Biomedical subjects

S Lenzen

Publications and source records attributed to S Lenzen.

At least 37 records · Page 2Linked to original sources

PubMed1999

Protection against the co-operative toxicity of nitric oxide and oxygen free radicals by overexpression of antioxidant enzymes in bioengineered insulin-producing RINm5F cells.

AIMS/HYPOTHESIS: The importance of different antioxidative enzymes for the defence of insulin-producing cells against the toxicity of nitric oxide (NO) was characterised in bioengineered RINm5F cells. METHODS: RINm5F insulin-producing cells stably overexpressing glutathione peroxidase (GPX), catalase (CAT) or Cu/Zn superoxide dismutase (SOD) were exposed to S-nitroso-N-acetyl-D,L-penicillamine (SNAP), sodium nitroprusside (SNP) and 3 morpholinosydnonimine (SIN-1), which generate both NO and reactive oxygen species, and to the polyamine/ NO, complex DETA/NO which generates NO alone. The viability of the cells was tested by the MTT assay. RESULTS: Overexpression of antioxidant enzymes provided significant protection against the toxicity of SNAP, SNP and SIN-1, with an individual specificity related to their chemical characteristics, but was without effect upon the toxicity of DETA/NO. Cells overexpressing GPX were well protected against SNP and SNAP, while CAT was most effective against SIN-1. SOD overexpression provided less protection against the toxicity of SNAP and SNP than overexpression of GPX but was more effective in protecting against SIN-1. Co-incubation of cells with NO donors and hydrogen peroxide or hypoxanthine and xanthine oxidase showed an overadditive synergism of toxicity. CONCLUSION/INTERPRETATION: The results emphasise the importance of a synergism between NO and reactive oxygen species for pancreatic beta-cell death. Such a synergism has also been observed after exposure of beta cells to cytokines. The component of the toxicity that is mediated by oxygen radicals can be suppressed effectively through overexpression of CAT, GPX or SOD or both.

Animals↗

PubMed1999

Nutrient-dependent distribution of insulin and glucokinase immunoreactivities in rat pancreatic beta cells.

Functional heterogeneity among pancreatic beta cells is a characteristic feature of the islets of Langerhans. Under physiological conditions, beta cells in the pancreas of fed rats exhibited heterogeneous immunohistochemical staining for insulin and glucokinase. Intracellular beta cell glucokinase staining was either faint or dense. In the pericapillary space beta cell glucokinase immunoreactivity had a polar orientation, with the highest density in cytoplasmic regions close to the blood vessels. Starvation resulted in a loss of heterogeneity with homogeneous insulin staining in all beta cells of the islets, and this was accompanied by a loss of heterogeneous glucokinase staining. The intracellular polarity of glucokinase staining in contact to blood vessels also disappeared after starvation. Refeeding resulted in the reappearance of intercellular heterogeneity. In dependence on the functional demand, the endocrine pancreas recruited insulin from beta cells according to a well-defined hierarchy, with an initial preferential mobilization of medullary beta cells. In the course of this process intracellular polarity of glucokinase staining reappeared in areas of the beta cell with functional contact to the GLUT2 glucose transporter in the plasma membrane. This can be regarded as the morphological correlate of an activation of the glucose signal recognition apparatus. Interestingly, the study also provides evidence that the changes in glucokinase distribution apparently preceded those in insulin distribution, which is in keeping with the central role of glucokinase as the glucose sensor of the pancreatic beta cell.

Animals↗

PubMed1999

Effect of superoxide dismutase, catalase, chelating agents, and free radical scavengers on the toxicity of alloxan to isolated pancreatic islets in vitro.

The effect of superoxide dismutase, catalase, metal-chelating agents and hydroxyl radical scavengers on the toxicity of alloxan to isolated ob/ob mouse pancreatic islets in vitro has been compared with the reported ability of such substances to protect against alloxan diabetes in vivo. Superoxide dismutase and catalase protected beta-cells of isolated pancreatic islets against alloxan cytotoxicity, as did the hydroxyl radical scavengers dimethyl sulfoxide (DMSO) and butanol. However, 1,3-dimethylurea and thiourea, that are recognised as effective hydroxyl radical scavengers and that protect animals against the diabetogenic effects of alloxan, were without effect. Similarly, desferrioxamine, that inhibits hydroxyl radical formation from alloxan in chemically defined systems, did not protect against alloxan toxicity. Diethylenetriamine pentaacetic acid, which does not inhibit hydroxyl radical formation from alloxan, also gave no significant protection. The results indicate a role for superoxide radical and hydrogen peroxide in the mechanism of toxicity of alloxan but do not support the involvement of the hydroxyl radical in this process. Alternative explanations must be sought for the ability of hydroxyl radical scavengers and metal-chelating agents to protect against alloxan toxicity in vivo.

1-Butanol↗

PubMed1999

Metabolic regulation, activity state, and intracellular binding of glucokinase in insulin-secreting cells.

Regulation of glucose-induced insulin secretion is crucially dependent on glucokinase function in pancreatic beta-cells. Glucokinase mRNA expression was metabolically regulated allowing continuous translation into enzyme protein. Glucokinase enzyme activity in the beta-cell was exclusively regulated by glucose. Using a selective permeabilization technique, different intracellular activity states of the glucokinase enzyme in bioengineered glucokinase-overexpressing RINm5F tissue culture cells were observed. These results could be confirmed in analogous experiments with dispersed islet cells. A diffusible glucokinase fraction with high enzyme activity could be distinguished from an intracellularly bound fraction with low activity. Glucose induced a significant long-term increase of the active glucokinase fraction. This effect was accomplished through the release of glucokinase enzyme protein from an intracellular binding site of protein character. The inhibitory function of this protein factor was abolished through proteolytic digestion or heat inactivation. Northern blot analyses revealed that this binding protein was not identical to the well-known liver glucokinase regulatory protein. This hitherto unknown new protein factor may have the function of a glucokinase regulatory protein in the pancreatic beta-cell, which may regulate glucokinase enzyme activity in a glucose-dependent manner.

Animals↗

PubMed1998

Polyamine modulation of mitochondrial calcium transport. I. Stimulatory and inhibitory effects of aliphatic polyamines, aminoglucosides and other polyamine analogues on mitochondrial calcium uptake.

In this study, the regulation of mitochondrial Ca2+ transport by polyamines structurally related to spermine and by analogous polycationic compounds was characterized. Similar to spermine, a number of amino groups containing cationic compounds exerted a dual effect on Ca2+ transport of isolated rat liver mitochondria: a decrease in Ca2+ uptake velocity and an enhancement of Ca2+ accumulation. In contrast to the effects of spermine and other aliphatic polyamines, however, the accumulation-enhancing effect of aminoglucosides, basic polypeptides, and metal-amine complexes turned into an inhibition of Ca2+ accumulation at higher concentrations. Within groups of structurally related compounds, the potency to decrease Ca2+ uptake velocity and to enhance Ca2+ accumulation correlated with the number of cationic charges. The presence of multiple, distributed cationic charges was a necessary, but not sufficient criterion for effects on mitochondrial Ca2+ transport, because cationic polyamines and basic oligopeptides which did not enhance mitochondrial Ca2+ accumulation could be identified. Spermine was not able to antagonize the blocking of Ca2+ uptake by ruthenium red, but rather showed an apparent synergism, which can be explained as a displacement of membrane-bound Ca2+ by spermine. The aminoglucosides, gentamicin and neomycin, but not the inactive polyamine bis(hexamethylene)-triamine, inhibited the binding of spermine to intact mitochondria. Apparently, the binding of spermine, gentamicin, and a number of polyamine analogues to low-affinity binding sites at mitochondria, which have low, but distinct structural requirements and which may correspond to phospholipid headgroups, indirectly influences the activity state of the mitochondrial Ca2+ uniporter. The ability of aminoglucosides to displace spermine from the mitochondria and to inhibit mitochondrial Ca2+ accumulation may contribute to the mitochondrial lesions, which are known to occur early in the course of aminoglucoside-induced nephrotoxicity.

Aminoglycosides↗

PubMed1998

Polyamine modulation of mitochondrial calcium transport. II. Inhibition of mitochondrial permeability transition by aliphatic polyamines but not by aminoglucosides.

In this study, the effects of polyamines and analogous compounds on mitochondrial permeability transition were characterized to distinguish between these effects and those on mitochondrial Ca2+ uptake, which are described in an accompanying report (Rustenbeck et al., Biochem Pharmacol 8: 977-985, 1998). When a transitional Ca2+ release from Ca2+-loaded mitochondria was induced by an acute increase in Ca2+ concentration in a cytosol-adapted incubation medium (Ca2+ pulse), this process was inhibited, but not abolished by spermine in the concentration range of 0.4 to 20 mM. The aminoglucoside, gentamicin, and the basic polypeptide, poly-L-lysine, which like spermine are able to enhance mitochondrial Ca2+ accumulation (preceding paper), had no or only a minimal inhibitory effect, while the aliphatic polyamine, bis(hexamethylene)triamine, which is unable to enhance mitochondrial Ca2+ accumulation, achieved a complete inhibition at 4 mM. The conclusion that the Ca2+ efflux was due to opening of the permeability transition pore was supported by measurements of mitochondrial membrane potential, ATP production, and oxygen consumption. Mg2+, a known inhibitor of mitochondrial membrane permeability transition, did not mimic the effects of spermine on mitochondrial Ca2+ accumulation, while ADP, the main endogenous inhibitor, showed both effects. However, a combination of spermine and ADP was significantly more effective than ADP alone in restoring low Ca2+ concentrations after a Ca2+ pulse. Two different groups of spermine binding sites were found at intact liver mitochondria, characterized by dissociation constants of 0.5 or 4.7 mM and maximal binding capacities of 4.6 or 19.7 nmol/mg of protein, respectively. In contrast to aminoglucosides, the aliphatic polyamine bis(hexamethylene)triamine did not displace spermine from mitochondrial binding sites. The total intracellular concentration of spermine in hepatocytes was measured to be ca. 450 microM and the free cytoplasmic concentration was estimated to be in the range of 10-100 microM. In conclusion, the enhancement of mitochondrial Ca2+ uptake by spermine is not an epiphenomenon of the inhibition of permeability transition. The physiological role of spermine appears to be that of an enhancer of mitochondrial Ca2+ accumulation rather than an inhibitor of permeability transition.

Aminoglycosides↗

PubMed1998

Molecular characterization of the glucose-sensing mechanism in the clonal insulin-secreting BRIN-BD11 cell line.

BRIN-BD11 cells represent a novel insulin-secreting cell line generated by electrofusion. Molecular characterization of these cells demonstrated the presence of mRNA and protein for the two key elements of the beta cell glucose-sensing system, GLUT2 and glucokinase. While levels of GLUT2 expression and 3-O-methyl-D-glucose equilibration were similar for both the BRIN-BD11 cell line and the parental control RINm5F cells, glucokinase expression was substantially higher in BRIN-BD11 cells. Expression of the two-component KATP channel complex, KIR6.2 and SUR1, was similar in both cells. However, while control RINm5F cells were completely unresponsive to glucose, BRIN-BD11 cells responded to physiological millimolar concentrations of this hexose sugar. These studies strongly suggest that the glucose-sensing ability of insulin-secreting cells is largely dictated by the level of glucokinase, as opposed to GLUT2, expression. Thus, BRIN-BD11 cells expressing the key attributes of the normal beta cell provide an interesting model for elucidation of regulatory principles of beta cell function.

3-O-Methylglucose↗

PubMed1998

Complementary action of antioxidant enzymes in the protection of bioengineered insulin-producing RINm5F cells against the toxicity of reactive oxygen species.

To determine the importance of different antioxidative enzymes for the defense status of insulin-producing cells, the effects of stable overexpression of glutathione peroxidase (Gpx), catalase (Cat), or Cu/Zn superoxide dismutase (SOD) in insulin-producing RINm5F cells on the cytotoxicity of hydrogen peroxide (H2O2), hypoxanthine/xanthine oxidase (H/XO), and menadione have been investigated. Single overexpression of Cat or Gpx provided less protection than the combined expression of Cat plus SOD or Cat plus Gpx, while single overexpression of SOD either had no effect on the toxicity of the test compounds or increased it. RINm5F cells were also susceptible to butylalloxan, a lipophilic alloxan derivative that is selectively toxic to pancreatic beta-cells. Overexpression of enzymes, both alone and in combination, did not protect against butylalloxan-induced toxicity while SOD overexpression increased it, as evident from a half maximally effective concentration (EC50) value. The addition of Cat to the culture medium completely prevented the toxic effects of H2O2 and H/XO but had no significant effect on the toxicity of menadione or butylalloxan. Extracellular SOD had no effect on the toxicity of any of the test compounds. The results of this study show the importance of a combination of antioxidant enzymes in protecting against the toxicity of reactive oxygen species. Thus, overexpression of Cat and Gpx, alone or in combination with SOD, by use of molecular biology techniques can protect insulin-producing cells against oxidative damage. This may represent a strategy to protect pancreatic beta-cells against destruction during the development of autoimmune diabetes and emphasizes the importance of optimal antioxidative enzyme equipment for protection against free radical-mediated diseases.

Alloxan↗

PubMed1997

Modulation of human glucokinase intrinsic activity by SH reagents mirrors post-translational regulation of enzyme activity.

The low-affinity glucose phosphorylating enzyme glucokinase plays a key role in the process of glucose recognition in pancreatic B-cells. To evaluate mechanisms of intrinsic regulation of enzyme activity human pancreatic B-cell and liver glucokinase and for comparison rat liver glucokinase were expressed in E. coli bacteria. A one-step purification procedure through metal chelate affinity chromatography revealed 58 kDa proteins with high specific activities in the range of 50 U/mg protein and K(m) values around 8 mM for the substrate D-glucose with a preference for the alpha-anomer. There were no tissue specific differences, no species differences in the electrophoretic mobility, and no differences of the kinetic properties of these well conserved enzymes. The deletion of the 15 tissue-specific NH2-terminal amino acids of the human glucokinase resulted in a catalytically active enzyme whose kinetic properties were not significantly different from those of the wild-type enzymes. The human and rat glucokinase isoforms were non-competitively inhibited by the sulfhydryl group reagents alloxan and ninhydrin with Ki values in the range of 1 microM. The inhibition of glucokinase enzyme activity was reversed by dithiothreitol with an EC50 value of 9 microM for alloxan and of 50 microM for ninhydrin. D-Glucose provided protection against alloxan-induced inhibition of human and rat glucokinase isoenzymes with half-maximal effective concentrations between 11 and 16 mM. The enzyme inhibition by alloxan was accompanied by a change in the electrophoretic mobility with a second lower molecular 49 kDa glucokinase band which can be interpreted as a compact glucokinase molecule locked by disulfide bonds. Quantification of free sulfhydryl groups revealed an average number of 3.6 free sulfhydryl groups per enzyme molecule for the native human glucokinase isoforms. Alloxan decreased the average number of free sulfhydryl groups to 1.9 per enzyme molecule indicating that more than one SH side group is oxidized by this compound. The extraordinary sensitivity of the SH side groups of the glucokinase may be a possible mechanism of enzyme regulation by interconversion of stable (active) and unstable (inactive) conformations of the enzyme. In pancreatic B-cells the glucose-dependent increase of reduced pyridine nucleotides may stabilize the enzyme in the 58 kDa form and provide optimal conditions for glucose recognition and glucose-induced insulin secretion.

Alloxan↗

PubMed1997

Comparative toxicity of alloxan, N-alkylalloxans and ninhydrin to isolated pancreatic islets in vitro.

The in vitro toxicity of the diabetogenic agent alloxan as documented by the induction of beta cell necrosis was studied in isolated ob/ob mouse pancreatic islets. The effect of alloxan has been compared with that of a number of N-alkyl alloxan derivatives and with that of the structurally related compound, ninhydrin. Alloxan and its derivatives were selectively toxic to pancreatic beta cells, with other endocrine cells and exocrine parenchymal cells being well preserved, even at high concentration. In contrast, ninhydrin was selectively toxic to pancreatic beta cells only at comparatively low concentration, destroying all islet cell types at high concentrations. The ultrastructural changes induced by all the test compounds in pancreatic beta cells in vitro were very similar to those observed during the development of alloxan diabetes in vivo. The relative toxicity of the various compounds to pancreatic beta cells in vitro was not, however, related to their ability to cause diabetes in vivo. Indeed, the non-diabetogenic substances ninhydrin, N-butylalloxan and N-isobutylalloxan were very much more toxic to isolated islets than the diabetogenic compounds alloxan and N-methylalloxan. These results suggest that the differences in diabetogenicity among alloxan derivatives are not due to intrinsic differences in the susceptibility of the pancreatic beta cells to their toxicity, but may reflect differences in distribution or metabolism. High concentrations of glucose protected islets against the harmful effects of alloxan and its derivatives, but not those of ninhydrin. Low levels of glucose, and non-carbohydrate nutrients, afforded little protection, indicating that the effect of glucose is not due to the production of reducing equivalents within the cell, 3-O-Methylglucose, which protects against alloan diabetes in vivo, did not protect against alloxan toxicity in vitro. Since 3-O-methylglucose is known to prevent uptake of alloxan by pancreatic beta cells, it appears that uptake of alloxan by the cell is not a prerequisite for the induction of beta cell necrosis.

Alloxan↗

PubMed1997

Relation between antioxidant enzyme gene expression and antioxidative defense status of insulin-producing cells.

Antioxidant enzyme expression was determined in rat pancreatic islets and RINm5F insulin-producing cells on the level of mRNA, protein, and enzyme activity in comparison with 11 other rat tissues. Although superoxide dismutase expression was in the range of 30% of the liver values, the expression of the hydrogen peroxide-inactivating enzymes catalase and glutathione peroxidase was extremely low, in the range of 5% of the liver. Pancreatic islets but not RINm5F cells expressed an additional phospholipid hydroperoxide glutathione peroxidase that exerted protective effects against lipid peroxidation of the plasma membrane. Regression analysis for mRNA and protein expression and enzyme activities from 12 rat tissues revealed that the mRNA levels determine the enzyme activities of the tissues. The induction of cellular stress by high glucose, high oxygen, and heat shock treatment did not affect antioxidant enzyme expression in rat pancreatic islets or in RINm5F cells. Thus insulin-producing cells cannot adapt the low antioxidant enzyme activity levels to typical situations of cellular stress by an upregulation of gene expression. Through stable transfection, however, we were able to increase catalase and glutathione peroxidase gene expression in RINm5F cells, resulting in enzyme activities more than 100-fold higher than in nontransfected controls. Catalase-transfected RINm5F cells showed a 10-fold greater resistance toward hydrogen peroxide toxicity, whereas glutathione peroxidase overexpression was much less effective. Thus inactivation of hydrogen peroxide through catalase seems to be a step of critical importance for the removal of reactive oxygen species in insulin-producing cells. Overexpression of catalase may therefore be an effective means of preventing the toxic action of reactive oxygen species.

Animals↗

PubMed1996

Limited proteolysis of human alpha2-HS glycoprotein/fetuin. Evidence that a chymotryptic activity can release the connecting peptide.

alpha2-HS glycoprotein is a major protein of human plasma whose function is still obscure. A proteolytically processed form of alpha2-HS glycoprotein lacking a segment of 40 amino acid residues bridging its heavy and light chain portions ("connecting peptide") has been described suggesting that this peptide is released by post-translational processing to fulfill biological role(s) of alpha2-HS glycoprotein. To test this hypothesis we investigated how the connecting peptide is released from the parental molecule by limited proteolysis. We developed monoclonal antibodies to various portions of the connecting peptide and its NH2-terminal flanking region which cross-react with the native alpha2-HS glycoprotein. Purified alpha2-HS glycoprotein from human plasma was subjected to limited proteolysis by proteinases including trypsin, chymotrypsin, elastase plasmin, kallikrein, thrombin, and renin. Immunoprint analysis of the proteolytic digests indicated that alpha2-HS glycoprotein is readily cleaved in its connecting peptide region. NH2-terminal amino sequence analysis of the generated fragments demonstrated that a single proteinase, chymotrypsin, cleaves the critical Leu-Leu bond flanking the NH2-terminal portion of the connecting peptide region. Most but not all of the other proteinase cleavage sites map to a short stretch of 9 residues located in the center portion of the connecting peptide region. Immunoprint analysis of plasma samples from patients with sepsis demonstrate that the connecting peptide region is cleaved under pathological conditions. Our results indicate that the connecting peptide and/or fragments thereof are readily releasable from alpha2-HS glycoprotein in vitro and in vivo.

Amino Acid Sequence↗

PubMed1996

Relation between accumulation of phospholipase A2 reaction products and Ca2+ release in isolated liver mitochondria.

A Ca(2+)-dependent stimulation of mitochondrial phospholipase A2 is often assumed to play a role in mitochondrial Ca2+ release. We sought to clarify this relation by measuring Ca2+ transport and determining phospholipase A2 reaction products from the same sample of isolated, incubated rat liver mitochondria. When mitochondria had accumulated and spontaneously released again Ca2+, most probably by membrane permeability transition, there was no increase of phospholipase A2 reaction products. However, when the incubation was continued after Ca2+ release, significant increases of the content of lysophosphatidylcholine and unesterified fatty acids could be seen. Quinacrine, an inhibitor of phospholipase A2 activity, prevented Ca2+ release and p-hydroxymercuribenzoic acid, an inhibitor of lysophospholipid reesterification, induced a fast release of Ca2+ from isolated mitochondria. Such effects are usually taken as indirect evidence for a participation of phospholipase A2 in mitochondrial Ca2+ release, but analysis of the mitochondrial lipids revealed that no significant changes of the mass of phospholipase A2 reaction products had occurred. These experiments suggest that the accumulation of phospholipase A2 reaction products in mitochondria is the consequence rather than the cause of the membrane permeability transition. Exogenous phospholipase A2 products, lysophosphatidylcholine and arachidonic acid, induced mitochondrial Ca2+ release after a time lag, which decreased with aging of the mitochondrial preparation. The amount of lysophosphatidylcholine taken up by the mitochondria from the incubation medium during these experiments was measured and compared to the amount of lysophosphatidylcholine produced endogenously by mitochondrial phospholipase A2. From these data it appears likely that the amount of lysophosphatidylcholine generated in the mitochondria after the permeability transition is sufficient to sustain the permeable state. An accumulation of mitochondrially generated phospholipase A2 reaction products after the permeability transition could thus be a decisive factor for the limited reversibility of the membrane permeability transition.

Animals↗

PubMed1996

Effect of metformin on SGLT1, GLUT2, and GLUT5 hexose transporter gene expression in small intestine from rats.

The effect of the antihyperglycaemic agent metformin was studied on gene expression of the energy-dependent sodium-hexose cotransporter (SGLT1) and the facilitative hexose transporters GLUT2 and GLUT5 in rat intestine. Metformin treatment (125 mg/kg body wt. twice daily for a period of 3 days) significantly increased SGLT1 gene expression in duodenum and jejunum. GLUT5 gene expression was increased by metformin treatment only in the jejunum. Gene expression of GLUT2 in the intestine was not significantly affected by metformin treatment. This increase in transporter gene expression offers the potential for increases in hexose uptake at the brush border membrane, and may compensate for other effects of the drug that have been suggested to decrease glucose uptake by SGLT1, as well as for metformin stimulation of glucose utilization by the intestinal mucosa.

Animals↗

PubMed1996

A plug-in minielectrode for measurements in stirred photometric cuvettes.

A device is described to perform potentiometric measurements with ion-sensitive electrodes in stirred photometric cuvettes. Its design allows to make additions to the reaction medium with microliter syringes during measurements. Originally, this plug-in electrode was designed to register the free Ca2+ concentrations in the incubation medium of mitochondrial suspensions during measurements of the free matrix Ca2+ concentration with a permeant fluorescent Ca2+ indicator. However, numerous other applications can be easily realized, such as the combination of mitochondrial light-scattering measurements and ion-transport measurements, the combination of the permeabilized cell technique with fluorescence measurements of intracellular organelles or simply the calibration of the fluorescence of Ca2+ indicators with a Ca2+ ion-sensitive minielectrode. Compared with the use of a second fluorescent indicator the use of an electrode has the advantage that the signal can be transformed into ion concentrations already during the measurements.

Aniline Compounds↗

PubMed1996

Loss of GLUT2 glucose transporter expression in pancreatic beta cells from diabetic Chinese hamsters.

The diabetic Chinese hamster is a well-established animal model for NIDDM with a defective glucose-induced insulin secretory response. In the pancreas of nondiabetic hamsters, the GLUT2 glucose transporter was localized in the plasma membrane of insulin-positive beta cells. At variance with the rat, immunoreactivity was also detected in the cytoplasm. Other islet cell types were not GLUT2 positive. GLUT2 immunoreactivity was already significantly reduced in beta cells from mildly diabetic animals in spite of a normal insulin immunoreactivity. In severely diabetic animals the majority of the beta cells had lost GLUT2 immunostaining. This observation was confirmed in a Western blot analysis of the GLUT2 protein in isolated pancreatic islets. Only beta cells that were densely immunostained for insulin were still GLUT2 positive. However, around 40% of the beta cells devoid of GLUT2 immunoreactivity were still insulin immunoreactive. Thus, the loss of GLUT2 immunoreactivity, which is an important component of the glucose recognition apparatus of the pancreatic beta cell, is an early indicator of beta cell dysfunction before the development of degenerative lesions or the loss of insulin immunoreactivity. GLUT2 loss may be important in the deterioration of glucose-induced insulin secretion in the diabetic Chinese hamster.

Animals↗

PubMed1996

Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues.

Using a sensitive Northern blot hybridization technique, gene expression of superoxide dismutase (SOD), catalase, and glutathione peroxidase was studied in pancreatic islets and for comparison in various other mouse tissues (liver, kidney, brain, lung, skeletal muscle, heart muscle, adrenal gland, and pituitary gland). Gene expression of the antioxidant enzymes was usually in the range of +/- 50% of that in the liver. Only in pancreatic islets gene expression was substantially lower. The levels of the cytoplasmic Cu/Zn SOD and the mitochondrial Mn SOD gene expression were in the range of 30-40% of those in the liver. Glutathione peroxidase gene expression was 15%, and catalase gene expression was not at all detectable in pancreatic islets. These low levels of antioxidant enzyme gene expression may provide an explanation for the extraordinary sensitivity of pancreatic beta cells towards cytotoxic damage by diabetogenic compounds and during the development of human and animal diabetes.

Animals↗

PubMed1996

Effects of sodium butyrate on glucose transporter and glucose-phosphorylating enzyme gene expression in RINm5F insulinoma cells.

RINm5F insulinoma cells show a defective physiological insulin secretory response to glucose stimulation. The short chain carbonic acid sodium butyrate induced a growth arrest during a 72-h tissue culture period. In contrast to control RINm5F cells, 2 mM glucose increased insulin secretion by more than 70% in these sodium butyrate-treated cells (1 mM) without any further increase of the secretory rate between 2 and 20 mM glucose. This effect of sodium butyrate on insulin secretion was assessed in comparison with its effect on gene expression of the GLUT1 and GLUT2 glucose transporter, hexokinase type I and type II, glucokinase and insulin. Sodium butyrate at a 1 mM concentration decreased GLUT1 gene expression by nearly 50%, but did not induce gene expression of the low-affinity GLUT2 glucose transporter above the detection limit. Furthermore, sodium butyrate increased glucokinase gene expression by more than 50% and hexokinase type II gene expression by more than 100%, while insulin gene expression was increased only by 24%. Hexokinase type II enzyme activity was increased by more than 100% without a concomitant significant change of the glucokinase enzyme activity. Sodium butyrate (2 mM) caused effects comparable with those of 1 mM sodium butyrate. Thus the improved insulin secretory responsiveness of RINm5F insulinoma cells after sodium butyrate treatment at low non-physiological millimolar glucose concentrations can be interpreted as a result of an increased hexokinase-mediated metabolic flux rate through the glycolytic chain.

Blotting, Northern↗