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N Fleischer

Publications and source records attributed to N Fleischer.

At least 37 records · Page 2Linked to original sources
PubMed1990

Regulation of insulin secretion from beta-cell lines derived from transgenic mice insulinomas resembles that of normal beta-cells.

Insulin secretory physiology has been characterized in tumor cell lines derived by primary culture of insulinomas that developed in transgenic mice expressing the large T-antigen of SV40 in pancreatic islet beta-cells. Cells in one of these lines, beta TC-3, contain large amounts of insulin (3100 +/- 294 ng/100 micrograms cellular protein). Constitutive release of insulin over 2 h in static incubation was low at 31.9 ng/100 micrograms protein and was increased 2-fold by glucose (16.7 mM) and 8-fold by depolarizing concentrations of potassium (45 mM). Isobutylmethylxanthine (IBMX; 0.5 mM) and forskolin (5 and 50 microM), which elevated cellular levels of cAMP, were ineffective as secretagogues, but dramatically potentiated glucose and potassium effects on insulin release (6.5- and 4-fold, respectively). A variety of other known insulin secretagogues stimulated insulin release in a manner analogous to their effects in normal islets. The sulfonylurea glipizide (1 microM) and the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (1 microM) stimulated insulin release 3.4- and 13.7-fold, respectively. The cholinergic agonist carbachol (2 microM) was ineffective alone, but potentiated glucose-induced insulin release 2.8-fold. Comparable stimulation of insulin release by glucose (16.7 mM) and glucose (16.7 mM) plus IBMX (0.5 mM) was noted with several other beta TC lines, which were derived independently from separate transgenic mice. Glucose- and glucose- plus IBMX (0.5 mM)-induced insulin release occurred progressively from 0.15-16.7 mM, indicating that insulin release from beta TC-3 cells occurred at much lower levels than that from normal islets. However, as in the normal islet, the glucose concentration dependency for insulin release was highly correlated (r = 0.93) with the glucose concentration dependency for glucose utilization (measured by 3H2O formation from [5-3H]glucose). This suggests that glucose induces insulin release from beta TC-3 cells by a mechanism similar to that in the normal islet. The high insulin content, the multifold stimulation of insulin release by a variety of secretagogues, their convenient propagation in culture, and the renewable source of these cell lines make the beta TC cells a convenient model for studies of beta-cell function.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Relationship of insulin secretion and glycemic response to dietary intervention in non-insulin-dependent diabetes.

Forty-two obese subjects with non-insulin-dependent diabetes mellitus had their plasma insulin, C peptide, and glucose levels measured after an overnight fast and in response to a 75-g oral glucose loading. Subjects were then prospectively followed up with dietary treatment, and the same measurements were repeated at 1 year. Although insulin values tended to be lower with greater fasting hyperglycemia at baseline, no correlation was observed among three parameters. However, near-normalization of glycemia (measured as the level of hemoglobin A1) was associated with significantly higher fasting and stimulated plasma insulin concentrations. Sixteen subjects were matched to each other for equivalent baseline hyperglycemia (by glycosylated hemoglobin) and divided into group 1 (normalization of the hemoglobin A1 value to 7.0% +/- 0.3% [mean +/- SE]) and group 2 (persistent hyperglycemia) (hemoglobin A1 value, 10.7% +/- 0.7% [mean +/- SE]). Before dietary therapy, the plasma insulin concentrations were twofold to threefold higher in group 1, and despite similar degrees of weight loss, group 2 failed to demonstrate improved glycemia. We concluded that the outcome of diet therapy for non-insulin-dependent diabetes mellitus is dependent on the duration of diabetes and endogenous insulin secretory reserve. There is a subgroup of patients with non-insulin-dependent diabetes mellitus in whom delayed dietary intervention may have a beneficial effect.

Blood Glucose↗
PubMed1989

Retrovirus-mediated expression of preprosomatostatin in rat pituitary GH3 cells: targeting of somatostatin to the regulated secretory pathway.

Somatostatin (SRIF) is a 14-amino acid peptide hormone that is synthesized as part of a larger precursor, prepro-SRIF, consisting of a signal peptide and a proregion of 80-90 amino acids; mature SRIF is located at the carboxyl-terminus of the precursor. We have used a recombinant retroviral expression vector encoding anglerfish prepor-SRIF-I to infect rat pituitary GH3 cells. The aim of these studies was to investigate the intracellular storage and secretion of the total pool of endogenous GH compared to that of SRIF. Several clonal lines of GH3 cells expressing high or low levels of SRIF were treated with TRH, forskolin, or depolarizing concentrations of potassium, and the levels of intracellular and secreted GH or SRIF were determined using highly sensitive RIAs. Approximately 65% of the total GH was secreted basally, whereas less than 20% of the SRIF-immunoreactive material was basally secreted. Forskolin treatment or potassium depolarization stimulated GH release, but only about 50% above basal levels. In contrast, SRIF secretion was stimulated approximately 5-fold in response to these secretagogues. Based on its lower basal rate of secretion compared to GH and its enhanced release in response to a variety of secretagogues, we conclude that the heterologously expressed SRIF is preferentially targeted to the regulated pathway in GH3 cells.

Animals↗
PubMed1987

Differential activation of type I and type II 3',5'-cyclic adenosine monophosphate-dependent protein kinases by growth hormone-releasing factor.

Rat GH-releasing factor (rGRF) stimulated GH release and intracellular cAMP accumulation in cultured rat anterior pituitary cells with EC50 values of approximately 10 and 150 pm, respectively. Consistent with an effect on cellular cAMP levels, rGRF stimulated the adenylate cyclase activity of rat anterior pituitary membranes with an EC50 value of approximately 60 pm. Using antisera directed against the regulatory subunits of type I and II cAMP-dependent protein kinases, these enzymes were immunoprecipitated from the cytosolic fraction of cultured cells in order to monitor the degree of their activation by rGRF. Both isoenzymes were rapidly activated in cells incubated with rGRF but with different kinetics; full activation of protein kinase I was evident within 3-5 min and activation of protein kinase II occurred between 5 and 15 min. The magnitude of activation was differentially regulated by rGRF in a concentration-dependent manner. Somatostatin only partially attenuated rGRF-stimulated GH release, cAMP accumulation, and adenylate cyclase activation. Somatostatin was effective in partially antagonizing activation of protein kinase II at all concentrations of rGRF and of protein kinase I only at intermediate concentrations of rGRF. The significance of this rGRF-induced differential activation of the two isoenzymes of cAMP-dependent protein kinase is discussed in terms of the multiple effects of rGRF on somatotropic cells of the rat anterior pituitary.

Animals↗
PubMed1987

Adrenocortical micronodular dysplasia, cardiac myxomas, lentigines, and spindle cell tumors. Report of a kindred.

In a family encompassing three generations, six of 11 evaluated members have two or three elements of a triad comprising adrenocortical micronodular dysplasia, mucocutaneous lentigines, and cardiac myxomas. Evaluation of the adrenals in affected members revealed characteristic pathologic lesions of micronodular adrenal hyperplasia and corticotropin-independent steroidogenesis that correlated with age, suggesting a progressive lesion that begins in early childhood. Since all subjects with micronodular hyperplasia and/or cardiac myxomas also had mucocutaneous lentigines, the skin lesions were markers for affected subjects. This family is one of the larger reported with this syndrome. Of special note was the finding of rare visceral tumors in affected family members, including melanocytic schwannomas and a fibrolamellar hepatoma, signaling another feature of the syndrome. Since 60% of this family encompassing three contiguous generations were affected, the syndrome appears to be inherited as an autosomal or X-linked dominant gene.

Adolescent↗
PubMed1986

Enhanced activation of cAMP-dependent protein kinase by rapid synthesis and degradation of cAMP.

Activation of cAMP-dependent protein kinase II by static and dynamic steady-state cAMP levels was studied by reconstituting an in vitro model system composed of hormone-sensitive adenylate cyclase, cyclic nucleotide phosphodiesterase, and cAMP-dependent protein kinase II. The rates of cAMP synthesis were regulated by incubating isolated membranes from AtT20 cells with various concentrations of forskolin. In the presence of 3-methylisobutylxanthine, the rate of protein kinase activation was proportional to the rate at which cAMP was synthesized, and there was a direct relationship between the degree of activation and the level of cAMP produced. The activation profiles of protein kinase generated in the presence of exogenous cAMP or cAMP produced by activation of adenylate cyclase in the absence of cAMP degradation were indistinguishable. Dynamic steady-state levels of cAMP were achieved by incubating the membranes with forskolin in the presence of purified cyclic nucleotide phosphodiesterase. Under these conditions, the apparent activation constant of protein kinase II for cAMP was reduced by 65-75%. This increased sensitivity to activation by cAMP was seen when phosphotransferase activity was measured directly in reaction mixtures containing membranes, protein kinase, and histone H2B or when regulatory and catalytic subunits were first separated by immunoprecipitation of holoenzyme and regulatory subunits with specific anti-serum. Our results are consistent with the hypothesis that rapid cAMP turnover may function as a mechanism for amplifying hormonal signals which use the cAMP-dependent protein kinase system.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Somatostatin inhibits corticotropin-releasing factor-stimulated adrenocorticotropin release, adenylate cyclase, and activation of adenosine 3',5'-monophosphate-dependent protein kinase isoenzymes in AtT20 cells.

The mechanisms by which somatostatin (SRIF) inhibits CRF-induced ACTH secretion from AtT20 cells were characterized by comparing the effects of SRIF on cAMP production, adenylate cyclase activity, and activation of cAMP-dependent protein kinase isoenzymes with its effects on ACTH release. In isolated membranes, CRF (100 nM) stimulated adenylate cyclase activity 4- to 5-fold. SRIF inhibited CRF-stimulated adenylate cyclase in a concentration-dependent manner. However, maximal inhibition was 50%. SRIF did not inhibit basal adenylate cyclase or forskolin-stimulated cyclase in the absence of guanine nucleotides and had only small effects on forskolin-stimulated cyclase when assayed in the presence of guanine nucleotides. CRF (100 nM) induced small rises (2-fold) in intracellular cAMP levels which produced maximal ACTH release. SRIF inhibited basal and CRF-stimulated ACTH release in a concentration-dependent manner, and there was a good correlation between inhibition of ACTH release and inhibition of the activation of cAMP-dependent protein kinases in these cells. Thus, the effect of SRIF on CRF-induced ACTH release appeared to result from its effect on inhibition of adenylate cyclase. In the presence of 3-methylisobutylxanthine (MIX), CRF increased cAMP levels 20-fold and activated a greater proportion of cAMP-dependent protein kinase, but did not stimulate ACTH release more than CRF alone. Under these conditions, SRIF (100 nM) inhibited cAMP accumulation by 90%. ACTH release was also inhibited, but higher concentrations of SRIF were required to block ACTH release compared to cells incubated in the absence of MIX. Sufficient cAMP levels were achieved so that activation of cAMP-dependent protein kinases was only partially blocked. There was still sufficient cAMP to activate cAMP-dependent protein kinase to an extent equal to that seen with CRF without MIX. Similar effects of SRIF on cAMP accumulation and protein kinase activation were seen when cells were stimulated with forskolin. Our results demonstrate that SRIF inhibits ACTH release from AtT20 cells by inhibiting hormone-sensitive adenylate cyclase and thereby prevents the activation of cAMP-dependent protein kinases. However, under conditions where cAMP-dependent protein kinases are still sufficiently active to induce ACTH secretion, high concentrations of SRIF can inhibit ACTH release by a mechanism independent of cAMP-dependent protein kinase.

1-Methyl-3-isobutylxanthine↗
PubMed1986

P19, a hormonally regulated phosphoprotein of peptide hormone-producing cells: secretagogue-induced phosphorylation in AtT-20 mouse pituitary tumor cells and in rat and hamster insulinoma cells.

P19, a group of 19,000 mol wt cytosolic proteins, with apparent isoelectric points of pI 5.9, pI 5.7, and pI 5.4, respectively, was identified in three peptide hormone-producing cell types: AtT20 mouse pituitary tumor cells, RIN-1122 rat insulinoma cells, and hamster insulinoma cells. Secretagogue-dependent phosphorylation of P19 was analyzed in 32P-labeled cells by two-dimensional electrophoresis and autoradiography. The results were quantitated by computer-assisted densitometry. Cellular levels of cAMP and hormone release were measured in parallel incubations. In addition to stimulating ACTH release, CRF raised the cellular level of cAMP and increased the 32P labeling of all three 19,000 mol wt proteins in AtT20 cells. Other agents known to act through cAMP, which included isoproterenol, forskolin, and 8-bromo-cAMP, mimicked the effect of CRF on both ACTH release and phosphorylation of P19. 12-O-Tetra-decanoylphorbol-13-acetate, a tumor-promoting phorbol ester, also stimulated both ACTH release and phosphorylation of P19. In contrast, although 40 mM K+ promoted ACTH release, it did not affect the phosphorylation of P19. Analogous findings were observed in insulinoma cells. Glucagon stimulated insulin release, increased cellular cAMP and promoted phosphorylation of P19 in RIN 1122 cells. 12-O-Tetradecanoylphorbol-13-acetate also enhanced insulin release and the phosphorylation of P19 in these cells. The results obtained with hamster insulinoma cells closely resembled the observations in RIN-1122 cells. In conclusion, P19, an apparently homologous set of cytosolic proteins, undergoes phosphorylation in three peptide hormone-producing cells in response to two groups of secretagogues, the effect of which is probably mediated, in one case, by cAMP-dependent protein kinase and, in the other, by protein kinase C. The data suggest the possibility that P19 participates in a secretory pathway activated by these two effector systems.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Immunological and molecular characterization of the cAMP-dependent protein kinases in AtT20 cells.

The properties of the cAMP-dependent protein kinases in AtT20 mouse pituitary tumor cells were characterized by a combination of immunological and biochemical techniques. Ninety per cent of the total cAMP-dependent protein kinase was in the 40,000 X g supernatant fraction. Protein kinases I and II were immunoprecipitated with specific antisera directed against their regulatory subunits. The immunoprecipitated kinases bound [3H]cAMP and were catalytically active when incubated with [gamma-32P]ATP-Mg and protamine or histone H2B. Immunoprecipitated protein kinases I and II bound [3H]cAMP with apparent Kb values of 1.5 and 15 nM, respectively. Regulatory subunit concentrations in AtT20 cells were measured by immunoprecipitation of [3H]cAMP-R complexes. R-I and R-II levels were 2.7 and 3.0 pmol of [3H]cAMP binding activity per mg of cytosolic protein, respectively, however, the ratio of protein kinase II to protein kinase I was 2.5 indicating the presence of a significant amount of free R-I. This was confirmed by DEAE-cellulose chromatography and the isolation of immunoreactive R-I devoid of protein kinase activity. A significant amount of R-I also coeluted with protein kinase II when AtT20 cell extracts were subjected to DEAE-cellulose chromatography. In quantitative immunoprecipitation experiments, 0.1 microliter of anti-brain R-II serum complexed up to 0.5 pmol of the [3H]cAMP-binding activity of protein kinase II prepared from bovine and rat brain, and AtT20 cells while 2 microliter of anti-brain R-II serum was required to precipitate an equal amount of protein kinase II from bovine skeletal muscle showing that the protein kinase II in AtT20 cells contained the neural-specific R-II subunit.

Affinity Labels↗
PubMed1984

Hormonal activation of the cAMP-dependent protein kinases in AtT20 cells. Preferential activation of protein kinase I by corticotropin releasing factor, isoproterenol, and forskolin.

The hormonal regulation of adenylate cyclase, cAMP-dependent protein kinase activation, and adrenocorticotropic hormone (ACTH) secretion was studied in AtT20 mouse pituitary tumor cells. Corticotropin releasing factor (CRF) stimulated cAMP accumulation and ACTH release in these cells. Maximal ACTH release was seen with 30 nM CRF and was accompanied by a 2-fold rise in intracellular cAMP. When cells were incubated with both 30 nM CRF and 0.5 mM 3-methylisobutylxanthine (MIX) cAMP levels were increased 20-fold, however, ACTH release was not substantially increased beyond release seen with CRF alone. The activation profiles of cAMP-dependent protein kinases I and II were studied by measuring residual cAMP-dependent phosphotransferase activity associated with immunoprecipitated regulatory subunits of the kinases. Cells incubated with CRF in the absence of MIX showed concentration-dependent activation of protein kinase I which paralleled stimulation of ACTH release. Protein kinase II was minimally activated. When cells were exposed to CRF in the presence of 0.5 mM MIX there was still a preferential activation of protein kinase I, although 50% of the cytosolic protein kinase II was activated. Complete activation of both protein kinases I and II was seen when cells were incubated with 0.5 mM MIX and 10 microM forskolin. Under these conditions cAMP levels were elevated 80-fold. CRF, isoproterenol, and forskolin stimulated adenylate cyclase activity in isolated membranes prepared from AtT20 cells. CRF and isoproterenol stimulated cyclase activity up to 5-fold while forskolin stimulated cyclase activity up to 15-fold. Our data demonstrate that ACTH secretion from AtT20 cells is mediated by small changes in intracellular levels of cAMP and activation of only a small fraction of the total cytosolic cAMP-dependent protein kinase in these cells is required for maximal ACTH secretion.

Adenylyl Cyclases↗
PubMed1983

Empty sella of normal size in Sheehan's syndrome.

This report describes the findings of computed tomography of the sella turcica region in 13 women in whom postpartum hypopituitarism developed in the absence of a pituitary tumor. Seven patients had the typical history of severe postpartum hemorrhage (Sheehan's syndrome). Computed tomographic scanning in these patients revealed absence (in six patients) or marked reduction (in one patient) of the amount of demonstrable pituitary tissue. The pituitary fossa was occupied by material with the density of cerebrospinal fluid, resulting in the appearance of an "empty sella." Sella size was within the normal range in six of the seven patients and enlarged in one. The six remaining patients lacked a characteristic history of vascular collapse, although several did have postpartum complications. The radiologic findings in four of these patients were similar to those seen in the patients with typical Sheehan's syndrome: one patient had a partially empty sella and another had normal results of computed tomographic scanning. Thus, 11 of 13 women with postpartum hypopituitarism were noted to have an empty or partially empty sella of normal size. This report represents the first radiologic documentation of Sheehan's syndrome as a cause of the secondary empty sella, with the characteristic finding being an empty sella of normal size.

Adult↗
PubMed1983

Role of Ca2+ in secretagogue-stimulated breakdown of phosphatidylinositol in rat pancreatic islets.

Breakdown of phosphatidylinositol (PI) has been shown to be increased during Ca2+-mediated stimulation of cellular responses in many systems and has been proposed to be involved in stimulus-secretion coupling. The effects on PI breakdown of insulin secretagogues that alter cellular Ca2+ or cyclic (c)AMP levels were investigated in perifused rat islets of Langerhans. Isolated islets were labeled with myo-[2-3H(N)]inositol and the efflux of 3H-labeled metabolites was monitored. Glucose (16.7 mM) greatly increased 3H release in a manner that paralleled the second phase of the insulin secretory response; by 60 min, the amount of [3H]PI in the islet decreased by 50%. Removal of Ca2+ from the perifusate or blockade of Ca2+ entry through the voltage-dependent channels by D600 (20 microM) abolished the glucose-induced increase in 3H efflux. Depolarization with 47 mM K+, which increases Ca2+ entry, stimulated protracted 3H and insulin release. Glucose-stimulated output of 3H was not prevented by epinephrine (1 microM) even though the insulin response was abolished. In contrast, 3H output was not affected by isobutylmethylxanthine (1 mM), known to raise cellular levels of cAMP, although insulin release was stimulated. These findings indicate that PI breakdown is not related to the exocytotic process since stimulation of insulin release and PI breakdown could be uncoupled, and that it is not associated with cAMP-mediated regulation of insulin release. PI breakdown in islets differs from the immediate, transient phenomenon reported in other systems in both its timing and requirement for Ca2+. It appears to result from the entry of Ca2+ and not to be the mechanism by which glucose initiates Ca2+ influx.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Diabetes susceptibility of BALB/cBOM mice treated with streptozotocin. Inhibition by lethal irradiation and restoration by splenic lymphocytes.

In genetically susceptible strains of mice, repeated injections of a subdiabetogenic dose of streptozotocin induces the development of progressive insulin-dependent hyperglycemia. We showed previously that host T-cell functions play an obligatory etiologic role in this experimental disease by demonstrating that the athymic nude mouse is resistant to diabetes induction unless its T-cell functions are reconstituted by thymus graft (Paik et al., Proc. Natl. Acad. Sci. USA 77:6129-33, 1980). Here we show that lethal irradiation of euthymic (+/nu) mice of BALB/cBOM background causes selective resistance of the mice to the diabetogenic effects of the multiple low doses of streptozotocin without affecting their sensitivity to a high pharmacologic dose of the toxin. We also show that reconstitution of the irradiated mice with splenic lymphocytes causes the restoration of diabetes susceptibility. Lethally irradiated mice thus represent a useful experimental model for analyzing the host functions involved in the development of this disease. These results provide an additional support for the hypothesis that the induction of diabetes in this model system is mediated by an autoimmune amplification mechanism.

Animals↗
PubMed1982

Insulin release and protein phosphorylation: possible role of calmodulin.

Both Ca2+ and cyclic AMP (cAMP) are implicated in the regulation of insulin release in the pancreatic beta cell. In hamster insulinoma cells used in our laboratory to study the mechanism of insulin release, Ca2+ and cAMP trigger secretion independently. Concomitant with stimulation of the secretory apparatus both cAMP and Ca2+ promote phosphorylation of distinct insulinoma cell proteins. Calmodulin may be involved in the stimulation of insulin release and protein phosphorylation induced by Ca2+ influx. The Ca2+-dependent protein kinase of the insulinoma cell is activated by exogenous calmodulin and blocked by trifluoperazine, and inhibitor of calmodulin action. This drug also inhibits glucose-induced insulin release in pancreatic islets. In insulinoma cells trifluoperazine blocks Ca2+ influx-mediated insulin release and protein phosphorylation with no effect on basal or cAMP-mediated insulin release and protein phosphorylation with no effect on basal or cAMP-mediated secretion. Inhibition of Ca2+ influx-mediated insulin release and protein phosphorylation occurs with nearly identical dose dependence. Inasmuch as trifluoperazine affects voltage-dependent Ca2+ uptake in insulinoma cells, an involvement of calmodulin cannot be directly inferred. The evidence suggests that protein phosphorylation may be involved in the activation of the secretory apparatus by both cAMP and Ca2+. It is proposed that stimulation of insulin release by cAMP and Ca2+ is mediated by cAMP-dependent protein kinase and calmodulin-dependent protein kinase, respectively.

Animals↗
PubMed1980

Ca2+-dependent protein phosphorylation and insulin release in intact hamster insulinoma cells. Inhibition by trifluoperazine.

Ca2+-dependent protein phosphorylation was studied in intact hamster insulinoma cells. Depolarizing concentrations of potassium which stimulate Ca2+ uptake and insulin release by these cells also increased phosphorylation of one peptide, Mr = 60,000 (P60). This was demonstrated by incubating 32P-labeled insulinoma cells in media containing 50 mM K+ followed by analysis of the cellular proteins by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and autoradiography. Potassium-induced phosphorylation of P60 was nearly half-maximal after 1 min and reached a plateau by 10 min. The enhanced 32P-labeling of P60 observed in the presence of 50 mM K+ was Ca2+-dependent since omission of extracellular Ca2+ or addition of the Ca2+ channel blocker alpha-isopropyl-alpha-[(N-methyl-N-homoveratryl)-gamma-aminopropyl]3,4,5-trimethoxyphenylacetonitrile hydrochloride prevented the effect. Glucagon (3 microM), which stimulates insulin release in a cAMP-dependent manner, had no effect on P60 phosphorylation. A possible involvement of calmodulin was explored in studies using trifluoperazine. The Ca2+-dependent increase in phosphorylation of P60 was prevented by trifluoperazine. Moreover, Ca2+ influx-mediated insulin release and P60 phosphorylation were inhibited at nearly identical concentrations of trifluoperazine. Half-maximal inhibition of potassium-induced insulin release and P60 phosphorylation was seen at 2.6 microM and 2.5 microM trifluoperazine, respectively. The data are consistent with a sequence of events involving Ca2+ influx, phosphorylation of P60 by a calmodulin-dependent protein kinase, and resultant insulin secretion.

Adenoma, Islet Cell↗