Matthias Elsner

Relative importance of transport and alkylation for pancreatic beta-cell toxicity of streptozotocin

Aims/hypothesis. The role of selective uptake and alkylation in the diabetogenic action of streptozotocin was investigated in bioengineered RINm5F insulin-producing cells, with different expression levels of the glucose transporter GLUT2, by comparing the toxicity of streptozotocin with that of four chemically related alkylating compounds, N-methyl-N-nitrosourea (MNU), N-ethyl-N nitrosourea (ENU), methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS). Methods. GLUT2 expressing RINm5F cells were generated through stable transfection of the rat glucose transporter GLUT2 cDNA under the control of the cytomegalovirus promoter in the pcDNA3 vector. Viability of the cells was determined using a microtitre plate-based 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Results. Cells expressing the glucose transporter GLUT2 were much more susceptible to streptozotocin toxicity than control cells due to the uptake of streptozotocin by this specific glucose transporter. In contrast, the GLUT2 expression had no effect upon the toxicity of MNU, ENU, MMS or EMS. Although the latter substances are, like streptozotocin, cytotoxic through their ability to cause DNA alkylation, they are not diabetogenic because they are not taken up through the glucose transporter GLUT2. Conclusion/interpretation. Our results are consistent with the central importance of selective uptake and alkylating activity in the mechanism of streptozotocin diabetogenicity. Alkylation of DNA leads to necrosis of pancreatic beta cells and thus to a state of insulin-dependent diabetes mellitus, well-known as streptozotocin diabetes in experimental diabetes research. [Diabetologia (2000) 43: 1528–1533]

Peroxisome generated hydrogen peroxide as important mediator of lipotoxicity in insulin-producing cells

OBJECTIVE Type 2 diabetes is a complex disease that is accompanied by elevated levels of nonesterified fatty acids (NEFAs), which contribute to β-cell dysfunction and β-cell loss, referred to as lipotoxicity. Experimental evidence suggests that oxidative stress is involved in lipotoxicity. In this study, we analyzed the molecular mechanisms of reactive oxygen species-mediated lipotoxicity in insulin-producing RINm5F cells and INS-1E cells as well as in primary rat islet cells. RESEARCH DESIGN AND METHODS The toxicity of saturated NEFAs with different chain lengths upon insulin-producing cells was determined by MTT and propidium iodide (PI) viability assays. Catalase or superoxide dismutase overexpressing cells were used to analyze the nature and the cellular compartment of reactive oxygen species formation. With the new H2O2-sensitive fluorescent protein HyPer H2O2 formation induced by exposure to palmitic acid was determined. RESULTS Only long-chain (>C14) saturated NEFAs were toxic to insulin-producing cells. Overexpression of catalase in the peroxisomes and in the cytosol, but not in the mitochondria, significantly reduced H2O2 formation and protected the cells against palmitic acid-induced toxicity. With the HyPer protein, H2O2 generation was directly detectable in the peroxisomes of RINm5F and INS-1E insulin-producing cells as well as in primary rat islet cells. CONCLUSIONS The results demonstrate that H2O2 formation in the peroxisomes rather than in the mitochondria are responsible for NEFA-induced toxicity. Therefore, we propose a new concept of fatty acid-induced β-cell lipotoxicity mediated via reactive oxygen species formation through peroxisomal β- oxidation.

Role of metabolically generated reactive oxygen species for lipotoxicity in pancreatic β-cells

Chronically elevated concentrations of non‐esterified fatty acids (NEFAs) in type 2 diabetes may be involved in β‐cell dysfunction and apoptosis. It has been shown that long‐chain saturated NEFAs exhibit a strong cytotoxic effect upon insulin‐producing cells, while short‐chain as well as unsaturated NEFAs are well tolerated. Moreover, long‐chain unsaturated NEFAs counteract the toxicity of palmitic acid. Reactive oxygen species (ROS) formation and gene expression analyses together with viability assays in different β‐cell lines showed that the G‐protein‐coupled receptors 40 and 120 do not mediate lipotoxicity. This is independent from the role, which these receptors, specifically GPR40, play in the potentiation of glucose‐induced insulin secretion by saturated and unsaturated long‐chain NEFAs. Long‐chain NEFAs are not only metabolized in the mitochondria but also in peroxisomes. In contrast to mitochondrial β‐oxidation, the acyl‐coenzyme A (CoA) oxidases in the peroxisomes form hydrogen peroxide and not reducing equivalents. As β‐cells almost completely lack catalase, they are exceptionally vulnerable to hydrogen peroxide generated in peroxisomes. ROS generation in the respiratory chain is less important because overexpression of catalase and superoxide dismutase in the mitochondria do not provide protection. Thus, peroxisomally generated hydrogen peroxide is the likely ROS that causes pancreatic β‐cell dysfunction and ultimately β‐cell death.

The LEW.1AR1/Ztm-iddm rat: a new model of spontaneous insulin-dependent diabetes mellitus.

Abstract.Aims/hypothesis: We describe a new Type I (insulin-dependent) diabetes mellitus rat model (LEW.1AR1/Ztm-iddm) which arose through a spontaneous mutation in a congenic Lewis rat strain with a defined MHC haplotype (RT1.Aa B/Du Cu).Methods: The development of diabetes was characterised using biochemical, immunological and morphological methods. Results: Diabetes appeared in the rats with an incidence of 20 % without major sex preference at 58 ± 2 days. The disease was characterised by hyperglycaemia, glycosuria, ketonuria and polyuria. In peripheral blood, the proportion of T lymphocytes was in the normal range expressing the RT6.1 differentiation antigen. Islets were heavily infiltrated with B and T lymphocytes, macrophages and NK cells with beta cells rapidly destroyed through apoptosis in areas of insulitis. Conclusion/interpretation: This Type I diabetic rat develops a spontaneous insulin-dependent autoimmune diabetes through beta cell apoptosis. It could prove to be a valuable new animal model for clarifying the mechanisms involved in the development of autoimmune diabetes. [Diabetologia (2001) 44: 1189–1196]

Importance of the GLUT2 glucose transporter for pancreatic beta cell toxicity of alloxan

Abstract Aims/hypothesis. We investigated the importance of the low affinity GLUT2 glucose transporter in the diabetogenic action of alloxan in bioengineered RINm5F insulin-producing cells with different expressions of the transporter. Methods. GLUT2 glucose transporter expressing RINm5F cells were generated through stable transfection of the rat GLUT2 cDNA under the control of the cytomegalovirus promoter in the pcDNA3 vector. Viability of the cells was determined using a microtitre plate-based 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Results. Cells expressing the GLUT2 transporter were susceptible to alloxan toxicity due to the uptake of alloxan by this specific glucose transporter isoform. The extent of the toxicity of alloxan was dependent upon the GLUT2 protein expression in the cells.The lipophilic alloxan derivative, butylalloxan, was toxic also to non-transfected control cells. Expression of the GLUT2 glucose transporter caused only a marginal increase in the toxicity of this substance. Butylalloxan, unlike alloxan itself, is not diabetogenic in vivo although, like the latter substance, it is beta-cell toxic in vitro through its ability to generate free radicals during redox cycling with glutathione. Conclusion/interpretation. Our results are consistent with the central importance of selective uptake of alloxan through the low affinity GLUT2 glucose transporter for the pancreatic beta-cell toxicity and diabetogenicity of this substance. Redox cycling and the subsequent generation of oxygen free radicals leads to necrosis of pancreatic beta cells and thus to a state of insulin-dependent diabetes mellitus, well-known as alloxan diabetes in experimental diabetes research.

Interaction between pro-inflammatory and anti-inflammatory cytokines in insulin-producing cells

Pro-inflammatory cytokines cause beta-cell dysfunction and death. The aim of this study was to investigate the interactions between different pro- and anti-inflammatory cytokines and their effects on apoptotic beta-cell death pathways. Insulin-producing RINm5F cells were exposed to different combinations of cytokines. Gene expression analyses of manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) were performed by real-time RT-PCR. Cell viability was measured by the MTT assay, NFkappaB activation using a SEAP reporter gene assay, protein expression by western blotting and caspase-3 activity using the DEVD cleavage method. IL-1beta, tumour necrosis factor alpha (TNFalpha) and a combination of all three pro-inflammatory cytokines increased while IFNgamma alone did not affect NFkappaB activity and iNOS gene and protein expression. Interestingly, the anti-inflammatory cytokines IL-4, IL-13 and IL-10 decreased IL-1beta-stimulated NFkappaB activation and iNOS expression. IL-1beta, TNFalpha and the pro-inflammatory cytokine combination also increased MnSOD gene and protein expression. But IL-4, IL-13 and IL-10 did not affect MnSOD expression and did not modulate IL-1beta-stimulated MnSOD expression. Caspase-3 activity was increased by IL-1beta and the pro-inflammatory cytokine combination, and to a lesser extent by TNFalpha. In contrast, IFNgamma had no effect on caspase-3 activity. IL-4, IL-13 and IL-10 decreased caspase-3 activity and increased viability of insulin-producing cells treated with pro-inflammatory cytokines. The anti-inflammatory cytokines counteracted the cytotoxic effects of pro-inflammatory cytokines in insulin-producing cells. This was achieved through the reduction of nitrosative stress. Thus, a balance between the anti-inflammatory and the pro-inflammatory cytokines is of crucial importance for the prevention of pancreatic beta-cell destruction.

Engineering of a Glucose-Responsive Surrogate Cell for Insulin Replacement Therapy of Experimental Insulin-Dependent Diabetes

Glucose responsiveness in the millimolar concentration range is a crucial requirement of a surrogate pancreatic beta cell for insulin replacement therapy of insulin-dependent diabetes. Novel insulin-secreting GK cell clones with millimolar glucose responsiveness were generated from an early-passage glucose-unresponsive RINm5F cell line. This line expressed constitutively both the K(ATP) channel and the GLUT2 glucose transporter; but it had a relative lack of glucokinase. Through overexpression of glucokinase, however, it was possible to generate glucose-responsive clones with a glucokinase-to-hexokinase ratio comparable to that of a normal pancreatic beta cell. This aim, on the other hand, was not achieved through overexpression of the GLUT2 glucose transporter. Raising the expression level of this glucose transporter into the range of rat liver, without correcting the glucokinase-to-hexokinase enzyme ratio, did not render the cells glucose responsive. These glucokinase-overexpressing RINm5F cells also stably maintained their molecular and insulin secretory characteristics in vivo. After implantation into streptozotocin diabetic immunodeficient rats, glucokinase-overexpressing cells retained their insulin responsiveness to physiological glucose stimulation under in vivo conditions. These cells represent a notable step toward the future bioengineering of a surrogate beta cell for insulin replacement therapy in insulin-dependent diabetes mellitus.

Mechanism underlying resistance of human pancreatic beta cells against toxicity of streptozotocin and alloxan

To the Editor: The diabetogenic agents, streptozotocin and alloxan induce insulin deficiency due to their selective pancreatic beta-cell toxicity [1]. However, there are significant species differences in the diabetogenicity of streptozotocin and alloxan [1]. While rodents are particularly prone to the diabetogenic action of these two diabetogens, humans are considered to be resistant [1]. Human pancreatic beta cells, in contrast to rodent beta cells, have been shown to be resistant to the toxic action of streptozotocin and alloxan in vitro [2, 3, 4]. RINm5F insulin-producing cells which do not constitutively express the GLUT2 glucose transporter are virtually resistant to the toxic action of streptozotocin and alloxan [5, 6]. Expression of the rat GLUT2 glucose transporter isoform rendered these cells sensitive and the toxicity of both streptozotocin and alloxan increased in parallel with the increase of the level of GLUT2 glucose transporter expression in this rat insulin-producing tissue culture cell line [5, 6]. Recently, it has been reported that after transplantation of human pancreatic islets into nude mice, beta cells are not damaged even after injection of high doses of streptozotocin [4] or alloxan [2, 3], while the beta cells of rat islets trans-planted concomitantly into the same animal are destroyed [2, 3, 4]. There are different potential explanations for this observation. Human beta cells could be more resistant to the toxic action of streptozotocin and alloxan either because they express the GLUT2 glucose transporter only to a very low extent [7, 8] (1–2% of the level expression in rat beta cells), or, alternatively, because streptozotocin and alloxan, in contrast to the rat GLUT2 glucose transporter isoform, are not taken up through the human GLUT2 glucose transporter isoform into the intracellular compartment where the toxins exert their cell-death action. We therefore cloned and expressed the human GLUT2 glucose transporter isoform, using a known methodology [5], in RINm5F insulin-producing tissue culture cells which constitutively do not express the GLUT2 glucose transporter isoform [5, 6]. In an experimental protocol using the MTT cytotoxicity assay [5] we could show that the toxicity of both streptozotocin and alloxan increased in dependence upon the human GLUT2 glucose transporter gene expression level in six different RINm5F cell clones (A–F), as measured by quantitative realtime PCR analyses using the Opticon real-time PCR cycler (MJ Research, San Francisco, Calif., USA) (Table 1). PCR was carried out with primer sets specific for human GLUT2 using SYBRGreen (Biozym, Hessisch Oldendorf, Germany) for fluorescence detection. There was a significant correlation (p<0.01; F-test) both for streptozotocin (r=0.987) and alloxan (r=0.948) between the decrease of the half maximally effective concentrations (EC50) for the toxins, at which 50% of the cells died, and the increase of the human GLUT2 glucose transporter expression level (Table 1). A calculation of the number of human GLUT2 mRNA molecules which must be expressed in insulin-producing cells in order to reduce the EC50 value for toxicity by 1 mmol/l showed that less GLUT2 mRNA molecules are required for this purpose in the case of the toxicity of alloxan (3331±228) than in the case of streptozotocin (4670±164). Thus, it can be concluded that the human GLUT2 glucose transporter shows an apparently greater affinity for alloxan than for streptozotocin. For purposes of comparison, we did a comparable calculation for the rat GLUT2 transporter using data which we published earlier [5, 6]. The result shows that also in the case of the rat GLUT2 transporter, alloxan (1213±96) requires less GLUT2 mRNA molecule expression than streptozotocin (3416±358) showing that not only the human GLUT2 glucose transporter but also the rat GLUT2 glucose transporter has a greater affinity for alloxan than for streptozotocin. In addition, it is evident that the expression of fewer rat GLUT2 mRNA molecules is required both in the case of alloxan (factor 2.8) and of streptozotocin (factor 1.4) to reduce the EC50 value for their toxicity by 1 mmol/l than in the case of the expression of the human GLUT2 glucose transporter. The results show that it is apparently the very low level of constitutive GLUT2 glucose transporter expression in the human beta cell [7, 8] rather than the inability of the human GLUT2 glucose transporter isoform to provide uptake of streptozotocin and alloxan into the intracellular compartment (Table 1) which is responsible for the extraordinarily high resistance of humans against the diabetogenic action of streptozotocin and alloxan. However, even if the human GLUT2 glucose transporter isoform would be more abundant in human pancreatic beta cells, the lower capacity for uptake of the toxins through the human GLUT2 glucose transporter isoform as compared to the rat transporter isoform would limit DOI 10.1007/s00125-003-1241-2 Received: 30 July 2003 / Revised: 11 September 2003 Published online: 12 November 2003

Reversal of Diabetes Through Gene Therapy of Diabetic Rats by Hepatic Insulin Expression via Lentiviral Transduction

Due to shortage of donor tissue a cure for type 1 diabetes by pancreas organ or islet transplantation is an option only for very few patients. Gene therapy is an alternative approach to cure the disease. Insulin generation in non-endocrine cells through genetic engineering is a promising therapeutic concept to achieve insulin independence in patients with diabetes. In the present study furin-cleavable human insulin was expressed in the liver of autoimmune-diabetic IDDM rats (LEW.1AR1/Ztm-iddm) and streptozotocin-diabetic rats after portal vein injection of INS-lentivirus. Within 5-7 days after the virus injection of 7 × 10(9) INS-lentiviral particles the blood glucose concentrations were normalized in the treated animals. This glucose lowering effect remained stable for the 1 year observation period. Human C-peptide as a marker for hepatic release of human insulin was in the range of 50-100 pmol/ml serum. Immunofluorescence staining of liver tissue was positive for insulin showing no signs of transdifferentiation into pancreatic β-cells. This study shows that the diabetic state can be efficiently reversed by insulin release from non-endocrine cells through a somatic gene therapy approach.

Antagonism Between Saturated and Unsaturated Fatty Acids in ROS Mediated Lipotoxicity in Rat Insulin-Producing Cells

Background/Aims: Elevated levels of non-esterified fatty acids (NEFAs) are under suspicion to mediate β-cell dysfunction and β-cell loss in type 2 diabetes, a phenomenon known as lipotoxicity. Whereas saturated fatty acids show a strong cytotoxic effect upon insulin-producing cells, unsaturated fatty acids are not toxic and can even prevent toxicity. Experimental evidence suggests that oxidative stress mediates lipotoxicity and there is evidence that the subcellular site of ROS formation is the peroxisome. However, the interaction between unsaturated and saturated NEFAs in this process is unclear. Methods: Toxicity of rat insulin-producing cells after NEFA incubation was measured by MTT and caspase assays. NEFA induced H2O2 formation was quantified by organelle specific expression of the H2O2 specific fluorescence sensor protein HyPer. Results: The saturated NEFA palmitic acid had a significant toxic effect on the viability of rat insulin-producing cells. Unsaturated NEFAs with carbon chain lengths >14 showed, irrespective of the number of double bonds, a pronounced protection against palmitic acid induced toxicity. Palmitic acid induced H2O2 formation in the peroxisomes of insulin-producing cells. Oleic acid incubation led to lipid droplet formation, but in contrast to palmitic acid induced neither an ER stress response nor peroxisomal H2O2 generation. Furthermore, oleic acid prevented palmitic acid induced H2O2 production in the peroxisomes. Conclusion: Thus unsaturated NEFAs prevent deleterious hydrogen peroxide generation during peroxisomal β-oxidation of long-chain saturated NEFAs in rat insulin-producing cells.