction of secretion by 10 mmol/l or 16.7 mmol/l glucose. At 3.3 mmol/l glucose, however, phorbol ester-induced secretion was augmented, and was characterized by a faster onset of secretion in glucose-sensitized islets relative to control islets. In contrast, a partial reduction in arachidonic acid (100 μmol/l)-induced insulin release was observed in glucose-sensitized islets in the absence of extracellular Ca²⁺. Increasing the Ca²⁺ concentration to 1.26 mmol/l in TCM 199 during the 22–24h exposure to glucose (16.8 mmol/l) led to inhibition of phase 1 and abolition of phase 2 of glucose (10 mmol/l, 16.7 mmol/l)-induced insulin secretion. In addition, this treatment abolished phorbol ester-induced and arachidonic acid-induced insulin secretion at 3.3 mmol/l glucose. Altogether, these data suggest that sensitization of insulin secretion is caused by a preferential down-regulation of the inhibitory effects of protein kinase C, leading to an increased first phase, and an increased coupling of glucose to the stimulatory effects of protein kinase C during the second phase of glucose-induced insulin secretion. Desensitization of insulin secretion appears to be a consequence of sustained Ca²⁺ influx, inducing extensive down-regulation of protein kinase C and also causing deleterious effects on islet cell function in protein kinase C-deprived islets.     

Effect of heat treatment on the ATPase activity of various sarcoplasmic reticulum preparations

Summary Ca²⁺-stimulated ATPase activity of sarcoplasmic reticulum (SR) preparations is activated after a short period of preincubation at temperatures between 40 and 45°C, but for temperatures higher than 48°C pronounced denaturation is observed. Heat denaturation is decreased if Mg²⁺ or K⁺ are present during heat treatment.Acknowledgments. This work was supported by research grants from Instituto de Alta Cultura (Grant No. CB/2) and the Calouste Gulbenkian Foundation. We are grateful to Drs.A. P. Carvalho andV. M. C. Madeira for many discussions and suggestions during the course of this work.     

In vitro effects of methionine-enkephalin,somatostatin and insulin on cultured gonadal cells of the snailHelix aspersa

Isolated snail gonadal cells were cultured in the presence of synthetic neuropeptides in order to determine the subsequent effect of these substances on gonadal synthetic activities. Gonadal cells were incubated for 24 h in concentrations of methionine-enkephalin, somatostatin and insulin ranging from 10^–4 M to 10^–9 M, in medium 199 supplemented with 6% Ultroser G. Synthesis of DNA and protein by the cultured cells were simultaneously estimated by measuring incorporation of³H thymidine and³⁵S methionine. The rate of labelled precursor incorporation was measured using the liquid scintillation technique. All substances tested exerted a dose-dependent effect. The synthetic activity of the cultured cells was highest when the concentration of the peptides added to the medium approximated the physiological levels. Methionine-enkephalin, somatostatin and insulin at 2×10^–8 M significantly increased³H thymidine incorporation, by 62%, 69% and 69% respectively, and protein synthesis by 42%, 57% and 57%, respectively. In the case of juvenile gonadal cultured cells, a similar increase in³H and³⁵S incorporation was registered for a 10^–7 M peptide concentration. Both lower and higher peptide concentrations inhibited³H thymidine and³⁵S methionine incorporation. Pharmacological studies suggest the existence of methionine-enkephalin and somatostatin-like receptors on snail gonadal cells. These results indicate that our gonadal cell culture model provides a useful tool for the study of the neuroendocrinological control of the activity of snail gonadal cells.     

Heat evolution of cultured human keratinocytes

Summary The heat production of normal and transformed human epidermal keratinocytes precultured in Petriperm ^tm tissue culture dishes was measured calorimetrically. For this purpose, the membrane at the bottom of the culture dish was cut out aseptically and put into a microcalorimeter vessel with the cell layer inwards. A continuous heat output of (83±12) pW/cell was measured for normal keratinocytes from a confluent primary culture. A value of (134±35) pW/cell was obtained when the transformed kerationocyte line SV-K14 was used. The method described in this paper is simple, leads to reproducible results, and can be easily adapted to the calorimetric study of other mammalian cells in vitro.     

Arachidonic acid signaling is involved in the mechanism of imidazoline-induced K<Subscript>ATP</Subscript> channel-independent stimulation of insulin secretion

The mechanism by which the novel, pure glucose-dependent insulinotropic, imidazoline derivative BL11282 promotes insulin secretion in pancreatic islets has been investigated. The roles of K_ATP channels, α₂-adrenoreceptors, the I₁-receptor-phosphatidylcholine-specific phospholipase (PC-PLC) pathway and arachidonic acid signaling in BL11282 potentiation of insulin secretion in pancreatic islets were studied. Using SUR1^(-/-) deficient mice, the previous notion that the insulinotropic activity of BL11282 is not related to its interaction with K_ATP channels was confirmed. Insulinotropic activity of BL11282 was not related to its effect on α₂-adrenoreceptors, I₁-imidazoline receptors or PC-PLC. BL11282 significantly increased [³H]arachidonic acid production. This effect was abolished in the presence of the iPLA₂ inhibitor, bromoenol lactone. The data suggest that potentiation of glucose-induced insulin release by BL11282, which is independent of concomitant changes in cytoplasmic free Ca²⁺ concentration, involves release of arachidonic acid by iPLA₂ and its metabolism to epoxyeicosatrienoic acids through the cytochrome P-450 pathway. Received 5 July 2007; received after revision 18 September 2007; accepted 20 September 2007     

Insulin effect in vitro on human erythrocyte plasma membrane

Summary The effect of porcine insulin has been tested in vitro on human erythrocyte plasma membrane (Na⁺–K⁺) and Mg²⁺-ATPase activities as well as on membrane fluidity. The results indicate that the hormonal treatment significantly inhibits (Na⁺–K⁺)-ATPase activity, and at the same time decreases membrane fluidity.This investigation has been supported by Consiglio Nazionale delle Richerche, Rome, Italy.     

Drug susceptibility of PCA in WBB6F1-W/Wv mice

Our previous study revealed that passive cutaneous anaphylaxis (PCA) can be produced in congenitally mast cell-deficient WBB6F1-W/W^v (abbreviated as W/W^v) mice on sensitization with undiluted or slightly diluted allogeneic and xenogeneic antisera but not on sensitization with allogeneic monoclonal immunoglobulin (Ig)E and IgG1 antibodies regardless of the antibody concentration [1]. In view of these findings, the present study was conducted to characterize PCA in this strain from its drug susceptibilities using mast cell-bearing WBB6F1-+/+ (abbreviated as +/+) and B6D2F1 mice as references. PCA in W/W^v mice mediated by a low dilution (1  4) of hyperimmune serum to bovine serum albumin of the B6D2F1 mouse origin was markedly suppressed by CV-6209, an antagonist of platelet-activating factor (PAF), but not by antihistamines such as cyproheptadine and oxatomide. In contrast, PCA in +/+ and B6D2F1 mice mediated by a high dilution (1  128) of the anti-serum (virtually by IgG1 antibody) was nearly completely suppressed by antihistamines but not by CV-6209. A remarkable difference between PCA in W/W^v and reference mice was also observed in the susceptibility to monoclonal anti mouse granulocyte (Gr-1) antibody PCA in W/W^v mice was potently suppressed by the 1- to 3-day pretreatment with this antibody but that in references was not at all. Putting these present results together with the previous finding that anti-granulocyte antibody greatly reduces circulatory Gr-1⁺ leukocytes, 1 to 3 days after the treatment [2], it is highly probable that PCA in W/W^v mice mediated by some antibody isotypes other than IgE and IgG1 is produced by PAF mainly released from Gr-1⁺ cells, while IgG1 antibody-mediated PCA in mast cell-bearing reference mice is evoked by histamine derived from mast cells. PCA homologous to that in W/W^v mice could also be produced in the reference mice on sensitization with undiluted or slightly diluted antiserum, when generalized blueing due to excess IgG1 antibody was removed by the oxatomide treatment be fore the antigen challenge. Received 10 December 1997; received after revision 2 February 1998; accepted 23 February 1998     

Retinoic acid enhances the proliferation of smooth muscle cells

Summary Retinoic acid (RA, 10^–5–10^–7 M) is shown to enhance the proliferation of cultured rat aortic smooth muscle cells (SMC). This effect is not connected with a synergistic action of RA together with serum mitogens. Moreover, the expression of L1, a surface antigen specific for modulated SMC entering the cell cycle, is amplified by RA treatment.