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Mitochondrial network functionality is vital for glucose-stimulated insulin secretion in pancreatic beta cells. Altered mitochondrial dynamics in pancreatic beta cells are thought to trigger the development of type 2 diabetes mellitus. Fission protein 1 (Fis1) might be a key player in this process. Thus, the aim of this study was to investigate mitochondrial morphology in dependence of beta cell function, after knockdown and overexpression of Fis1. We demonstrate that glucose-unresponsive cells with impaired glucose-stimulated insulin secretion (INS1-832/2) showed decreased mitochondrial dynamics compared with glucose-responsive cells (INS1-832/13). Accordingly, mitochondrial morphology visualised using MitoTracker staining differed between the two cell lines. INS1-832/2 cells formed elongated and clustered mitochondria, whereas INS1-832/13 cells showed a homogenous mitochondrial network. Fis1 overexpression using lentiviral transduction significantly improved glucose-stimulated insulin secretion and mitochondrial network homogeneity in glucose-unresponsive cells. Conversely, Fis1 downregulation by shRNA, both in primary mouse beta cells and glucose-responsive INS1-832/13 cells, caused unresponsiveness and significantly greater numbers of elongated mitochondria. Overexpression of FIS1 in primary mouse beta cells indicated an upper limit at which higher FIS1 expression reduced glucose-stimulated insulin secretion. Thus, FIS1 was overexpressed stepwise up to a high concentration in RINm5F cells using the RheoSwitch system. Moderate FIS1 expression improved glucose-stimulated insulin secretion, whereas high expression resulted in loss of glucose responsiveness and in mitochondrial artificial loop structures and clustering. Our data confirm that FIS1 is a key regulator in pancreatic beta cells, because both glucose-stimulated insulin secretion and mitochondrial dynamics were clearly adapted to precise expression levels of this fission protein., (© 2016 Society for Endocrinology.)

Mutations in mitochondrial DNA (mtDNA) cause a variety of pathologic phenotypes. In this study, we used conplastic mouse strains to characterize the impact of a mtDNA mutation in the Atp8 gene on β-cell function, reactive oxygen species (ROS) generation, β-cell mass, and glucose metabolism in response to high-fat diet (HFD). In comparison with B6-mt(AKR) controls, the B6-mt(FVB) strain carries a point mutation of the mtDNA-coded Atp8 gene (ATP synthase), leading to a fragmentated mitochondrial phenotype. Isolated pancreatic islets from 3-month-old B6-mt(FVB) mice showed increased mitochondrial generation of ROS, reduced cellular ATP levels, reduced glucose-induced insulin secretion, higher susceptibility to palmitate stress, and pathological morphology of mitochondria. ROS generation in β-cells was not affected by changes of the ambient glucose concentrations. Feeding a HFD for 3 months resulted in impaired glucose tolerance in B6-mt(FVB) mice but not in B6-mt(AKR) controls. In B6-mt(FVB) animals, glucose intolerance positively correlated with gain of body weight. Serum insulin levels and β-cell mass significantly increased in B6-mt(FVB) mice after a 3-month HFD. The data indicate that the mutation in the Atp8 gene induces mitochondrial dysfunction in β-cells with concomitant impairment of secretory responsiveness. This mitochondrial dysfunction induced a higher susceptibility to metabolic stressors, although this effect appeared not strictly linked to nutrient-induced ROS generation. The Atp8 gene mutation caused mitochondrial dysfunction, apparently stimulating an adaptive increase of β-cell mass in response to HFD, whereas mitochondrial ROS might have had an supportive role.

Significant progress has been made in recent years in the characterisation of the signal pathways of beta cell dysfunction and death in the pathogenesis of type 2 diabetes. Glucolipotoxicity acts as an exogenous factor whereas oxidative stress and endoplasmic reticulum stress may result from the processes of signal recognition and stimulated secretion within the beta cell. The pharmacological stimulation of secretion may thus appear to be a double-edged sword: it counteracts hyperglycaemia, but may do so at the expense of beta cell mass. So, in the long run, insulinotropic glucose-lowering drugs might do more harm than good. However, much of this logic is derived by analogy from the long-held assumption that beta cell hypersecretion imposed by insulin resistance causes the absolute secretion deficit in the later course of type 2 diabetes. In this concept the beta cell has a secondary role and loss of beta cell mass is necessary for the manifestation of type 2 diabetes. Recent studies have shown that a secretion deficit can exist well before insulin resistance and that major genetic risk factors concern beta cell function. Also, the evidence for a beta cell toxic effect of insulinotropic drugs is currently inconclusive. Assuming that the insulin secretion deficit is of pathogenetic importance in a network with insulin resistance as an aggravating factor, an insulinotropic glucose-lowering drug may do more good than harm if it relieves the beta cell from the stress of glucose overstimulation and does so without inducing hypoglycaemia.

The glucose sensor enzyme glucokinase plays a pivotal role in the regulation of glucose-induced insulin secretion in pancreatic beta-cells. Activation of glucokinase represents a promising concept for the treatment of type 2 diabetes. Therefore, we analyzed the glucokinase activation through its physiological interaction partner, the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) and the resulting effect on glucose metabolism in insulin-producing cells. In RINm5F-GK-PFK-2/FBPase-2 cells stably overexpressing glucokinase plus islet PFK-2/FBPase-2, colocalization between both enzymes as well as elevation of glucokinase activity were significantly increased at a stimulatory glucose concentration of 10 mmol/liter. RINm5F-GK-PFK-2/FBPase-2 cells showed under this culture condition a significant increase in glucose utilization and in the ATP/ADP ratio compared with RINm5F-GK cells, which only overexpress glucokinase. Also glucose-induced insulin secretion was elevated in RINm5F-GK-PFK-2/FBPase-2 cells in comparison to RINm5F-GK cells. Furthermore, pyruvate accumulation and lactate production in RINm5F-GK-PFK-2/FBPase-2 cells were significantly lower at both 10 and 30 mmol/liter glucose than in RINm5F-GK and RINm5F cells. The significant improvement of glucose metabolism after PFK-2/FBPase-2 overexpression is apparently not exclusively the result of high glucokinase enzyme activity. Stabilization of the closed glucokinase conformation by PFK-2/FBPase-2 may not only activate the enzyme but also improve metabolic channeling in beta-cells.

Using the metabolic stimulus-secretion coupling of pancreatic beta cells as an example, this review illustrates how new strategies in the treatment of type 2 diabetes mellitus can be developed from the results of basic research. Metabolic stimulus-secretion coupling presupposes the metabolizing of those stimuli of insulin secretion that have the properties of nutritional substances. Changes in the ATP/ADP ratio within the beta cells will then trigger the release of insulin granules from them. Glucokinase, a glucose phosphorylating enzyme, functions as a metabolic glucose sensor, which couples changes in physiological glucose concentration in the pancreatic beta cells and in the liver to the intermediary metabolism, i.e. glycolysis, the citrate cycle and respiratory-chain phosphorylation. In this way insulin secretion and hepatic metabolism are positively influenced. Several pharmaceutical companies (Roche, Merck, Astra-Zeneca, Lilly) have recently developed first examples of glucokinase-activating compounds and demonstrated in animal models their efficacy in the treatment of type 2 diabetes mellitus. These glucokinase activators prevent glucokinase from changing into a catalytically inactive structure. They also increase glucose affinity of the enzyme and stabilize a catalytically active form of glucokinase proteins. In this way glucokinase activators increase glucose-induced insulin secretion and inhibit hepatic glucogenesis. Glucokinase activators are an interesting innovation in the future treatment of type 2 diabetes, because their action on beta cells and the liver is caused by changes in blood glucose concentration.

Aims/hypothesis: Free radicals generated in mitochondria play a crucial role in the toxic effects of cytokines upon insulin-producing cells. This study therefore investigated the role of manganese superoxide dismutase (MnSOD) in cytokine-mediated toxicity in insulin-producing cells., Methods: MnSOD was either stably overexpressed (MnSODsense) or stably suppressed (MnSODantisense) in insulin-producing RINm5F cells. Cell viability was quantified after incubation with different chemical reactive oxygen species (ROS) generators and with cytokines (IL-1beta alone or a mixture of IL-1beta, TNF-alpha and IFN-gamma). Additionally, cell proliferation and endogenous MnSOD protein expression were determined after exposure to cytokines., Results: After incubation with hydrogen peroxide (H(2)O(2)) or hypoxanthine/xanthine oxidase no significant differences were observed in viability between control and MnSODsense or MnSODantisense clones. MnSOD overexpression reduced the viability of MnSODsense cells after exposure to the intracellular ROS generator menadione compared with control and MnSODantisense cells. MnSODsense cells also showed the highest susceptibility to cytokine toxicity with more than 75% loss of viability and a significant reduction of the proliferation rate after 72 h of incubation with a cytokine mixture. In comparison with control cells (67% viability loss), the reduction of viability in MnSODantisense cells was lower (50%), indicating a sensitising role of MnSOD in the progression of cytokine toxicity. The cell proliferation rate decreased in parallel to the reduction of cell viability. The MnSOD expression level after exposure to cytokines was also significantly lower in MnSODantisense cells than in control or MnSODsense cells., Conclusions/interpretation: The increase of the mitochondrial imbalance between the superoxide- and the H(2)O(2)-inactivating enzyme activities corresponds with a greater susceptibility to cytokines. Thus optimal antioxidative strategies to protect insulin-producing cells against cytokine toxicity may comprise a combined overexpression of H(2)O(2)-inactivating enzymes or suppression of MnSOD activity.

The association of glucokinase with insulin secretory granules has been shown by cell microscopy techniques. We used MIN6 insulin-secretory cells and organelle fractionation to determine the effects of glucose on the subcellular distribution of glucokinase. After permeabilization with digitonin, 50% of total glucokinase remained bound intracellularly, while 30% was associated with the 13,000g particulate fraction. After density gradient fractionation of the organelles, immunoreactive glucokinase was distributed approximately equally between dense insulin granules and low-density organelles that cofractionate with mitochondria. Although MIN6 cells show glucose-responsive insulin secretion, glucokinase association with the granules and low-density organelles was not affected by glucose. Subfractionation of the insulin granule components by hypotonic lysis followed by sucrose gradient centrifugation showed that glucokinase colocalized with the granule membrane marker phogrin and not with insulin. PFK2 (6-phosphofructo-2-kinase-2/fructose-2,6-bisphosphatase)/FDPase-2, a glucokinase-binding protein, and glyceraldehyde phosphate dehydrogenase, which has been implicated in granule fusion, also colocalized with glucokinase after hypotonic lysis or detergent extaction of the granules. The results suggest that glucokinase is an integral component of the granule and does not translocate during glucose stimulation.

Aims/hypothesis: Cytokines are important humoral mediators of beta cell destruction in autoimmune diabetes. The aim of this study was to identify novel cytokine-induced genes in insulin-producing RINm5F cells, which may contribute to beta cell death or survival., Methods: A global gene expression profile in cytokine-exposed insulin-producing RINm5F cells was achieved by automated restriction fragment differential display PCR. The expression of selected candidate genes was confirmed by real-time RT-PCR analysis., Results: Exposure of RINm5F cells to IL-1beta or to a cytokine mixture (IL-1beta, TNF-alpha, IFN-gamma) for 6 h resulted in the differential expression of a functional gene cluster. Apart from the well-known up-regulation of the cytokine-responsive genes iNOS, NF-kappaB, MnSOD and Hsp70, several genes that belong to the functional cluster of the endocytotic pathway were identified. These endocytotic genes comprised: clathrin, megalin, synaptotagmin and calcineurin, which were up-regulated by IL-1beta or the cytokine mixture. In contrast, the expression of the calcineurin inhibitor CAIN and of the GDP/GTP exchange protein Rab3 was down-regulated by cytokines. Other up-regulated cytokine-responsive genes were: agrin, murine adherent macrophage protein mRNA ( MAMA) and transport-associated protein ( TAP1/MTP), whereas the plasma membrane calcium ATPase ( PMCA) 2 and PMCA 3 genes were down-regulated by cytokines., Conclusions/interpretation: Our results indicate that genes of the endocytotic pathway are regulated by pro-inflammatory cytokines. This might affect the density of cytokine receptors at the beta cell surface and concomitantly the sensitivity of the cells to cytokine toxicity. A better understanding of the functional cross-talk between endocytotic and cytokine signalling pathways could further the development of novel strategies to protect pancreatic beta cells against toxic effects of pro-inflammatory cytokines.

Insulin-producing cells show very low activity levels of the cytoprotective enzymes catalase, glutathione peroxidase, and superoxide dismutase. This weak antioxidative defense status has been considered a major feature of the poor resistance against oxidative stress. Therefore, we analyzed the protective effect of a combined overexpression of Cu,ZnSOD or MnSOD together with different levels of catalase. Catalase alone was able to increase the resistance of transfected RINm5F insulin-producing tissue culture cells against H(2)O(2) and HX/XO, but no protection was seen in the case of menadione. In combination with an increase of the MnSOD or Cu,ZnSOD expression, the protective action of catalase overexpression could be further increased and extended to the toxicity of menadione. Thus, optimal protection of insulin-producing cells against oxidative stress-mediated toxicity requires a combined overexpression of both superoxide- and hydrogen peroxide-inactivating enzymes. This treatment can compensate for the constitutively low level of antioxidant enzyme expression in insulin-producing cells and may provide an improved protection in situations of free radical-mediated destruction of pancreatic beta cells in the process of autoimmune diabetes development.

Proinflammatory cytokines (interleukin-1beta [IL-1beta], tumor necrosis factor-alpha [TNF-alpha], and gamma-interferon [IFN-gamma]) initiate a variety of signal cascades in pancreatic beta-cells that affect the expression level of genes involved in both the destruction and the protection of the beta-cell. The generation of nitric oxide (NO) via the inducible NO synthase (iNOS) and oxygen free radicals play a key role in cytokine-mediated beta-cell destruction. Within these signal cascades, the activation of the transcription factor nuclear factor-kappaB (NF-kappaB) is crucial, and many cytokine-sensitive genes contain binding sites for this transcription factor in their promoter regions. The aim of this study was to characterize the cytokine-mediated activation of NF-kappaB and the subsequent expression of iNOS protein in insulin-producing RINm5F cells with an improved antioxidant defense status by overexpression of the cytoprotective enzymes catalase (Cat), glutathione peroxidase (Gpx), and the cytoplasmic Cu/Zn superoxide dismutase (Cu/ZnSOD). RINm5F cells with diverse mitochondrial antioxidative defense status were generated by stable overexpression of MnSOD constructs in sense (MnSOD sense) and antisense orientation (MnSOD antisense). Cytokine-induced (IL-1beta or cytokine mix consisting of IL-1beta + TNF-alpha + IFN-gamma) activation of NF-kappaB in RINm5F cells was reduced by >80% through overexpression of MnSOD. The activity of the iNOS promoter remained at basal levels in cytokine-stimulated MnSOD sense cells. In contrast, the suppression of MnSOD gene expression in cytokine-stimulated MnSOD antisense cells resulted in a threefold higher activation of NF-kappaB and a twofold higher activation of the iNOS promoter as compared with control cells. The iNOS protein expression was significantly reduced after a 6- and 8-h cytokine incubation of MnSOD sense cells. The low activity level of MnSOD in RINm5F MnSOD antisense cells increased the iNOS protein expression in particular during the early phase of cytokine-mediated toxicity. Cat, Gpx, and the cytoplasmic Cu/ZnSOD did not affect the activation of NF-kappaB and the iNOS promoter. In conclusion, the overexpression of MnSOD, which inactivates specifically mitochondrially derived oxygen free radicals, significantly reduced the activation of NF-kappaB in insulin-producing cells. As a consequence of this protective effect in the early cytokine signaling pathways, the induction of iNOS, an important event in the beta-cell destruction process, was also significantly reduced. The results provide evidence that mitochondrially derived reactive oxygen species (ROS) play a critical role in the activation of the cytokine-sensitive transcription factor NF-kappaB. Overexpression of MnSOD may thus be beneficial for beta-cell survival through suppression of oxygen free radical formation, prevention of NF-kappaB activation, and iNOS expression.

The Psammomys obesus (sand rat) is a well-established model of nutritionally induced non-insulin-dependent type-2 diabetes. When fed a high-energy (HE) diet, the diabetes-prone animals develop hyperinsulinaemia and hyperglycaemia. Within 1 week, all animals become hyperinsulinaemic. However, a loss of immunostaining for insulin as well as for the GLUT2 glucose transporter in the plasma membrane and the glucokinase in the cytoplasm of the pancreatic beta cells became evident only when the animals subsequently developed hyperglycaemia. After 1 week of HE diet feeding, the pancreatic beta-cell volume was reduced by one-third in hyperglycaemic Psammomys. Insulin immunostaining as well as GLUT2 glucose transporter immunostaining in the plasma membrane and glucokinase immunostaining in the cytoplasm were reduced by more than 50%. After 3 weeks of HE diet feeding, all changes observed after 1 week were even more pronounced, with reductions in the range of 70-95%. The reduction of the total beta-cell volume of the pancreas due to beta-cell death and the diminution of insulin content of the remaining beta cells in the islets during the HE diet feeding was accompanied by a parallel fall of the pancreas insulin content. For all changes observed, there was a significant correlation with the increase of the blood glucose concentration (r>0.9) but not with the increase of the plasma insulin concentration (r>0.2). Thus, increasing glycaemia appears to be the factor responsible for the deterioration of the pancreatic beta-cell function and the resulting loss of the insulin secretory capacity in Psammomys. The final result of this development is an irreversible diabetic state due to the feeding of the HE diet.

The role of lactate dehydrogenase (LDH) in the generation of the metabolic signal for insulin secretion was studied after stable overexpression in INS-1 and RINm5F insulin-producing cells. INS-1 cells with a 25-fold overexpression of LDH-A, the highest level achieved, showed a 20-30% decrease in the glucose oxidation rate at glucose concentrations above 5 mM when compared with control cells, whereas values were unchanged at lower glucose concentrations. Lactate release increased in parallel with a decrease in the glucose oxidation rate. However, the INS-1 cell glucose-induced insulin secretory response, together with the rate of glucose utilization, were not significantly affected by LDH-A overexpression. Despite 3-fold overexpression of LDH-A in glucose-unresponsive RINm5F cells, there was no change in insulin secretion, glucose metabolism or lactate production in these cells. Exogenously added pyruvate and lactate potentiated glucose-stimulated insulin secretion in INS-1 cells, an effect that was abolished after LDH-A overexpression. Both compounds significantly decreased glucose oxidation rates in control cells. After overexpression of LDH-A in INS-1 cells, the effects of pyruvate and lactate on glucose oxidation were diminished. On the other hand, after LDH-A overexpression, both glycolytic metabolites decreased the glucose utilization rate at 5 mM glucose. The present data suggest that the level of LDH expression in insulin-secreting cells is critical for correct channelling of pyruvate towards mitochondrial metabolism. Interestingly, glucokinase-mediated glycolytic flux was decreased after LDH-A overexpression. Thus preferential channelling of glucose towards aerobic metabolism by glucokinase may be determined, at least in part, by the low level of constitutive expression of LDH-A in pancreatic beta-cells. In conclusion, the level of LDH expression in insulin-secreting cells is an important determinant of the physiological insulin-secretory capacity, and also determines how pyruvate and lactate affect insulin secretion.

Nitric oxide (NO) and reactive oxygen species (ROS) are crucial elements in cytokine-mediated beta-cell destruction. In insulin-producing RINm5F cells, overexpression of cytoprotective enzymes provides significant protection against the synergistic toxicity of NO and ROS. We therefore examined whether overexpression of catalase (Cat), glutathione peroxidase (Gpx), and Cu/Zn superoxide dismutase (SOD) can provide protection for bioengineered RINm5F cells against cytokine-mediated toxicity. A 72-h exposure of RINm5F control cells to interleukin-1beta (IL-1beta) alone or a combination of IL-1beta, tumor necrosis factor-alpha, and gamma-interferon resulted in a time- and concentration-dependent decrease of cell viability in the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cytotoxicity assay. Although IL-1beta alone caused only a moderate reduction of viability in the range of 25%, the cytokine mixture induced a significant loss of viability of >75%. This increased toxicity of the cytokine mixture compared with that of IL-1beta alone could be explained by a higher rate of NO generation within the early 24-48 h incubation period that would favor the toxic synergism of NO and oxygen free radicals. Overexpression of Cat, Gpx, and Cu/Zn SOD protected against toxicity of the cytokine mixture but not against that of IL-1beta alone. The reduction of cytokine-mediated toxicity was evident also because of an increased proliferation rate and a drastic decrease in the cell death rate. The improved antioxidant defense status did not prevent the activation of iNOS after cytokine exposure. However, RINm5F cells overexpressing cytoprotective enzymes showed a significantly lower level of ROS-damaged protein residues. Thus, protection through Cat, Gpx, and Cu/Zn SOD overexpression was apparently because of an inactivation of ROS generated in the signal cascades of the cytokines. Overexpression of cytoprotective enzymes thus represents a feasible strategy to protect insulin-producing cells against cytokine-mediated cytotoxicity.

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.

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.

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.

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.

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.

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.

Aims/hypothesis. Thyroid hormones reduce glucose tolerance in animals and humans. This effect is accompanied by a reduction in the beta-cell volume of the pancreas. Methods. We studied the underlying mechanism using terminal UTP nick end labelling (TUNEL) and caspase-3 to analyse apoptosis and BrdU labelling of beta-cell proliferation. Results. The reason for the reduction of the beta-cell volume of the pancreas after thyroxine treatment is apparently an increased rate of beta-cell apoptosis by an increase of TUNEL and caspase-3 positive rat beta cells. In parallel, thyroxine treatment increased the rate of apoptosis in rat pancreatic ductal cells which are considered to contribute to the pool of stem cells from which insulin-producing beta cells originate. The effects of thyroid hormone treatment are reversible through an increase of the beta-cell replication rate when thyroxine is withdrawn as documented by an increase of the BrdU labelling index. Conclusion/interpretation. An increased rate of beta-cell death due to apoptosis causes a decrease of insulin content and glucose-induced insulin secretion from the pancreas in hyperthyroidism. The resulting reduction of beta cells in the pancreas can provide an explanation for the decrease of glucose tolerance in hyperthyroidism. [ABSTRACT FROM AUTHOR]

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] [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]