Triphenylmethylphosphonium uptake by pancreatic islet cells

Uptake of triphenylmethylphosphonium cation (TPMP⁺) was studied in pancreatic islet cells. Islets rich in β-cells were prepared from non-inbred ob/ob-mice and incubated with [³H]TPMP⁺ and l-[1-¹⁴C]glucose. Conjoined with the Nernst equation, the values for TPMP⁺ uptake in excess of the extracellular (l-glucose) space predicted membrane electric potentials far from those previously recorded with intracellular electrodes. Improved agreement with the electrode data was achieved by correcting for assumed voltage-independent binding of TPMP⁺; plausible correction terms were derived from the kinetics of TPMP⁺ efflux and from the uptake of [³H]TPMP⁺ in islets treated with non-radioactive TPMP⁺ at such a high concentration (50 μM) as to abolish the glucose oxidation. In whole islets the magnitude of the TPMP⁺-derived potentials decreased with increasing extracellular K⁺ in the range 5.9–130 mM, and was diminished by 20 mM d-glucose or 0.5 mM 2,4-dinitrophenol, but not by 20 mM 3-O-methyl-d-glucose, 20 mM d-mannoheptulose alone, or 10 μM chlorotetracycline. The effect of d-glucose was not observed in the presence of d-mannoheptulose and was diminished when 130 mM NaCl in the medium was replaced by sodium isethionate. The magnitude of TPMP⁺ uptake and the effects of K⁺ and dinitrophenol were reproduced with dispersed islet cells from ob/ob-mice and with whole islets of normal inbred NMRI-mice; the d-glucose effect was reproduced with NMRI-mouse islets. The results support our previous hypotheses that the depolarizing and insulin-releasing actions of d-glucose are in part mediated by electrodiffusion mechanisms involving K⁺ and Cl^?.     

Alloxan cytotoxicity in vitro. Inhibition of rubidium ion pumping in pancreatic beta-cells.

Exposing micro-dissected pancreatic islets of non-inbred ob/ob mice to 2-5 mM-alloxan for 10 min decreased the ability of the islets to accumulate Rb+. Rb+ accumulation in pieces of exocrine pancreas was unaffected by alloxan. When islets were treated with alloxan in the presence of 2-20 mM-D-glucose, the Rb+-accumulating ability was protected in a dose-dependent manner. The protective action of D-glucose was reproduced with 3-O-methyl-D-glucose but not with L-glucose or D-mannoheptulose; mannoheptulose prevented D-glucose from exerting its protective action. The inhibition of Rb+ accumulation was due to a decreased inward pumping, since alloxan did not affect Rb+ efflux from pre-loaded islets. The inhibitory effect of alloxan had a latency of about 1 min, as revealed by experiments with dispersed islet cells in suspension. Alloxan-treated islets showed only a marginal decrease in ATP and no change in glucose 6-phosphate concentration. Although alloxan slightly decreased the hydrolysis of ATP in a subcellular fraction enriched in plasma membranes, this effect could not be attributed to a ouabain-sensitive adenosine triphosphatase. The plasma membranes exhibited a K+-activated hydrolysis of p-nitrophenyl phosphate; this enzyme activity too was insensitive to alloxan. Glucose may protect the univalent-cation pump by preventing permeation of alloxan via a path coupled to the hexose-transport system. Inhibition of the pump may be fundamental to the induction of alloxan-diabetes.     

Spontaneous cytotoxicity of macrophages against pancreatic islet cells

Activated peritoneal macrophages were found to lyse syngeneic [3H]leucine-labeled pancreatic islet cells or rat insulinoma cells after 15 h of coculture at 37 degrees C. Lysis was verified by electron microscopic analysis. Islet cell lysis was dependent on the T:E ratio and was comparable with P815 and L929 tumor cells used as targets. The cytotoxic activity was localized in the adherent fraction of Corynebacterium parvum activated peritoneal cells and was destroyed by incubation of cells with macrophage-toxic silica particles. Syngeneic thyrocytes and hepatocytes were found to be resistant to the cytolytic action of activated macrophages. It has been shown previously that macrophages contribute to pancreatic islet inflammation. The present in vitro analysis demonstrates that macrophages can function as effector cells in islet destruction.     

Artefactual and true uptake of labelled sucrose by rat pancreatic islet cells

Labelled sucrose is currently used as an extracellular marker in pancreatic islets or isolated islet cells. Contamination of [6,6'(n)-3H]sucrose by tritiated hexose results in severe overestimation of the extracellular space. The relative magnitude of this overestimation depends on such factors as the temperature and length of incubation and the presence or absence of metabolized unlabelled hexose. Even with uncontamined [U-14C]sucrose, a restricted amount of the disaccharide is apparently taken up, hydrolyzed and further metabolized in the islet cells. The uptake of sucrose by islet cells may reflect the phylogenic maintenance of a physiological attribute of these primitively intestinal epithelial cells.     

Magnesium uptake by pancreatic islet cells is modulated by stimulators and inhibitors of the B-cell function

28Mg2+ uptake by rat islets was measured during incubation with various stimulators or inhibitors of insulin release. D-Glucose induced a dose-dependent increase in 28Mg2+ uptake after 10 min or 120 min. The threshold concentration was around 6 mM and the maximum effect was observed with 15-20 mM glucose. After 120 min 28Mg2+ uptake was also stimulated by the metabolized sugars mannose, N-acetylglucosamine or glyceraldehyde, was unaffected by the non-metabolized or poorly metabolized L-glucose, galactose, 3-O-methylglucose, 2-deoxyglucose, fructose or mannoheptulose and was inhibited by glucosamine. The effect of glucose was markedly impaired by mannoheptulose, glucosamine, aminooxyacetate and NH4Cl, but was only partially decreased by D600 or diazoxide, which were ineffective in a glucose-free medium. Tolbutamide or KCl slightly increased 28Mg2+ uptake. Alanine, leucine alone or with glutamine, and ketoisocaproate also stimulated 28Mg2+ uptake, whereas arginine and lysine decreased it. These changes in 28Mg2+ uptake, brought about by various modifiers of the B-cell function, are thus similar but not identical to the changes in Ca2+ uptake, and are not the consequence of insulin release. The stimulatory effect of glucose requires glucose metabolism by islet cells, but is only partially due to depolarization of the B-cell membrane.     

Protein phosphorylation in permeabilized pancreatic islet cells.

A system of digitonin-permeabilized islet cells was developed to characterize Ca2+- and calmodulin-dependent protein phosphorylation further and to determine whether activation of this membrane-bound process was sufficient for initiation of Ca2+-stimulated insulin secretion. The efficacy of digitonin in permeabilizing the plasma membrane was assessed by Trypan Blue exclusion, by extracellular leakage of lactate dehydrogenase, and by permeability to [gamma-32P]ATP. This treatment did not detectably alter the ultrastructure of the permeabilized cells. Digitonin was equally effective when presented to islet cells that had been previously dispersed or directly to intact isolated islets. The Ca2+- and calmodulin-dependent phosphorylation of endogenous membrane-bound substrates could be demonstrated in the permeabilized cells incubated with [gamma-32P]ATP. This activity displayed characteristics that were similar to those described for the protein kinase measured in subcellular fractions and was dependent on addition of exogenous calmodulin, indicating that calmodulin had been removed from the kinase by permeabilization of the cells. Ca2+-dependent insulin release by the digitonin-permeabilized islet was demonstrated, with half-maximal release occurring at 0.1 microM-free Ca2+ and maximal secretion at 0.2 microM-free Ca2+. Under these conditions, calmodulin did not further enhance insulin release, although a stimulatory effect of calmodulin was observed in the absence of free Ca2+. These studies indicate that the permeabilized-islet model will be useful in dissecting out the factors involved in Ca2+-activated insulin secretion.     

Nicotinamide modulation of rat pancreatic islet cell responsiveness in vitro.

The influence of nicotinamide (NA), a highly suitable precursor substrate for NAD synthesis in various tissues, on islet cell responsiveness was determined. After a 30 minute perifusion with this compound, nicotinamide, in a dose-dependent manner, potentiated glucose-induced insulin secretion. Maximal potentiation (approximately 250%) was observed at 20 mM NA and the threshold for potentiation was 3 mM. In the absence of glucose, NA did not affect basal secretion rates. Mannoheptulose blocked the primary stimulant action of glucose and the potentiating effects of NA. NA did not alter the rate of glucose usage by isolated islets. These results further underscore the possible importance of pyridine nucleotides in stimulated secretion.     

Redifferentiation of insulin-secreting cells after in vitro expansion of adult human pancreatic islet tissue

Cellular replacement therapy holds promise for the treatment of diabetes mellitus but donor tissue is severely limited. Therefore, we investigated whether insulin-secreting cells could be differentiated in vitro from a monolayer of cells expanded from human donor pancreatic islets. We describe a three-step culture protocol that allows for the efficient generation of insulin-producing cell clusters from in vitro expanded, hormone-negative cells. These clusters express insulin at levels of up to 34% that of average freshly isolated human islets and secrete C-peptide upon membrane depolarization. They also contain cells expressing the other major islet hormones (glucagon, somatostatin, and pancreatic polypeptide). The source of the newly differentiated endocrine cells could either be indigenous stem/progenitor cells or the proliferation-associated dedifferentiation and subsequent redifferentiation of mature endocrine cells. The in vitro generated cell clusters may be efficacious in providing islet-like tissue for transplantation into diabetic recipients.     

The mechanism of sugar uptake by sugarcane suspension cells

Sugarcane cell suspensions took up sugar from the medium at rates comparable to or greater than sugarcane tissue slices or plants in the field. This system offers an opportunity for the study of kinetic and energetic mechanisms of sugar transport in storage parenchyma-like cells in the absence of heterogeneity introduced by tissues. The following results were obtained: (a) The sugar uptake system was specific for hexoses; as previously proposed, sucrose was hydrolyzed by an extracellular invertase before the sugar moieties were taken up; no evidence for multiple sugar uptake systems was obtained. — (b) Uptake of the glucose-analog 3-O-methylglucose (3-OMG) reached a plateau value with an intracellular concentration higher than in the medium (approximately 15-fold). — (c) There was a balance of influx and efflux during steady state; the rate of exchange influx was lower than the rate of net influx; the K_m value was higher (70 M) than for net influx (24 M); the exchange efflux is proposed to be mediated by the same transport system with a K_m value of approximately 2.6 mM for internal 3-OMG; the rate of net efflux of hexoses was less than a third of the rate of exchange efflux. — (d) The uptake of hexoses proceeded as proton-symport with a stoichiometry of 0.87 H⁺ per sugar; during the onset of hexose transport there was a K⁺ exit of 0.94 K⁺ per sugar for charge compensation. (It was assumed that the real stoichiometries are 1 H⁺ and 1 K⁺ per sugar.) The K_m values for sugar transport and sugar-induced proton uptake were identical. Sucrose induced proton uptake only in the presence of cell wall invertase. — (e) There was no net proton uptake with 3-OMG by cells which were preloaded with glucose though there was significant sugar uptake. It is assumed, therefore, that the exit of hexose occurs together with protons. — (f) The protonmotive potential of sugarcane cells corresponded to about 120 mV: pH-gradient 1.1 units, membrane potential of-60 mV (these values increased if vacuolar pH and membrane potential were also considered). It was abolished by uncouplers, and the magnitude of the components depended on the external pH value. We present evidence for the operation of a proton-coupled sugar transport system in cell suspensions that were derived from, and have characteristics of, storage parenchyma. The quantitative rates of sugar transport suggest that the role of this transport system is not limiting for sugar storage.Abbreviations 3-OMG 3-O methylglucose - DMO 5,5-dimethyl-2, 4-oxazolidinedione - TPP tetraphenylphosphonium chloride - CCCP carbonyl cyanide, m-chlorophenylhydrozane     

Mechanism of amino Acid uptake by sugarcane suspension cells

The amino acid carriers in sugarcane suspension cells were characterized for amino acid specificity and the stoichiometry of proton and potassium flux during amino acid transport.

Amino acid transport by sugarcane cells is dependent upon three distinct transport systems. One system is specific for neutral amino acids and transports all neutral amino acids including glutamine, asparagine, and histidine. The uptake of neutral amino acids is coupled to the uptake of one proton per amino acid; one potassium ion leaves the cells for charge compensation. Histidine is only taken up in the neutral form so that deprotonation of the charged imidazole nitrogen has to occur prior to uptake. The basic amino acids are transported by another system as uniport with charge-compensating efflux of protons and potassium. The acidic amino acids are transported by a third system. Acidic amino acids bind to the transport site only if the distal carboxyl group is in the dissociated form (i.e. if the acidic amino acid is anionic). Two protons are withdrawn from the medium and one potassium leaves the cell for charge compensation during the uptake of acid amino acids. Common to all three uptake systems is a monovalent positively charged amino acidproton carrier complex at the transport site.

     

Increased cytokine-induced cytotoxicity of pancreatic islet cells from transgenic mice expressing the Src-like tyrosine kinase GTK

BACKGROUND: The loss of beta cells in type 1 diabetes may involve protein kinases because they control cell growth, differentiation, and survival. Previous studies have revealed that GTK, a Src-like protein tyrosine kinase expressed in beta cells (also named Bsk/Iyk), regulates multiple responses including growth and survival of rat insulinoma cells (RINm5F) and differentiation of neuronal PC12 cells. In the present study, we have generated a transgenic mouse expressing a kinase active GTK mutant (GTK-Y504F) under the control of the rat insulin I promoter to establish a role of GTK in beta cells. MATERIALS AND METHODS: Control and GTK-transgenic CBA mice were used for determination of in vivo glucose tolerance and the relative insulin-positive area. Isolated islets from both groups were cultured in the absence and presence of cytokines and insulin secretion, viability and protein expression were assessed. RESULTS: The beta-cell mass of GTK-transgenic mice was increased as a consequence of a larger pancreas and an increased relative beta-cell area. Islets isolated from the transgenic animals exhibited an enhanced glucose-induced insulin release and reduced viability in response to cytokines that could not be explained by higher levels of nitric oxide (NO) compared with control islets. Extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK), and Akt were all activated by cytokines, but GTK-transgenic islets contained higher basal levels of phosphorylated ERK1/2 and lower basal levels of phosphorylated p38 compared with the control islets. The total amount of activated MAPKs was, however, higher in the cytokine-stimulated transgenic islets compared with the control islets due to increased levels of phospho-ERK1/2. Moreover, the proline-rich tyrosine kinase (PYK) 2 (also named RAFTK/CAK beta/CADTK) levels were elevated in response to a 24-hr exposure to cytokines in control islets but not in the GTK-transgenic islets. CONCLUSIONS: These results suggest that although GTK increases the beta-cell mass, it also enhances islet cell death in response to cytokines and may thus be involved in the beta-cell damage in type 1 diabetes.     

Effect of intracellular alkalinization on pancreatic islet calcium uptake and insulin secretion.

Microdissected beta-cell-rich pancreatic islets of ob/ob mice were used in studies of the relationship between intracellular pH (pHi) and 45Ca2+ uptake and insulin release. Stepwise increases in extracellular pH (pHo) from 6.80 to 8.00 resulted in a parallel, although less pronounced, elevation of pHi from 7.24 to 7.69. Experimental conditions that alkalinize the islet cell interior, i.e. addition of 5 mM-NH4+, sudden withdrawal of extracellular bicarbonate buffer or increase in pHo, induced insulin secretion in the absence of other types of secretory stimulation (1 mM-D-glucose). Intracellular acidification by lowering pHo below 7.40 or sudden addition of bicarbonate buffer did not induce insulin secretion. The removal of extracellular bicarbonate buffer, increase in pHo from 7.40 to 8.00, or the addition of 5 mM-L-5-hydroxytryptophan or 5 mM-NH4+, which all alkalinize the islet cells and induce insulin secretion, also increased the La3+-non-displaceable 45Ca2+ uptake in the presence of 1 mM-D-glucose. The results suggest that intracellular alkalinization in beta-cells can trigger insulin secretion. Taken together with the fact that D-glucose increases pHi in the islet cells, the results also point to the possibility that alkalinization may be a link in the stimulus-secretion coupling sequence in beta-cells.     

Absence of T cell tolerance to pancreatic islet cells.

To examine whether the lack of self-tolerance to beta cells is responsible for the development of type I diabetes in nonobese diabetic (NOD) mice, we attempted to induce T cell responses to cells from the islets of Langerhans. The data show that all NOD mice, irrespective of age, sex, and disease progression, possess islet cell-specific CD4+, MHC class II-restricted T cells. Both primary and secondary proliferative responses to islet cells were readily induced. The activation of T cells required presentation of islet cell Ag by APC in the responding lymph node cell population. Cells from other tissues, e.g., salivary gland, adrenal gland, and spleen, failed to activate autologous T lymphocytes. T cells specific for other Ag did not respond to islet cells, indicating that the proliferation is not the result of nonspecific stimulation by islet cell products. The presence of islet cell-reactive T cells is, however, not unique to NOD mice, because similar T cell reactivity was also demonstrated in non-diabetes-prone mouse strains. Hence, self-tolerance to islet cells appears to be absent. The results indicate a normal occurrence of islet cell-reactive T cells in both diabetes-prone as well as non-diabetes-prone mice. Thus, the lack of tolerance cannot be the initial cause of diabetes, but the activation of such autoreactive T cells may be important for the development of the disease.     

Metabolic labelling and partial characterization of glycophospholipids in pancreatic islet cells.

Insulin action is thought to be mediated by an inositol-, glucosamine- and galactose-containing oligosaccharide liberated by phosphodiesterase hydrolysis of a glycosyl-phosphatidylinositol. This oligosaccharide inhibits insulin biosynthesis and secretion in pancreatic islets. In the present study, two main glycolipids (peak I and II) were resolved by sequential TLC of lipids extracted from islet cells labelled with tritiated glucosamine, galactose or myristate. The two glycolipids displayed comparable sensitivity to beta-galactosidase but differed from one another by their sensitivity to phosphatidylinositol-specific phospholipase C. Moreover, structural heterogeneity within each peak was suggested by their partial resistance to nitrous acid deamination. These findings support the presence in islet cells of glycolipids similar to those currently considered as a possible postreceptor target for insulin in other cell types.     

Initial uptake and insulin releasing action of chloromercuribenzene-p-sulphonic acid (CMBS) in suspensions of pancreatic islet cells

The effects of chloromercuribenzene-p-sulphonic acid on dispersed cells prepared from beta-cell-rich ob/ob-mouse islets were studied. 1) Chloromercuribenzene-p-sulphonic acid at concentrations of 0.1 mmol/l or higher diminished cell viability which was partially counteracted by increasing concentrations of bovine serum albumin. 2) The uptake of 203Hg-chloromercuribenzene-p-sulphonic acid after incubation for 4 seconds or longer showed that most of the non-toxic concentrations of chloromercuribenzene-p-sulphonic acid was bound to the cell within 40 seconds. Maximal uptake was achieved after 3 minutes of incubation. The uptake of radioactive chloromercuribenzene-p-sulphonic acid was inhibited by bovine serum albumin. 3) The dynamics of insulin release from perifused dispersed beta-cells embedded in fibrin showed a maximal 40--50-fold stimulation by 0.03 mmol/l chloromercuribenzene-p-sulphonic acid within 10 minutes of perifusion. 4) Scanning electron microscopy of beta-cells revealed no major changes in the cell surface under conditions of maximal binding and insulin releasing effects of chloromercuribenzene-p-sulphonic acid. These data support the concept that the ability of chloromercuribenzene-p-sulphonic acid to induce insulin release is related to its initial binding to the beta-cell surface. The binding of chloromercuribenzene-p-sulphonic acid and the subsequent release of insulin seem to occur without major changes in beta-cell surface morphology.     

Nutrient-induced changes in the pH of pancreatic islet cells

Fluorescein rapidly accumulates in rat pancreatic islets exposed to fluorescein diacetate. The influence of environmental agents upon cellular pH was examined in fluorescein-labelled islets by recording their fluorescence intensity at 520 nm after excitation at 490 and 435 nm, respectively. Glucose caused a rapid, sustained and dose-related increase in cellular pH. Another nutrient secretagogue, 2-ketoisocaproic acid, also increased cellular pH. The stimulation of islet cells by non-nutrient secretagogues, e.g. by glibenclamide or in response to an increase in extracellular K+ concentration, decreased cellular pH, indicating that the nutrient-induced increase in cellular pH is not merely a consequence of stimulated Ca2+ inflow and/or insulin release. In either the presence of amiloride or absence of bicarbonate, glucose decreased cellular pH. These results strongly suggest that the acidification of islet cells which can be expected from the increased metabolism of glucose in glucose-stimulated islets is normally masked and overcome by stimulation of such processes as Na+/H+ and HCO3-/Cl- exchange.     

The uptake of homologous ribonucleic acid by rat-liver parenchymal cells in suspension

1. Native or partially degraded RNA derived from intact rat liver, or from the parenchymal-cell or the non-parenchymatous fraction of liver, has been shown to be transported into rat parenchymal cells in suspension, without prior degradation to acid-soluble components, when the cell suspension is incubated with the RNA at 37 degrees . The amount of RNA of exogenous origin present in the parenchymal cells in an acid-precipitable form increased rapidly up to 30-60min., after which it gradually decreased, indicating intracellular degradation to acid-soluble components of the RNA taken up by the cells. 2. The RNA taken up by the parenchymal cells from the medium, and the acid-soluble products of its degradation within the cells, could be released back into the medium. 3. The RNA of exogenous origin present in acid-precipitable form in the parenchymal cells represented up to 5% of the RNA of the cells after 60min. of incubation. 4. When the concentration of RNA in the medium was less than 200mug./ml., over 10% of the RNA was transported in an acid-precipitable form in 60min. into the parenchymal cells incubated at a concentration of 2.3x10(6)/ml. 5. Ribonuclease inhibited the uptake of exogenous RNA by the parenchymal cells, whereas 2,4-dinitrophenol, sodium azide, protamine sulphate and polyvinyl sulphate had no significant effect. 6. The uptake of exogenous RNA by liver slices proceeded at a rate which was 4-20% of that obtained in the parenchymal-cell suspensions; the RNA taken up did not appear to become degraded, unlike that taken up by the cell suspensions. 7. It is concluded that dispersion of liver tissue to a suspension of single cells increases the permeability of the parenchymal cells to macromolecular RNA and creates conditions that lead to a rapid degradation of the RNA taken up.     

Destruction of pancreatic islet cells by cytotoxic T lymphocytes in nonobese diabetic mice

Proliferation of islet-associated leukocytes occurred when isolated islets from 20-wk-old female nonobese diabetic (NOD) mice were cultured with 10 U/ml rIL-2 for 7 days. Co-culture of these leukocytes with freshly isolated islets from 6- to 8-wk-old NOD donors in the presence of 1 U/ml rIL-2 produced islet structural deformation within 24 h and islet cytolysis within 48 h. Three lines of evidence suggest that these leukocytes were composed mainly of CTL specific for islet cells. First, morphologically, these proliferating cells adhered to NOD islets at 6 h and killed islets within 48 h of culture, but these phenomena could not be observed in the other tissues from NOD mice. These islet-derived cells were cytotoxic to NOD islet cells in a 51Cr-release assay, whereas no appreciable cytotoxicity was observed when NOD Con A-induced splenic blasts or fibroblasts were used as targets. Second, a flow cytometric analysis showed that these cells consisted of 97% Thy-1.2, 69% Lyt-2, 8% L3T4, and 4% asialo-GM1-positive cells, whereas Mac-1-positive cells could not be seen in these assays. After treatment with anti-Thy-1.2 or Lyt-2 mAb and C, these cells lost their activity to lyse NOD islet cells. However, these cells still had a full killing activity after the depletion of L3T4 or asialo GM1-positive cells. Third, islet cells from BALB/c, DBA/2, and B10.GD mice which share the same H-2K Ag with NOD mice were susceptible to cytolytic activity of these cells, whereas islet cells from NON, C57BL/6, C57BL/10, and C3H mice remained intact. Furthermore, anti-Kd antibody was capable of blocking this cytolysis. These results suggest that CTL expressing Thy-1.2 and Lyt-2 phenotypes appear to recognize the islet cell Ag with the restriction of MHC class I Kd, and then destroy NOD islet cells.