ABSTRACT

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.

Oxidative folding of nascent proteins in the endoplasmic reticulum (ER), catalysed by one or more members of the protein disulfide isomerase family and the sulfhydryl oxidase ER oxidoreductin 1 (ERO1), is accompanied by generation of hydrogen peroxide (H₂O₂). Because of the high rate of insulin biosynthesis and the low expression of H₂O₂-inactivating enzymes in pancreatic β cells, it has been proposed that the luminal H₂O₂ concentration might be very high. As the role of this H₂O₂ in ER stress and proinsulin processing is still unsolved, an ER-targeted and luminal-active catalase variant, ER-Catalase N244, was expressed in insulin-secreting INS-1E cells. In these cells, the influence of ER-specific H₂O₂ removal on cytokine-mediated cytotoxicity and ER stress, insulin gene expression, insulin content and secretion was analysed. The expression of ER-Catalase N244 reduced the toxicity of exogenously added H₂O₂ significantly with a threefold increase of the EC₅₀ value for H₂O₂. However, the expression of cytokine-induced ER stress genes and viability after incubation with β cell toxic cytokines (IL1β alone or together with TNFα+IFNγ) was not affected by ER-Catalase N244. In control and ER-Catalase N244 expressing cells, insulin secretion and proinsulin content was identical, while removal of luminal H₂O₂ reduced insulin gene expression and insulin content in ER-Catalase N244 expressing cells. These data show that ER-Catalase N244 reduced H₂O₂ toxicity but did not provide protection against pro-inflammatory cytokine-mediated toxicity and ER stress. Insulin secretion was not affected by decreasing H₂O₂ in the ER in spite of a reduced insulin transcription and processing.

Free intracellular ferrous iron (Fe^{2 +}) is essential for the generation of the extremely toxic hydroxyl radicals, which contribute to β-cell destruction by cytokines. Therefore the expression of the different divalent metal transporter 1 (Dmt1) isoforms and ferritin (Ft) subunits, responsible for iron import and chelation, was analyzed under pro-inflammatory conditions (IL1β alone or together with TNFα+IFNγ). The Dmt1 isoforms (1A/1B and +IRE/−IRE) and the total Dmt1 expression in insulin-producing cells (RINm5F and INS-1E), in primary rat islets and, for comparison, in the neuroendocrine PC12 cell line were quantified by qRT-PCR. In addition, the expression of the light (L-Ft) and heavy Ft (H-Ft) subunits and the mitochondrial Ft isoform (Mtft) in insulin-producing cells under control conditions and after cytokine treatment was estimated. The 1B isoform was the predominant Dmt1 mRNA in all insulin-producing cells, accounting for almost 100% of the 1A/1B isoform expression. For the IRE variants, +IRE expression was higher than −IRE expression. Pro-inflammatory cytokines accelerated the expression of Dmt1 isoforms significantly with an overall 2.5- to 3-fold increase in the total Dmt1 expression. In contrast, the expression of the iron-buffering ferritin subunits L- and H-Ft was unaffected by IL1β and only slightly induced by the cytokine mixture. Mtft expression was also not increased. Dmt1 expression was significantly elevated through pro-inflammatory cytokines, whereas Ft expression was marginally increased. This imbalance between the increased iron transport capacity and the almost unaffected iron storage capacity can foster cytokine-mediated formation of hydroxyl radicals and thus pro-inflammatory cytokine toxicity through elevated free iron concentrations.

Pancreatic beta-cells have low activities of the antioxidant enzyme catalase. Nitric oxide interacts with the haem group of catalase inhibiting its activity. We have studied the activity of catalase in beta-cells under conditions mimicking prediabetes and in which nitric oxide is generated from cytokine treatment in vitro. We also studied whether there is regulation of catalase enzyme activity by nitric oxide at the protein or gene expression level. RINm5F insulin-producing cells, treated for 24 h with cytokines, showed increased medium nitrite production (17+/-2.2 vs 0.3+/-0.2 pmol/ micro g protein) and significantly decreased cellular catalase activity (42.4+/-4.5%) compared with control cells. A similar reduction was seen in catalase-overexpressing RIN-CAT cells and in rat or human pancreatic islets of Langerhans. Catalase activity was also suppressed by the long-acting nitric oxide donor diethylenetriamine/nitric oxide adduct (Deta-NO) and this inhibition was reversible. The inhibition of catalase activity by cytokines in RINm5F cells was significantly reversed by the addition of the nitric oxide synthase 2 (NOS2) inhibitors nitro monomethylarginine or N-(3-(aminomethyl)benzyl)acetamidine (1400W). Protein expression was found to be unchanged in cytokine- or Deta-NO-treated RINm5F cells, while mRNA expression was marginally increased. We have shown that inhibition of catalase activity by cytokines is nitric oxide dependent and propose that this inhibition may confer increased susceptibility to cytokine- or nitric oxide-induced cell killing.