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Biomedical subjects

S Lenzen

Publications and source records attributed to S Lenzen.

At least 55 records · Page 3Linked to original sources
PubMed1996

Dissociation between effects of polyamines on mitochondrial calcium uptake and mitochondrial permeability transition by elongation of polyamine methylene backbone.

The aliphatic polyamine bis(hexamethylene)triamine, a spermidine analogue, was found to lack the enhancing effect on mitochondrial Ca(2+) accumulation which is typical for the natural polyamines, spermidine and spermine. However, like spermine and spermidine, this compound had a significant inhibitory effect on the Ca(2+) and Pi-induced mitochondrial permeability transition. By chromatographic determination of the amount of polyamines bound to mitochondria after a 2 min incubation, it was found that the binding affinity was spermine > or = bis(hexamethylene)triamine > spermidine. There was no competition between binding of spermine and of bis(hexamethylene)triamine. It is concluded that membrane binding sites of bis(hexamethylene)triamine are different from those of spermine. This different membrane interaction is apparently caused by the elongated hydrophobic spans in bis(hexamethylene)triamine and leads to a loss of the effect on Ca(2+) uptake but leaves intact the inhibitory effect on membrane permeability transition.

Animals↗
PubMed1995

Effects of glucose refeeding and glibenclamide treatment on glucokinase and GLUT2 gene expression in pancreatic B-cells and liver from rats.

The mutual role of glucose and insulin in the regulation of glucokinase and GLUT2 glucose transporter gene expression in pancreatic B-cells and liver has been studied in vivo in the rat. Glucokinase mRNA was quantified by competitive reverse-transcriptase PCR analysis, and GLUT2 mRNA by Northern-blot analysis in total RNA fractions. As in the liver, glucokinase mRNA decreased by 64% in pancreatic B-cells after starvation for 2 days and was induced 3-fold by short-term treatment (1 h) of the rats with oral glucose (4 g/kg body wt.). In contrast the sulphonylurea compound glibenclamide (0.1 mg/kg body wt.) did not significantly stimulate glucokinase gene expression in pancreatic B-cells. But glibenclamide caused a 4-fold increase of glucokinase mRNA in liver which was abolished by concomitant administration of diazoxide, a drug which antagonizes glibenclamide stimulated insulin secretion. GLUT2 gene expression was decreased by 50% in pancreatic B-cells and liver after starvation of the rats for 2 days. Neither short-term treatment (1 h) with glucose nor glibenclamide resulted in a significant increase of GLUT2 gene expression in pancreatic B-cells and liver. The results suggest that it is glucose which stimulates glucokinase gene expression in pancreatic B-cells whereas the transcriptional regulation of the glucokinase gene in liver is directed by insulin.

Animals↗
PubMed1995

Degradation of dansyl polyamines on high-performance thin-layer chromatographic plates.

Using high-performance thin-layer chromatography with in situ quantitation to measure dansylated polyamines in the range of 1-20 pmol, we found that dansylated polyamines apparently react with the silica gel of the plates. The fluorescence of the dansyl polyamines diminished with increase in the time interval between application of a sample to the plate and start of the chromatographic separation. Conversely, the fluorescence at the site of application increased with the length of the time interval, indicating the formation of polar reaction products. If this reaction is not accounted for, considerable errors in quantitation of dansyl polyamines may occur.

Chromatography, Thin Layer↗
PubMed1995

Gene therapy of diabetes mellitus--aims, methods and future prospects.

Progress in pancreatic islet research and molecular biology techniques has opened the perspective for gene therapy of diabetes mellitus. This review will focus on the prerequisites for the generation of genetically engineered insulin-producing cells and current methods of gene transfer. A short account of the genetically modified insulin-secreting cell lines generated so far is also given. Although still experimental each of these new insulin-secreting cell lines is a step forward on the way to gene therapy of human diabetes.

Animals↗
PubMed1994

Lipid composition of glucose-stimulated pancreatic islets and insulin-secreting tumor cells.

The effect of glucose stimulation (25 mM for 5 min) on the phospholipid and neutral lipid composition of isolated pancreatic islets was studied to find out whether there is a change in the mass of potential lipid mediators or modulators of insulin secretion. For comparison, the lipid compositions of homogenates and subcellular fractions from RINm5F insulin-secreting tumor cells and of glucose-stimulated streptozotocin/nicotinamide-induced islet cell tumors were analyzed. After separation of the lipid extract into a neutral and an acidic fraction by anion-exchange chromatography, lipids were separated by high-performance thin-layer chromatography and quantitated by in situ densitometry of the cupric sulfate-charred bands. In glucose-stimulated islets, the molar percentages of phosphatidic acid (PA) and of phosphatidylinositol were significantly increased (3.1 vs. 4.7 mol% and 8.6 vs. 11.8 mol%), while those of all other phospholipids and neutral lipids, including 1,2-diacylglycerol, were not significantly changed. In stimulated islet cell tumors, an increase of PA was visible in the microsomal fraction, and there was an increase of lysophosphatidylcholine in the mitochondrial fraction. However, in both tumoral tissues, particularly in RINm5F cells, the lipid distribution pattern showed abnormalities which can be regarded as a loss of differentiation and which limit the usefulness of these tissues for the study of the physiological regulation of lipid metabolism during glucose stimulation. In conclusion, the data are in accordance with a role of PA early in stimulus-secretion coupling. The well-known stimulation of phospholipid synthesis in pancreatic islets during glucose-induced insulin secretion does not result in an increase in the total phospholipid mass.

Adenoma, Islet Cell↗
PubMed1993

Insulin secretion, insulin content and glucose phosphorylation in RINm5F insulinoma cells after transfection with human GLUT2 glucose-transporter cDNA.

The insulin-secretory response to glucose is defective in the RINm5F insulin-producing tumour cell line. Stable transfection with human low-affinity GLUT2 glucose-transporter cDNA revealed a significant improvement in stimulus-secretion coupling in these insulinoma cells. 3-O-Methylglucose uptake increased 10-fold in the concentration range 10-20 mM, whereas non-transfected control cells were unresponsive. Northern-blot analysis revealed a 7-fold increase in expression of the insulin gene in the GLUT2-transfected RINm5F cell clone T1. In contrast, glucokinase and GLUT1 glucose-transporter mRNA gene expression were not affected by transfection with GLUT2 glucose-transporter cDNA. The insulin content of transfected RINm5F cells was 7-fold higher after tissue culture at high glucose concentrations than in non-transfected controls. GLUT2-transfected RINm5F cells also regained insulin-secretory responsiveness toward high glucose concentrations. Tissue culture for 72 h in 20 mM glucose induced glucokinase activity in the GLUT2-transfected RINm5F clone T1, raising the glucokinase/hexokinase phosphorylation ratio from 0.2 to 0.6. The experiments demonstrate that an increased glucose uptake via a low-affinity glucose transporter and an increased metabolic flux rate are important factors in the induction of insulin-gene expression and glucokinase activity and thus improved glucose-induced biosynthesis and secretion of insulin in RINm5F insulinoma cells.

3-O-Methylglucose↗
PubMed1993

Effect of spermine on mitochondrial matrix calcium in relation to its enhancement of mitochondrial calcium uptake.

The mechanism of spermine-induced enhancement of mitochondrial Ca2+ uptake was explored using the fluorescent Ca2+ indicator Fluo-3/AM to measure the free matrix Ca2+ concentration. Simultaneously, the extramitochondrial Ca2+ concentration was registered by a Ca(2+)-ion selective electrode. Spermine lowered the extramitochondrial steady state Ca2+ concentration and at the same time induced a decrease of the intramitochondrial Ca2+ concentration. However, there is a concentration-dependent reversal of the stimulatory action of spermine, which may be explained by the existence of a second, low-affinity binding site for spermine which mediates an inhibition of uptake in spite of the existence of an inwardly directed Ca2+ gradient.

Aniline Compounds↗
PubMed1993

The relationship between the physicochemical properties and the biological effects of alloxan and several N-alkyl substituted alloxan derivatives.

Alloxan causes diabetes in experimental animals through its ability to destroy the insulin-secreting B-cells of the pancreas. Alloxan is hydrophilic and chemically unstable; it is reactive toward thiols, undergoing redox cycling in the presence of glutathione and oxidizing protein-bound thiol groups, as reflected by inhibition of the thiol enzymes, hexokinase and glucokinase. It is apparently also selectively taken up by the GLUT-2 glucose transporter in the pancreatic B-cell membrane. In order to investigate which, if any, of these physicochemical properties are important in the toxic action of alloxan, we have examined seven N-alkyl substituted alloxan derivatives of various diabetogenic activity. Hydrophilicity was identified as a factor essential for diabetogenicity. Stability, rate of redox cycling and reactivity toward thiol groups were not correlated with diabetogenicity. Selective uptake by the GLUT-2 glucose transporter is not a prerequisite for the diabetogenicity of alloxan derivatives.

Alloxan↗
PubMed1993

Differential regulation of glucokinase and GLUT-2 glucose transporter gene expression in pancreas and liver from neonatal and 16 day old rats.

Glucokinase and GLUT-2 high Km glucose transporter gene expression were studied in pancreas and liver from 2 day old neonatal and 16 day old rats. In pancreas tissue the glucokinase gene was transcribed into a 2.8 kb and a 4.4 kb mRNA species. However, while the 2.8 kb transcript was expressed already in pancreas from 2 day old neonatal rats, distinct expression of the 4.4 kb transcript occurred only in the pancreas from 16 day old rats. GLUT-2 glucose transporter was expressed already in the pancreas from 2 day old neonatal rats, albeit to a lesser extent than in the pancreas from 16 day old rats. Liver glucokinase gene was not expressed in 2 day old neonatal rats but in 16 day old neonatal rats, whereas the GLUT-2 glucose transporter gene was expressed both in the liver from 2 day old neonatal and 16 day old rats. The differences between glucokinase gene expression in neonatal pancreas and neonatal liver provide an indication for a different function of this enzyme in the two tissues. The immature insulin secretory response to glucose in neonatal pancreas may be related to a deficient glucokinase and probably also to a reduced GLUT-2 glucose transporter gene expression.

Aging↗
PubMed1992

Effects of alloxan and ninhydrin on mitochondrial Ca2+ transport.

Alloxan at millimolar concentrations slightly inhibited the velocity of Ca2+ uptake by isolated rat liver mitochondria irrespective of the free Ca2+ concentration between 1 and 10 microM and was an effective concentration-dependent stimulator of mitochondrial Ca2+ efflux. Ninhydrin also slightly inhibited the velocity of mitochondrial Ca2+ uptake but only at free Ca2+ concentrations above 5 microM. However, ninhydrin was a strong stimulator of mitochondrial Ca2+ efflux even at micromolar concentrations, 10-50 times more potent than alloxan. The mitochondrial membrane potential was reduced 10-20% at most by alloxan and ninhydrin. Alloxan and ninhydrin also stimulated Ca2+ efflux from isolated permeabilized liver cells. When isolated intact liver cells had been pre-incubated with alloxan or ninhydrin before permeabilization of the cells the ability of spermine to induce mitochondrial Ca2+ uptake was abolished. Glucose provided the typical protection against the effects of alloxan on mitochondrial Ca2+ transport only in experiments with intact cells but not in experiments with permeabilized cells or isolated mitochondria. Therefore glucose protection is apparently due to inhibition of alloxan uptake into the cell. Glucose provided no protection against effects of ninhydrin under any of the experimental conditions. Thus both alloxan and ninhydrin are potent stimulators of Ca2+ efflux by isolated mitochondria but very weak inhibitors of the velocity of mitochondrial Ca2+ uptake. The direct effects of ninhydrin on mitochondrial Ca2+ efflux may contribute to the cytotoxic action of this agent whereas the direct effects of alloxan on mitochondrial Ca2+ transport require concentrations which are too high to be of relevance for the induction of the typical pancreatic B-cell toxic effects of alloxan. However, the effects on mitochondrial Ca2+ transport during incubation of intact cells which may result from the generation of cytotoxic intermediates during alloxan xenobiotic metabolism may well contribute to the pancreatic B-cell toxic effect of alloxan.

Alloxan↗
PubMed1992

Dual effect of spermine on mitochondrial Ca2+ transport.

1. A dual effect of the polyamine spermine on Ca2+ uptake by isolated rat liver, brain and heart mitochondria could be demonstrated by using a high-resolution system for studying mitochondrial Ca2+ transport. Depending on the experimental situation, spermine had an inhibiting or accelerating effects on mitochondrial Ca(2+)-uptake rate, but invariably increased the mitochondrial Ca2+ accumulation. 2. Both effects were concentration-dependent and clearly discernible on the basis of their different kinetic characteristics. For mitochondria from all three tissues the half-maximally effective concentration for inhibition of the initial rate of Ca2+ uptake was approx. 180 microM, whereas that for the subsequent stimulation of Ca2+ accumulation was approx. 50 microM. 3. Acceleration of the initial uptake rate could be seen when the mitochondria were preloaded with spermine during a 2 min preincubation period and thereafter incubated in a medium without spermine. 4. When such spermine-preloaded mitochondria were incubated in a spermine-containing medium, the increase in Ca(2+)-accumulation capacity was maintained in spite of an unchanged rate of Ca2+ uptake. 5. Mg2+ interacted with the effects of spermine in a differential manner, enhancing the initial inhibition of the rate of mitochondrial Ca2+ uptake and diminishing the subsequent stimulation of mitochondrial Ca2+ accumulation. 6. This dual effect of spermine on mitochondrial Ca2+ transport resolves the apparent paradox that a polycationic compound can act as a stimulator of Ca2+ uptake.

Animals↗
PubMed1992

Inhibition of aconitase by alloxan and the differential modes of protection of glucose, 3-O-methylglucose, and mannoheptulose.

Alloxan inhibited aconitase with a half maximal inhibitory concentration of 0.5 mM in sonically disrupted and 2.3 mM in intact isolated liver mitochondria. For dialuric acid the half maximal inhibitory concentrations were 1.1 mM and 2.5 mM, respectively. Ninhydrin and N-ethylmaleimide (NEM) also inhibited aconitase with half maximal inhibitory concentrations in the submillimolar range and t-butylhydroperoxide (BuOOH) in the millimolar range, which, however, were not different for disrupted and intact mitochondria. Only the aconitase substrate citrate, but not glucose provided protection of the enzyme against inhibition. In intact liver cells the half maximal inhibitory concentration for alloxan was 6.8 mM. Again, dialuric acid and BuOOH were less potent inhibitors while ninhydrin and NEM were more potent inhibitors of aconitase in intact liver cells. In intact liver cells, glucose and 3-O-methylglucose, but not mannoheptulose and citrate provided protection against alloxan inhibition. The results show that aconitase is not an enzyme particularly sensitive towards alloxan inhibition and thus apparently not a primary site for mediation of alloxan toxicity as it is the glucokinase. This makes a primary site of alloxan action in the mitochondria extremely unlikely. On the other hand the results demonstrate that both the intact mitochondrial and plasma membrane as uptake barriers provide protection against alloxan toxicity. In addition the results clearly show, that 3-O-methylglucose provides protection against alloxan action only at the level of the plasma membrane through inhibition of alloxan uptake into the cell, while the site of protection of mannoheptulose is only the sugar binding site of the glucokinase. In contrast, glucose is shown here to be the only sugar with a dual protective effect both through inhibition of alloxan uptake through the plasma membrane like 3-O-methylglucose and through protection of the glucokinase sugar binding site against alloxan inhibition of the enzyme like mannoheptulose. In the light of these results the unique protective potency of glucose as compared to that of other sugars is not surprising.

3-O-Methylglucose↗
PubMed1992

Effect of lysophospholipids, arachidonic acid and other fatty acids on regulation of Ca2+ transport in permeabilized pancreatic islets.

The immediate reaction products of PLA2-mediated hydrolysis of phospholipids were tested for their ability to induce Ca2+ mobilization from internal stores in permeabilized ob/ob mouse pancreatic islets. Lysophospholipids and unsaturated fatty acids increased the free Ca2+ concentration in the incubation medium of permeabilized ob/ob mouse pancreatic islets. The potency of the lysophospholipids decreased in the following order: lysophosphatidylcholine = lysophosphatidylglycerol much greater than lysophosphatidylinositol greater than lysophosphatidylserine much greater than lysophosphatidylethanolamine. Arachidonic acid and palmitoleic acid had a potency comparable to lysophosphatidylinositol, while palmitic acid was ineffective. The Ca(2+)-mobilizing effect of inositol-1,4,5-trisphosphate (IP3) in permeabilized islet cells was additive to the lysophospholipid effect, indicating different sites of action. Both Ca(2+)-mobilizing effects were counteracted by the polyamine spermine, while the presence of Mg2+ shifted the Ca2+ concentrations to higher levels. Since not only an activation of a phospholipase C but also an activation of a phospholipase A2 with subsequent generation of lysophospholipids and free fatty acids is reported to occur in glucose-induced insulin secretion, the interaction of the phospholipase C reaction product IP3 with a lysophospholipid or an unsaturated fatty acid may affect the extent and duration of the rise in the free cytoplasmic Ca2+ concentration responsible for initiation of insulin secretion.

Animals↗
PubMed1991

Regulation of glucokinase and GLUT-2 glucose-transporter gene expression in pancreatic B-cells.

Glucokinase (EC 2.7.1.2) is the signal-recognition enzyme in pancreatic B-cells for initiation of glucose-induced insulin secretion. We show here that both the glucokinase and glucose-transporter GLUT-2 genes are regulated physiologically. Fasting decreased B-cell glucokinase and glucose-transporter GLUT-2 mRNA in pancreatic B-cells as well as in liver, whereas refeeding induced expression of both genes. In pancreatic B-cells a approximately 4.4 kb glucokinase-related mRNA was detectable, in addition to the 2.8 kb form. This approximately 4.4 kb glucokinase transcript was drastically decreased during refeeding. The 2.8 kb mRNA, which is typical for pancreatic B-cells, was accompanied after refeeding by a 2.4 kb mRNA species typical for liver glucokinase. Starvation primarily decreased the 2.8 kb pancreatic B-cell glucokinase mRNA species. The concordant regulation of both genes may represent the basis for the physiological regulation of glucose-induced insulin secretion at a transcriptional level.

Animals↗
PubMed1991

Structural requirements of lysophospholipid-regulated mitochondrial Ca2+ transport.

Analogues of lysophosphatidylcholine, including PAF (platelet-activating-factor) and HePC (an experimental anticancer drug), were studied for their influence on mitochondrial Ca2+ transport and membrane potential. Lysophospholipids released Ca2+ from mitochondria and reduced the maximal Ca2+ uptake. The structure-activity relations indicate that deprotonated head groups like phosphocholines yield active compounds while partially protonated head groups like phosphoethanolamines are essentially inactive. Structural requirements for the apolar part of the molecules were acyl or alkyl chain lengths of less than 18 carbon atoms at the C1-position of the glycerol backbone and residues of small size and/or low polarity at the C2-position. Choline lysophospholipids, but not ethanolamine lysophospholipids, may therefore induce mitochondrial Ca2+ efflux and become mediators of ischaemic tissue damage where dysregulated phospholipase A2 activity and an impairment of mitochondrial function are supposed to play a crucial role.

Animals↗
PubMed1991

Thiol-group reactivity, hydrophilicity and stability of alloxan, its reduction products and its N-methyl derivatives and a comparison with ninhydrin.

The diabetogenic agent, alloxan, is a hydrophilic and chemically unstable compound. The logarithm of the octanol/water partition coefficient of alloxan was found to be -1.86; its half-life at pH 7.4 and 37 degrees in phosphate buffer was 1.5 min. The partition coefficients and half-lives of the alloxan reduction products, alloxantin and dialuric acid, were very similar to those of the parent compound; N-methylalloxan and N,N'-dimethylalloxan were less hydrophilic but more unstable. Ninhydrin was found also to be hydrophilic although this compound, in contrast to alloxan and its derivatives, was quite stable in aqueous solution. Alloxan and its N-methyl derivatives were reduced by thiols and in the presence of glutathione and cysteine, rapid redox cycling occurred, with formation of 'active oxygen' species; no such reaction was observed, however, with ninhydrin. Comparatively slow redox cycling was recorded with alloxan derivatives and dithiothreitol although rapid cycling occurred with ninhydrin and this dithiol. Such differences may explain why ninhydrin does not share with alloxan a selective toxic effect upon the pancreatic B-cell.

Alloxan↗
PubMed1991

Purification and characterization of a chicken egg white cystatin variant expressed in an Escherichia coli pIN-III-ompA system.

A synthetic gene coding for a chicken egg white cystatin variant was cloned and expressed using the pIN-III-ompA Escherichia coli expression system. After osmotic shock of the E. coli cells, the cysteine proteinase inhibitor was isolated from periplasm and purified by S-carboxymethylpapain affinity chromatography. The resulting inhibitory material was characterized by SDS/PAGE, reversed-phase HPLC, peptide mapping and amino acid sequencing. The recombinant variant chicken AEF-[S1----M, M29----I, M89----L]cystatin shows strong inhibitory activity and displays Ki values in the complex with papain, actinidin and cathepsin B similar to those found for natural chicken cystatin. The purified variant showed a native-chicken-cystatin-like conformational state, as determined by NMR spectroscopy, if the NMR data of 15N-labelled recombinant inhibitor were compared with those of the natural inhibitor.

Amino Acid Sequence↗