High glucose and hydrogen peroxide increase c-Myc and haeme-oxygenase 1 mRNA levels in rat pancreatic islets without activating NFκB
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Hyperglycaemia and the pro-inflammatory cytokine IL-1β induce similar alterations of beta cell gene expression, including up-regulation of c-Myc and haeme-oxygenase 1. These effects of hyperglycaemia may result from nuclear factor-kappa B (NFκB) activation by oxidative stress. To test this hypothesis, we compared the effects of IL-1β, high glucose, and hydrogen peroxide, on NFκB DNA binding activity and target gene mRNA levels in cultured rat islets.
Rat islets were pre-cultured for 1 week in serum-free RPMI medium containing 10 mmol/l glucose, and further cultured in glucose concentrations of 5–30 mmol/l plus various test substances. Islet NFκB activity was measured by ELISA and gene mRNA expression was measured by RT-PCR.
IL-1β consistently increased islet NFκB activity and c-Myc, haeme-oxygenase 1, inducible nitric oxide synthase (iNOS), Fas, and inhibitor of NFκB alpha (IκBα) mRNA levels. In comparison, 1- to 7-day culture in 30 mmol/l instead of 10 mmol/l glucose stimulated islet c-Myc and haeme-oxygenase 1 expression without affecting NFκB activity or iNOS and IκBα mRNA levels. Fas mRNA levels only increased after 1 week in 30 mmol/l glucose. Overnight exposure to hydrogen peroxide mimicked the effects of 30 mmol/l glucose on haeme-oxygenase 1 and c-Myc mRNA levels without activating NFκB. On the other hand, the antioxidant N-acetyl-l-cysteine inhibited the stimulation of haeme-oxygenase 1 and c-Myc expression by 30 mmol/l glucose and/or hydrogen peroxide.
In contrast to IL-1β, high glucose and hydrogen peroxide do not activate NFκB in cultured rat islets. It is suggested that the stimulation of islet c-Myc and haeme-oxygenase 1 expression by 30 mmol/l glucose results from activation of a distinct, probably oxidative-stress-dependent signalling pathway.
KeywordsAntioxidant Glucose toxicity NFκB Oxidative stress Pancreatic beta cell
Glucose-stimulated insulin secretion
Inhibitor of κB alpha
inducible nitric oxide synthase
Nuclear factor of kappa B chain
Protein kinase C β2
TATA-box binding protein
Type 2 diabetes results from the combination of insulin resistance and defective glucose-stimulated insulin secretion (GSIS) [1, 2]. This beta cell dysfunction seems to result from a moderate reduction in beta cell mass [3, 4] and profound functional alterations of the remaining beta cells [5, 6]. In contrast, type 1 diabetes is characterised by selective immune destruction of beta cells and subsequent lack of insulin secretion .
Chronic hyperglycaemia induces phenotypic alterations of rodent beta cells, including GSIS abnormalities, reduced expression of specific genes (loss of beta cell differentiation), hypertrophy, slight increase in apoptosis, and increased expression of genes normally repressed in fully differentiated beta cells [6, 8, 9, 10, 11, 12, 13, 14]. These phenotypic alterations may contribute to the progressive worsening of GSIS in type 2 diabetes [15, 16]. In comparison, in vitro exposure to inflammatory cytokines involved in the pathogenesis of type 1 diabetes, e.g. IL-1β, induces similar alterations of the beta cell phenotype [7, 17, 18]. These effects of IL-1β largely result from receptor-mediated activation of the transcription factor nuclear factor of kappa B chain (NFκB) [19, 20].
Among the genes induced by IL-1β and supraphysiological glucose concentrations, the transcription factor c-Myc stimulates apoptosis more than proliferation in rodent beta cells [21, 22, 23], whereas the antioxidant enzyme haeme-oxygenase 1 (HO1) improves beta cell survival under various stressful conditions [17, 24, 25, 26]. In vitro, overnight culture of rat islets in the presence of 30 mmol/l glucose (G30) instead of 10 mmol/l glucose (G10) markedly stimulated expression of HO1 and c-Myc at the mRNA and protein levels in a Ca2+- and cyclic AMP-dependent manner without affecting c-Myc or HO1 mRNA stability [27, 28]. Of note, culture in glucose concentrations lower than G10 (G2–G5), a stressful condition for rat beta cells, also stimulated islet HO1 and c-Myc expression. These V-shaped glucose concentration response curves for changes in islet c-Myc and HO1 expression with a minimum at G10 are similar to that of beta cell death . Whereas the stimulation of beta cell apoptosis by prolonged culture in low glucose may be due to activation of AMP-dependent protein kinase and stimulation of c-Myc expression under these conditions [23, 30], it has been suggested that the deleterious effects of supraphysiological glucose concentrations on the beta cell phenotype result from NFκB activation by oxidative stress [12, 31, 32, 33, 34] or by induction of production and autocrine actions of IL-1β .
The aim of this study was to evaluate the role of NFκB activation in the changes of beta cell gene expression by high glucose concentrations. Therefore, we tested the effects of high glucose on both NFκB DNA binding activity and the mRNA levels of HO1, c-Myc and other NFκB target genes in cultured rat islets. These effects were compared to those of IL-1β, a positive control for NFκB activation and stimulation of NFκB target gene expression, and to those of hydrogen peroxide (H2O2), a positive control for oxidative stress. The effects of glucose and H2O2 were tested with and without addition of the antioxidant N-acetyl-l-cysteine (NAC).
Materials and methods
Human IL-1β was kindly provided by Dr. R.E. Aurigemma (National Cancer Institute Biological Resources Branch Preclinical Repository, Rockville, MD, USA), and diluted, aliquoted and stored as recommended. NAC, H2O2 and aminoguanidine hydrochloride were purchased from Merck (Darmstadt, Germany), Acros Organics (Geel, Belgium) and Sigma (St. Louis, MO, USA) respectively. The inhibitors of p38MAPK (SB203580) and of ERK activation (PD98059) were purchased from Campro scientific (Veenendaal, The Netherlands) and Research Biochemical International (Natick, MA, USA) respectively.
Islet isolation and culture
Male Wistar rats (180–200 g) were obtained from the animal facility of the Faculty of Medicine at the University of Louvain (Brussels, Belgium). Their pancreatic islets were isolated by collagenase digestion of the gland, separated from the digest by density gradient centrifugation, and handpicked under a stereomicroscope . They were then pre-cultured for 1 week at 37°C in serum-free RPMI 1640 medium (Gibco, Grand Island, NY, USA) containing G10 and 5 g/l BSA (fraction V, Roche, Basel, Switzerland), 100 IU/ml penicillin, and 100 μg/ml streptomycin in the presence of 5% CO2. Large islets (>150- to 200-μm diameter) that frequently develop central necrosis were discarded. After preculture, the islets were further cultured in the same medium containing 5 mmol/l glucose (G5), G10 or G30 and various test substances. This test period lasted from 15 min up to 8 days, so that the total duration of culture never exceeded 15 days. In all cases, the medium was renewed after 1 day, then every other day. At the end of the culture, a portion of the medium was withdrawn for insulin secretion measurement, and the islets were processed for measurement of NFκB DNA binding activity or mRNA levels.
All experiments were conducted in accord with accepted standards of humane animal care and were approved by the Institutional Committee on Animal Experimentation from the Faculty of Medicine of the University of Louvain.
Measurement of islet NFκB DNA binding activity
After culture, batches of 100 islets were washed three times in ice-cold PBS (10 mmol/l phosphate buffer, pH 7.5, 150 mmol/l NaCl), followed by rapid centrifugation for 4 min at 1,000 g. The TransAM NFκB ELISA kit (Active Motif, Rixensart, Belgium) was then used for whole islet cell extract preparation and measurement of NFκB (p65) DNA-binding activity according to the manufacturer’s instructions. A 30-min exposure to 50 IU/ml IL-1β in the presence of G10 was used as a positive control condition for islet NFκB activation. The TransAM NFκB ELISA kit was validated by comparing its results with those obtained by electrophoretic mobility shift assay using an NFκB consensus oligonucleotide . Comparable results were obtained with both methods in insulin-producing RINm5 cells exposed to IL-1β for 30 min. Moreover, the signal intensity of NFκB activation detected by the ELISA kit was proportional to the number of IL-1β-treated cells in RINm5 cells, INS-1 cells and primary beta cells (25, 50 and 100×103 cells) (data not shown).
RNA extraction and cDNA synthesis
Islet total RNA was extracted and reverse transcribed into cDNA, as previously described [27, 28], using random hexamers and 200 units of M-MLV Reverse Transcriptase RNase H− Point Mutant (Promega, Madison, WI, USA).
Duplex radioactive PCR
TATA-box binding protein (TBP) was used as a housekeeping gene. The islet HO1 and c-Myc:TBP mRNA ratios were determined by simultaneous amplification of c-Myc or HO1 plus TBP islet cDNAs using a validated radioactive hot-start PCR protocol. The primer sequences, reaction conditions and PCR validation experiments have been reported previously [27, 28]. The PCR products were separated on a 6% PAGE and the amount of [α-32P] dCTP incorporated in each amplimer was quantified with a Cyclone Storage Phosphor System (Packard, Meriden, CT, USA). The ratio “gene/TBP” was calculated for each test condition, and normalised to the same ratio in control islets cultured in G10 within the same experimental series. Relative to total RNA, TBP mRNA levels remained constant under our different experimental conditions, unless otherwise specified.
Real-time fluorescent PCR
Sequences of oligonucleotide primers and reaction conditions for real-time PCR, and characteristics of PCR products
Standard curve cDNAb
IL-1β treated islets
IL-1β treated islets
Except for Fas primers , pairs of primers were designed using Hybsimulator 4.0 software (Advanced Gene Computing Technologies, Irvine, CA, USA). The specificity of sense and antisense primers (Table 1) was checked by BLAST search on the Genbank database. For each gene studied, the size of the amplicon corresponded to the expected one, based on published sequences.
Measurement of insulin secretion
Insulin concentrations in the culture media were assayed by radioimmunoassay using rat insulin as a standard .
Results are means±SEM for the indicated number of independent experiments. Statistical analysis was performed using unpaired Student’s t-test when only two groups were compared, one-way ANOVA followed by a Newman–Keuls test, or two-way ANOVA as indicated. Differences were considered significant at a p value of less than 0.05.
Time-dependent effect of high glucose on islet HO1 and c-Myc mRNA levels
Comparison of the effects of 30 mmol/l glucose and IL-1β on islet HO1 and c-Myc/TBP mRNA ratios (relative to 10 mmol/l glucose)
Experimental series 1
Experimental series 2
G10 (18 h), n=8
G30 (18 h), n=8
G10+IL-1β (6 h), n=3
G10 (1 week), n=6–7
G30 (1 week), n=6–7
Effect of high glucose on NFκB (p65) DNA binding activity
Effects of high glucose on islet mRNA levels of various NFκB-dependent genes
Comparison of the effects of 30 mmol/l glucose, IL-1β and hydrogen peroxide on islet mRNA levels of TBP and various NFκB target genes (relative to 10 mmol/l glucose)
Experimental series 1
Experimental series 2
G30/G10 (18 h), n=8a
G10+IL-1β/G10 (6 h), n=3
G10+H2O2 /G10 (18 h), n=4
G30/G10 (1 week), n=6–7
Effects of antioxidants on islet HO1 and c-Myc mRNA induction by G30
Effects of hydrogen peroxide on islet HO1 and c-Myc mRNA levels
After 1-week preculture in G10 and further culture for 18 h in G10 plus increasing concentrations of H2O2, the islet HO1 and c-Myc/TBP mRNA ratios were unaffected by 0.2–1 μmol/l H2O2, but were increased by 5 μmol/l H2O2 (n=3, p<0.01 vs G10 alone, data not shown). Higher concentrations of H2O2 (10–25 μmol/l) induced macroscopical alterations of the islets, reduced the amount of total RNA recovered per islet, and decreased the mRNA expression of TBP relative to the input of cDNA (data not shown). These alterations prevented reliable determination of changes in islet HO1 and c-Myc mRNA levels after islet treatment with a high concentration of H2O2. In a subsequent series of experiments, the stimulatory effect of 5 μmol/l H2O2 on islet HO1 and c-Myc mRNA levels was synergistic with that of G30, and was abrogated by 1 mmol/l NAC (Fig. 3b and c).
Effects of H2O2 on islet NFκB (p65) DNA binding activity and mRNA levels of various NFκB-dependent genes
Effect of high glucose, H2O2 and IL-1β on proIL-1β mRNA expression
After 1-week preculture, islet proIL-1β mRNA levels were not increased by further culture in G30 or G10+5 μmol/l H2O2. In contrast, a 30-min exposure to 50 IU/ml IL-1β markedly increased islet proIL-1β mRNA levels (Table 3).
Effect of p38MAPK and ERK pathway inhibition on islet HO1 and c-Myc mRNA expression
This study was performed with rat islets precultured for 1 week in serum-free RPMI medium containing G10. This preculture is routinely used in our lab because it preserves glucose stimulus-secretion coupling events and islet cell morphology  while allowing islet recovery from the stress of isolation [27, 28]. As such, it is an important condition to detect an effect of high glucose on islet expression of stress-induced genes such as c-Myc and HO1, two genes that are early markers of alterations induced by high glucose in beta cell gene expression [11, 12, 27, 28].
Our results demonstrate that the in vitro stimulation of rat islet HO1 and c-Myc mRNA expression by supraphysiological glucose occurs without activation of the p65 subunit of NFκB, which has been previously shown to be the crucial component of the activated NFκB complex in pancreatic beta cells [41, 42]. Accordingly, islet mRNA expression of NFκB target genes such as iNOS and IκBα were not affected by overnight and 1-week culture in G30 as compared to G10, whereas Fas was increased after 1 week only. These results make it unlikely that other NFκB subunits (p50, c-Rel) migrate to the nucleus and compensate for the lack of p65 activation under these culture conditions. In contrast with G30, IL-1β consistently and markedly increased islet NFκB DNA binding activity and mRNA expression of all NFκB target genes tested under our culture conditions, in agreement with studies on the effects of IL-1β in purified rat beta cells [17, 19, 20]. Therefore, our results do not support our previous hypothesis that the stimulation of islet HO1 and c-Myc expression by high glucose might result from NFκB activation .
It could be argued that we failed to detect an effect of glucose on rat islet NFκB DNA binding activity because this activation is transient. However, islet HO1 and c-Myc expression were maximally induced after 18–24 h and remained elevated after several days of culture in G30, whereas islet NFκB activity was not increased after 15 min up to 8 days of culture in high glucose. It could also be argued that the sensitivity of the assay was too low. However, glucose did not affect islet mRNA levels of iNOS and IκBα, two NFκB target genes induced about 700- and 15-fold by IL-1β. It is therefore unlikely that high glucose would activate islet NFκB without inducing a detectable increase in iNOS and IκBα mRNA levels. Finally, the lack of glucose effect on islet NFκB DNA binding activity was confirmed in rat islets exposed to G30 instead of G10 for 1–4 days in serum-containing RPMI, and in FACS-purified rat beta cells (attached to poly-lysine-coated dishes) exposed to high glucose for 12–24 h, thereby excluding the possibility that our results are due to the absence of serum or lack of islet cell attachment during culture. In the latter type of experiments, NFκB activation was evaluated by immunofluorescent determination of the cytoplasmic and nuclear localisation of NFκB, as previously described . Thus, after 12–24 h of culture in G28 vs G5–G10, NFκB remained localised in the cytoplasm, whereas it was mainly localised in the nucleus after IL-1β treatment (cytoplasmic localisation in 99.6±0.4 and 99.2±0.5% of the cells cultured for 24 h in G10 and G30 respectively, n=3, p>0.05; nuclear localisation in 95.6±2.2 vs 0.4±0.4% of the cells exposed to IL-1β vs controls, n=3, p<0.001).
Interestingly, hydrogen peroxide reproduced the effect of G30 on islet HO1 and c-Myc mRNA expression without activating NFκB, while the antioxidant NAC reduced or abrogated these effects of G30 and/or hydrogen peroxide. Although an increase in production of reactive oxygen species was not directly demonstrated under our particular culture conditions, these data nevertheless suggest that oxidative stress is involved in the stimulation of islet HO1 and c-Myc mRNA expression by hyperglycaemia, but through the activation of transcription factor(s) other than NFκB. Although it is usually accepted that oxidative stress activates NFκB, such a causal link has been recently challenged . Besides NFκB DNA binding sites, rat hmox1 and c-Myc gene promoters contain consensus sequences for binding of other transcription factors activated by oxidative stress, i.e. AP1, CREB, ATF4, HIF, SRE, etc. [45, 46]. In this regard, while the stimulation of c-Myc expression by high glucose could result from the activation of PKCβ2 , our preliminary results indicate that the stimulation of islet HO1 expression may involve glucose activation of both p38MAPK and ERK .
A role of oxidative stress in the stimulation of islet HO1 and c-Myc expression by G30 is compatible with a recent study showing that culture in high glucose increases reactive oxygen species production in rat pancreatic islets . It is also compatible with earlier reports on the beneficial effect of antioxidants, including NAC, on beta cell glucose toxicity in models of type 2 diabetes [32, 33, 49, 50]. It should be noted, however, that very high NAC or cysteine concentrations can be detrimental (pro-oxidant?) to pancreatic beta cells .
It has recently been reported that NFκB activation and subsequent stimulation of beta cell apoptosis in isolated human islets cultured for a few days in high glucose result from an increase in beta cell production of IL-1β with autocrine effects . These observations, together with similarities between the effects of cytokines and high glucose on beta cell gene expression, have led to the proposal that type 2, like type 1 diabetes, is a chronic inflammatory disease . Our finding that NFκB activity (using the same ELISA kit as in ) is not affected by glucose in cultured rat islets markedly contrasts with these results. Another difference with regard to the observations in human islets is that proIL-1β mRNA expression by rat islets was unaffected by G30 or hydrogen peroxide although it was induced by IL-1β. The stimulatory effect of IL-1β on its own expression was not reproduced in FACS-purified rat beta cells  (Liu and Eizirik, unpublished data), and could thus occur in non-beta cells of the islet. Whatever the cell type involved in this response, these results indicate that, under our experimental conditions, G30 did not stimulate IL-1β release by rat islets.
The reasons for the discrepancy between our and the above-mentioned studies are unclear. We consider the species difference (rat vs human) an unlikely explanation, because prolonged culture in G30 instead of G10 induces similar alterations of glucose-stimulated-insulin secretion in rat and human islets [9, 10, 40]. Moreover, in three well characterised human islet preparations containing more than 50% insulin-positive beta cells, culture in 28 instead of 5.6 mmol/l glucose for 2 or 7 days failed to induce mRNA expression of the NFκB target genes Fas and IL-1β, indicating that NFκB was not activated in human islets under these culture conditions (Welsh et al., preliminary observations). Of note, Fas and IL-1β mRNAs were detected at 30- and 100-fold higher levels respectively in stimulated human dendritic cells. Regarding the differences in culture conditions, we have shown that glucose does not activate rat islet NFκB in various culture systems (with or without serum, floating or attached whole islets, dispersed cells attached to poly-lysine coated dishes). Our current hypothesis is that culturing human islets spread on an extracellular matrix  permits long-term survival/proliferation of non-endocrine cells that inevitably contaminate human islet preparations . It is possible that hyperglycaemia activates the p65 subunit of NFκB, in a manner dependent on oxidative stress, in duct cells, endothelial cells, or fibroblasts [55, 56, 57]. In contrast, our culture of floating islets in serum-free medium allows optimal survival of endocrine cells in the absence of contaminating cell types, as judged by optical and electron microscopy of islet sections . The conserved islet structure (floating round islets with central beta cells and peripheral non-beta cells) should also preserve beta cell interactions with their neighbouring beta cells and with components of the extracellular matrix . Further studies are definitely required before one can conclude that one culture system is more appropriate than the other for the in vitro study of islet cell (patho)physiology.
We thank A. Dannau and F. Knockaert for expert technical help, and I. Kharroubi for helping with the determination of NFκB activation by immunohistochemistry. This work was supported by Grants 3.4552.98, 1.5.182.04 and 3.4514.03 from the Fonds National de la Recherche Scientifique (Brussels), the Interuniversity Poles of Attraction Program (P5/17)-Belgian Science Policy, Grant ARC 00/05-260 from the General Direction of Scientific Research of the French Community of Belgium, and a Johnson and Johnson Type 2 Diabetes Research Grant award from the European Foundation for the Study of Diabetes. J.C. Jonas is Senior Research Associate of the Fonds National de la Recherche Scientifique (Brussels).