cAMP-signaling pathway acts in selective synergism with glucose or tolbutamide to increase cytosolic Ca2+ in rat pancreatic beta-cells

The effect of repeated cocaine administration on serotonin2 (5-HT2) receptor function was examined in male rats. Rats were fitted with indwelling jugular catheters and subsequently received cocaine (15 mg/kg, i.p., b.i.d.) or saline for 7 days. Rats were challenged with the 5-HT2 agonist DOI (25, 100, 400 micrograms/kg, i.v.) or saline 42 hr and 8 days after cessation of chronic treatment. Serial blood samples were collected at various times after DOI challenge and analyzed for prolactin levels. DOI-induced head shakes and skin jerks were examined concurrently in the same subjects. After 42 hr of withdrawal, the stimulatory effects of DOI on prolactin release and shaking behavior were significantly enhanced in cocaine-treated rats. Conversely, the skin jerk response to DOI was not altered by prior cocaine exposure. After 8 days of withdrawal, the prolactin and head shake responses to DOI were still potentiated in cocaine-treated rats, but this effect was no longer statistically significant. The data indicate that chronic cocaine enhances the sensitivity of 5-HT2 receptor mechanisms. Our findings further suggest the possibility that altered 5-HT2 receptor function may be involved in the mood disturbances experienced by abstinent cocaine addicts.     

Glucose and tolbutamide induce apoptosis in pancreatic beta-cells. A process dependent on intracellular Ca2+ concentration

High concentrations of glucose are considered to be toxic for the pancreatic beta-cell. However, the mechanisms underlying beta-cell dysfunction and resulting cell death are not fully characterized. In the present study we have demonstrated that incubation of pancreatic islets and beta-cells from ob/ob mice and Wistar rats with glucose induced a process of apoptotic beta-cell death, as shown by DNA laddering, TdT-mediated dUTP-biotin nick end-labeling (TUNEL) technique, and by using DNA-staining dye HOECHST 33342. The obtained results show that the percentage of apoptotic cells was dependent on glucose concentration, being minimal at 11 mM glucose. At a concentration of 100 microM, aurintricarboxylic acid, an inhibitor of endonuclease activity, almost completely inhibited apoptosis triggered by 17 mM glucose. We have also shown that long term incubation with 100 microM sulfonylurea, tolbutamide, triggered apoptosis in pancreatic beta-cells. The process of beta-cell death induced by high glucose concentration and tolbutamide were Ca2+-dependent, because introduction to the culture medium of 50 microM D-600 or 200 microM diazoxide, which blocked glucose- and tolbutamide-induced [Ca2+]i increase, inhibited apoptosis. Thus, this study shows for the first time that high glucose concentrations and tolbutamide induce apoptosis in pancreatic beta-cells, and that this process is Ca2+-dependent.     

Differences in glucose transporter gene expression between rat pancreatic alpha- and beta-cells are correlated to differences in glucose transport but not in glucose utilization

Glucose exerts inverse effects upon the secretory function of islet alpha- and beta-cells, suppressing glucagon release and increasing insulin release. This diverse action may result from differences in glucose transport and metabolism between the two cell types. The present study compares glucose transport in rat alpha- and beta-cells. beta-Cells transcribed GLUT2 and, to a lesser extent, GLUT 1; alpha-cells contained GLUT1 but no GLUT2 mRNA. No other GLUT-like sequences were found among cDNAs from alpha- or beta-cells. Both cell types expressed 43-kDa GLUT1 protein which was enhanced by culture. The 62-kDa beta-cell GLUT2 protein was converted to a 58-kDa protein after trypsin treatment of the cells without detectable consequences upon glucose transport kinetics. In beta-cells, the rates of glucose transport were 10-fold higher than in alpha-cells. In both cell types, glucose uptake exceeded the rates of glucose utilization by a factor of 10 or more. Glycolytic flux, measured as D-[5(3)H]glucose utilization, was comparable in alpha- and beta-cells between 1 and 10 mmol/liter substrate. In conclusion, differences in glucose transporter gene expression between alpha- and beta-cells can be correlated with differences in glucose transport kinetics but not with different glucose utilization rates.     

Triphenyltin chloride induces glucose intolerance by the suppression of insulin release from hamster pancreatic beta-cells

An increased activity of phospholipase A2 has been observed in the plasma of patients with uremia. This enzyme converts phosphatidylcholine to lysophosphatidylcholine (LPC), an inhibitor of platelet aggregation. We measured the levels of plasma phospholipids including LPC, and platelet aggregation in 7 patients with uremia. Platelet response to agonists was defective, mainly with collagen (p < 0.001). The patients' levels of LPC in plasma were similar to those of controls (109.7 +/- 41.6 vs. 80.4 +/- 16.8 nmol/ml) and did not correlate with the platelet response to adenosine diphosphate (r = -0.51). The amount of phosphatidylcholine was increased with respect to normal plasma (1,041.0 +/- 201.8 vs. 760.8 +/- 142.7 nmol/ml, p < 0.01), while the levels of other phospholipids were normal. These results do not suggest a participation of plasma LPC in the genesis of the platelet defect observed in patients with uremia.     

Protein kinase Ciota activity is necessary for Bcr-Abl-mediated resistance to drug-induced apoptosis

K562 chronic myelogenous leukemia cells are highly resistant to chemotherapeutic drugs, such as taxol, that induce cell death by apoptosis. This resistance is mediated by the chimeric tyrosine kinase oncogene Bcr-Abl. However, little is known about the mechanism by which Bcr-Abl protects K562 cells from apoptosis. We recently demonstrated that expression of PKCiota is necessary for the resistance of K562 cells to taxol-induced apoptosis (Murray, N. R., and Fields, A. P. (1997) J. Biol. Chem. 272, 27521-27524). We now demonstrate that treatment of K562 cells with taxol leads to sustained activation of PKCiota. In contrast, Bcr-Abl-negative HL60 myeloid leukemia cells, which are sensitive to taxol-induced apoptosis, do not exhibit sustained PKCiota activation in response to taxol. Treatment of K562 cells with tyrphostin AG957, a selective Bcr-Abl inhibitor, blocks taxol-induced PKCiota activation and sensitizes these cells to taxol-induced apoptosis, indicating that PKCiota is a relevant downstream target of Bcr-Abl-mediated resistance. Furthermore, expression of constitutively active PKCiota by adenovirus-mediated gene transfer rescues AG957-treated K562 cells from taxol-induced apoptosis. Taken together, these results demonstrate that both Bcr-Abl and PKCiota activity are necessary for apoptotic resistance in K562 cells. Furthermore, they identify PKCiota as a critical downstream target of Bcr-Abl that is sufficient to mediate the anti-apoptotic effects of Bcr-Abl.     

Effects of extracellular calcium on electrical bursting and intracellular and luminal calcium oscillations in insulin secreting pancreatic beta-cells

The extracellular calcium concentration has interesting effects on bursting of pancreatic beta-cells. The mechanism underlying the extracellular Ca2+ effect is not well understood. By incorporating a low-threshold transient inward current to the store-operated bursting model of Chay, this paper elucidates the role of the extracellular Ca2+ concentration in influencing electrical activity, intracellular Ca2+ concentration, and the luminal Ca2+ concentration in the intracellular Ca2+ store. The possibility that this inward current is a carbachol-sensitive and TTX-insensitive Na+ current discovered by others is discussed. In addition, this paper explains how these three variables respond when various pharmacological agents are applied to the store-operated model.     

Mouse pancreatic beta-cells exhibit preserved glucose competence after disruption of the glucagon-like peptide-1 receptor gene

Previous work suggested that glucagon-like peptide 1 (GLP-1) can acutely regulate insulin secretion in two ways, 1) by acting as an incretin, causing amplification of glucose-induced insulin release when glucose is given orally as opposed to intravenous glucose injection; and 2) by keeping the beta-cell population in a glucose-competent state. The observation that mice with homozygous disruption of the GLP-1 receptor gene are diabetic with a diminished incretin response to glucose underlines the first function in vivo. Isolated islets of Langerhans from GLP-1 receptor -/- mice were studied to assess the second function in vitro. Absence of pancreatic GLP-1 receptor function was observed in GLP-1 receptor -/- mice, as exemplified by loss of [125I]GLP-1 binding to pancreatic islets in situ and by the lack of GLP-1 potentiation of glucose-induced insulin secretion from perifused islets. Acute glucose competence of the beta-cells, assessed by perifusing islets with stepwise increases of the medium glucose concentration, was well preserved in GLP-1 receptor -/- islets in terms of insulin secretion. Furthermore, neither islet nor total pancreatic insulin content was significantly changed in the GLP-1 receptor -/- mice when compared with age-and sex-matched controls. In conclusion, mouse islets exhibit preserved insulin storage capacity and glucose-dependent insulin secretion despite the loss of functional GLP-1 receptors. The results demonstrate that the glucose responsiveness of islet beta-cells is well preserved in the absence of GLP-1 receptor signaling.     

Rat and human pancreatic islet cells contain a calcium ion independent phospholipase A2 activity selective for hydrolysis of arachidonate which is stimulated by adenosine triphosphate and is specifically localized to islet beta-cells

The recent demonstration that myocardial Ca(2+)-independent phospholipase A2 exists as a complex of catalytic and regulatory polypeptides that is modulated by ATP has suggested a novel mechanisms through which alterations in glycolytic flux can be coupled to the generation of eicosanoids which facilitate insulin secretion. To determine the potential relevance of this mechanism, we examined the kinetic characteristics, substrate specificities, and cellular locus of phospholipase A2 activity in pancreatic islets. Rat pancreatic islets contain a Ca(2+)-independent phospholipase A2 activity which is optimal at physiologic pH, preferentially hydrolyzes phospholipid substrates containing a vinyl ether linkage at the sn-1 position, and prefers arachidonic acid compared to oleic acid in the sn-2 position. Rat islet Ca(2+)-independent phospholipase A2 activity is inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one and is stimulated by ATP. Purification of beta-cells from dispersed pancreatic islet cells by fluorescence-activated cell sorting demonstrated that beta-cells (but not non-beta-cells) contain Ca(2+)-independent, ATP-stimulated phospholipase A2 activity. Remarkably, clonal RIN-m5f insulinoma cells, which possess a defect in glucose-induced insulin secretion, contain a Ca(2+)-independent phospholipase A2 which is not modulated by alterations in ATP concentration. Collectively, these results and those of an accompanying paper [Ramanadham et al. (1993) Biochemistry (following paper in this issue)] implicate Ca(2+)-independent phospholipase A2 as a putative glucose sensor which can couple alterations in glycolytic metabolism to the generation of biologically active eicosanoids and thereby facilitate glucose-induced insulin secretion.     

Activation of stimulus-secretion coupling in pancreatic beta-cells by specific products of glucose metabolism. Evidence for privileged signaling by glycolysis

The energy requirements of most cells supplied with glucose are fulfilled by glycolytic and oxidative metabolism, yielding ATP. In pancreatic beta-cells, a rise in cytosolic ATP is also a critical signaling event, coupling closure of ATP-sensitive K+ channels (KATP) to insulin secretion via depolarization-driven increases in intracellular Ca2+ ([Ca2+]i). We report that glycolytic but not Krebs cycle metabolism of glucose is critically involved in this signaling process. While inhibitors of glycolysis suppressed glucose-stimulated insulin secretion, blockers of pyruvate transport or Krebs cycle enzymes were without effect. While pyruvate was metabolized in islets to the same extent as glucose, it produced no stimulation of insulin secretion and did not block KATP. A membrane-permeant analog, methyl pyruvate, however, produced a block of KATP, a sustained rise in [Ca2+]i, and an increase in insulin secretion 6-fold the magnitude of that induced by glucose. These results indicate that ATP derived from mitochondrial pyruvate metabolism does not substantially contribute to the regulation of KATP responses to a glucose challenge, supporting the notion of subcompartmentation of ATP within the beta-cell. Supranormal stimulation of the Krebs cycle by methyl pyruvate can, however, overwhelm intracellular partitioning of ATP and thereby drive insulin secretion.     

Electron microscopic study of intercalated duct cells in the chicken pancreatic islet and effects of tolbutamide administration

Intercalated duct cells are present in the alpha and beta islets of chicken. The intercalated duct cells adhere to each other via intercellular junctional complexes at the apical side, projecting many microvilli and a few cilia into the lumen. They also extend slender cytoplasmic processes between the islet endocrine cells. These intercalated cells appear to have a stellate form, and to wrap their cytoplasm around endocrine cells. Administration of tolbutamide led to increased electron density in the cytoplasm of intercalated duct cells. Lysosomes are present in these cells in various numbers and sizes and tend to increase with time after administration of tolbutamide. These observations suggested that the intercalated ducts not only pass through the islet, but also play a role in supplying islet cells.     

Effects of sulphonylureas on the volume-sensitive anion channel in rat pancreatic beta-cells

OBJECTIVE: This study's aim was to determine whether maintenance therapy with terbutaline administered by pump prolongs gestation in women after treatment with intravenous magnesium sulfate tocolysis for suspected preterm labor. STUDY DESIGN: Consenting women with a singleton gestation and intact membranes who had uterine contractions and >1 cm cervical dilation, 80% effacement, or progressive cervical change and whose contractions were successfully arrested with intravenous magnesium were randomly assigned to receive either terbutaline or normal saline solution placebo by subcutaneous infusion pump. Pump therapy was administered with a standardized protocol. Pump therapy was discontinued and parenteral magnesium was resumed if recurrent preterm labor developed while women were on the therapeutic regimen at <34 weeks' gestation and no contraindication for tocolysis existed. If recurrent labor was arrested, pump therapy was restarted according to the original treatment group. A sample size of 48 women was required to detect a 2-week intergroup difference in mean time to delivery. Analyses were based on intent to treat. RESULTS: Fifty-two women received terbutaline (n = 24) or placebo (n = 28). At random assignment the groups were similar with respect to age, race, parity, previous preterm delivery, gestational age, and cervical examination. Overall there was a 1-day difference in mean time to delivery between the groups (terbutaline 29 +/- 22 days and placebo 28 +/- 23 days, P = .78). There were no differences in the rates of preterm delivery at <34 and <37 weeks' gestation. Neonatal outcomes were similar. CONCLUSIONS: Maintenance terbutaline therapy administered by pump does not prolong gestation in women successfully treated for suspected preterm labor.     

The effects of tolbutamide on the myocardium in experimental diabetes

The effects of chronic tolbutamide treatment were examined in a diabetic animal model in which abnormal myocardial function and composition have previously been demonstrated. Eight diabetic dogs were given tolbutamide 250 mg/day orally and compared with seven untreated diabetics, five healthy dogs receiving tolbutamide, and eight normal controls. After one year, resting hemodynamic studies in the intact anesthetized state showed that treated diabetic dogs had a significantly higher left ventricular end-diastolic pressure of 12.1+/-1.3 mm Hg associated with normal end-diastolic volume, compared to 6.1+/-0.8 mm Hg in untreated diabetics (P less than 0.01) and 6.3+/-0.5 in normals. Stroke work and ejection fraction were similar to normals. Acute volume expansion revealed a larger rise of left ventricular end-diastolic pressure in treated and untreated diabetics than normals, without a significant stroke volume response in treated diabetics. Enhanced stiffness of myocardium appeared to be related to interstitial accumulation of periodic acid-Schiff staining material, further intensified in treated diabetics by triglyceride accumulation observed on electron microscopy and by chemical analysis. Thus treatment of diabetes with tolbutamide, despite improved glucose tolerance, effected further reduction of left ventricular function and altered morphology of myocardium.     

Selective activation of JNK1 is necessary for the anti-apoptotic activity of hILP

The balance between the inductive signals and endogenous anti-apoptotic mechanisms determines whether or not programmed cell death occurs. The widely expressed inhibitor of apoptosis gene family includes three closely related mammalian proteins: c-IAP1, c-IAP2, and hILP. The anti-apoptotic properties of these proteins have been linked to caspase inhibition. Here we show that one member of this group, hILP, inhibits interleukin-1beta-converting enzyme-induced apoptosis via a mechanism dependent on the selective activation of c-Jun N-terminal kinase 1. These data demonstrate that apoptosis can be inhibited by an endogenous cellular protein by a mechanism that requires the activation of a single member of the mitogen-activating protein kinase family.     

Phosphorylation of triciribine is necessary for activity against HIV type 1

Triciribine (TCN) is a tricyclic nucleoside with known antineoplastic and antiviral activity. It is a potent and selective inhibitor of HIV-1 and HIV-2, including strains known to be resistant to AZT or TIBO. TCN is phosphorylated to its 5'-monophosphate (TCN-P) by intracellular adenosine kinase (AK), but is not converted to di- or triphosphates. We now report that 5'-phosphorylation is requisite for the activity of TCN against HIV-1. CEM cells incubated with TCN at concentrations ranging from 0.1 to 330 microM gave intracellular TCN-P concentrations from 27 to 775 microM, respectively. There was no difference in the amount of intracellular TCN-P detected in uninfected compared with HIV-1-infected CEM cells. The antiviral effect of TCN against HIV-1 was strongly antagonized by the AK inhibitor 5-iodotubercidin (ITu). In contrast, TCN and ITu only exhibited additive cytotoxicity. The 5'-deoxy analog of TCN, which cannot be phosphorylated, had no antiviral effect against HIV-1 at a concentration more than 100 times higher than the IC50 of TCN. Similarly, TCN was not active against HIV-1 in an AK-deficient cell line (AA-2) at concentrations shown to inhibit the virus by >95% in CEM cells. Consistent with its AK-deficient phenotype, this cell line phosphorylated TCN to only 3% of the extent observed in CEM cells. We conclude that TCN must be phosphorylated to TCN-P for activity against HIV-1.     

A role for Ca2+-sensitive nonselective cation channels in regulating the membrane potential of pancreatic beta-cells

The incretin hormones, glucagon-like peptide 1 and pituitary adenylyl cyclase-activating polypeptide, are proposed to activate a maitotoxin (MTX)-sensitive, Ca2+-dependent nonselective cation current in pancreatic beta-cells and insulinoma cells. This MTX-sensitive current is present in human beta-cells as well as in mouse and rat beta-cells, and is accompanied by a rise in cytosolic Ca2+ in voltage-clamped cells in which the activation of voltage-dependent Ca2+ channels is prevented. Activation of the nonselective cation current is inhibited by reduction of disulfide bonds with intracellular, but not extracellular, dithiothreitol, and is also abolished by intracellular dialysis with trypsin. The nonselective cation channels that carry this current have a conductance of about 30 pS, with Na+ as the major extracellular cation. We estimate that these cation channels are expressed on beta-cells at a density similar to that of ATP-sensitive potassium channels (K(ATP) channels) and exhibit spontaneous activity at basal glucose concentrations. We propose that this spontaneous cation channel activity constitutes at least part of the depolarizing background conductance that permits changes in the activity of K(ATP) channels to regulate the resting potential of beta-cells.     

Repair of O6-methylguanine in rat pancreatic beta-cells after exposure to N-methyl-N-nitrosourea

Because of the possible involvement of O6-methyldeoxyguanosine as a cytotoxic and carcinogenic lesion in pancreatic beta-cells, studies were undertaken to assess the ability of rat beta-cells to repair this DNA lesion. Primary cultures of neonatal rat beta-cells were shown to contain very low levels of O6-methylguanine-DNA-methyltransferase activity, the predominant mechanism for repairing O6-methyldeoxyguanosine in mammalian cells. However, using a 32P-endlabeling assay to measure O6-methyldeoxyguanosine in cells after exposure to N-methyl-N-nitrosourea, it was determined that rat beta-cells repaired O6-methyldeoxyguanosine to a substantial extent over a 24-h period. To elucidate the mechanism of O6-methyldeoxyguanosine repair in the virtual absence of constitutive O6-methylguanine-DNA-methyltransferase expression, studies were performed to determine if O6-methylguanine-DNA-methyltransferase expression was enhanced in N-methyl-N-nitrosourea-treated beta-cells. No increase in O6-methylguanine-DNA-methyltransferase activity was detected 24 or 48 h after exposure. However, Northern blot analysis showed a two- to threefold elevation in O6-methylguanine-DNA-methyltransferase messenger RNA levels in beta-cells 12 and 24 h after N-methyl-N-nitrosourea treatment. This finding is the first demonstration of a change in O6-methylguanine-DNA-methyltransferase messenger RNA levels in a cell type with low constitutive activity.     

Type IB secretory phospholipase A2 is contained in insulin secretory granules of pancreatic islet beta-cells and is co-secreted with insulin from glucose-stimulated islets

People, now in their fifties and sixties, who were children during the Nazi Holocaust in WWII, endured persecution, massive traumatization, the constant risk of being killed, as well as the violent loss of (most of) their family members. They have internalized the resulting ongoing confusion and conflicts as to whether they should be alive or dead. This is maintained as an integral part of the child component of their compound personality, described in this paper. During the three years of the psychotherapy group, on which we focused here, these issues were expressed in different ways, such as suicide threats, occasional intolerance to physically remaining in the group, and outbursts of annihilating rage at the therapists. The confusion and conflicts about the legitimacy and risks of their survival came to a head during the termination process we insisted upon. Much attention has also been given to the intricacies of our countertransference--further complicated by our own connection to the Holocaust. We learned, and described, just how essential it is to acknowledge and process this countertransference in order to both contain the intense affects of anxiety, rage, and mourning in the groups, and enable them to be safely expressed. We imagine similar dynamics can be expected in group therapy with other populations who suffered massive, man-made traumatization.     

The influence of transportation stress on selected nutritional parameters to establish the necessary minimum period for adaptation in rat feeding studies

The optimal length of the adaptation period after transportation of rats, to be used in nutritional studies, was investigated in this study. After intracontinental transportation of rats by car and by air to and from the laboratory for a total period of 15 h, measurements were carried out for a period of 3 weeks after transport. Control and transported animals were housed in the same laboratory before and after transportation. During transport the animals had access to food and water. As blood collection could also cause stress, a factorial design was carried out with transport and blood collection as main factors. Transport or blood collection did not cause significant effects on the following parameters: body weight, growth, clinical observation, and blood enzyme activities of LDH and ASAT. Water intake was significantly increased after transport. Food intake did not show consistent effects after transport or blood collection. Unexpectedly, blood corticosterone levels were significantly lower in the transported animals at day 1 after transport. After 3 days these levels were back to normal. Blood glucose, blood free fatty acids and blood urea nitrogen concentrations were incidentally decreased, whereas total cholesterol levels showed an incidental rise in the transported rats. The open-field behaviour test revealed no clear-cut results concerning the effects of transport or blood collection on faeces production, rearing and ambulation. Our results indicate that after intracontinental transport, an adaptation period of 3 days appears to be sufficient for rats to be used in nutritional studies.