Inhibitor of differentiation proteins protect against oxidative stress by regulating the antioxidant–mitochondrial response in mouse beta cells
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Oxidative stress is implicated in beta cell glucotoxicity in type 2 diabetes. Inhibitor of differentiation (ID) proteins are transcriptional regulators induced by hyperglycaemia in islets, but the mechanisms involved and their role in beta cells are not clear. Here we investigated whether or not oxidative stress regulates ID levels in beta cells and the role of ID proteins in beta cells during oxidative stress.
MIN6 cells were cultured in H2O2 or ribose to induce oxidative stress. ID1, ID3 and small MAF proteins (MAFF, MAFG and MAFK) were inhibited using small interfering RNA. Isolated islets from Id1 −/−, Id3 −/− and diabetic db/db mice were used.
ID1–4 expression was upregulated in vivo in the islets of diabetic db/db mice and stimulated in vitro by ribose and H2O2. Id1/3 inhibition reduced the expression of multiple antioxidant genes and potentiated oxidative stress-induced apoptosis. This finding was associated with increased levels of intracellular reactive oxygen species, altered mitochondrial morphology and reduced expression of Tfam, which encodes a mitochondrial transcription factor, and respiratory chain components. Id1/3 inhibition also reduced the expression of small MAF transcription factors (MafF, MafG and MafK), interacting partners of nuclear factor, erythroid 2-like 2 (NFE2L2), master regulator of the antioxidant response. Inhibition of small MAFs reduced the expression of antioxidant genes and potentiated oxidative stress-induced apoptosis, thus recapitulating the effects of Id1/3 inhibition.
Our study identifies IDs as a novel family of oxidative stress-responsive proteins in beta cells. IDs are crucial regulators of the adaptive antioxidant–mitochondrial response that promotes beta cell survival during oxidative stress through a novel link to the NFE2L2–small MAF pathway.
KeywordsApoptosis Beta cell Islets of Langerhans Oxidative stress Reactive oxygen species Type 2 diabetes
Bone morphogenetic protein
Electron transport chain
Heme oxygenase 1
Inhibitor of differentiation
Nuclear factor, erythroid 2-like 2
Reactive oxygen species
Small interfering RNA
Superoxide dismutase 2
Transcription factor A, mitochondrial
Beta cell failure in type 2 diabetes is characterised by dysfunctional insulin secretion and reduced beta cell mass, which has been linked to an increased rate of apoptosis [1, 2, 3]. Several lines of evidence underscore a role for chronic hyperglycaemia—termed ‘glucotoxicity’—in increased beta cell apoptosis. Thus, elevated glucose concentrations trigger apoptosis in cultured islets and beta cell lines [4, 5, 6, 7, 8] and in animal models of type 2 diabetes [9, 10, 11].
Oxidative stress has been proposed as a central mechanism of hyperglycaemia-induced beta cell demise [12, 13]. Elevated glucose or ribose levels have been shown to increase reactive oxygen species (ROS) production in beta cells [8, 14, 15, 16], and islets of diabetic mice exhibit higher ROS content, mitochondrial dysfunction and oxidative damage [15, 17, 18, 19].
The vulnerability of beta cells to oxidative stress may be owed to their low expression of several key antioxidant genes, namely Gpx1, Sod1–2 and catalase . However, other genes of the antioxidant arsenal either display strong expression in beta cells (e.g. other Gpx isoforms, Hmox1, Srxn1, Prdxs, G6pdx), or are markedly upregulated under oxidative stress conditions [6, 7, 21, 22]. The signalling pathways that regulate redox status and antioxidant gene expression in beta cells are only partially understood. Nuclear factor, erythroid 2-like 2 (NFE2L2, also known as NRF2) is a primary regulator of the antioxidant response and its activation has been shown to protect beta cells against oxidative damage . NFE2L2 activation of antioxidant gene expression requires its dimerisation with small MAF proteins (MAFF, MAFG and MAFK) [24, 25, 26].
The inhibitor of differentiation (ID) proteins are transcriptional regulators that play important roles in both physiology (e.g. development) and pathology (e.g. tumourigenesis). They have been proposed to act as repressors of basic helix-loop-helix transcription factors thereby modulating cell differentiation and proliferation [27, 28]. Previous reports have demonstrated that the expression of ID1 and ID3 is induced by glucose stimulation in human islets and beta cell lines [29, 30]. Further studies link the upregulation of Id1 mRNA levels to hyperglycaemia in islets of diabetic mice  and suggest an influence of Id1 expression in insulin secretion . However, the mechanisms underlying the induction of ID proteins by hyperglycaemia and their precise role in beta cell pathophysiology are not clear.
Here, we demonstrate that IDs are novel oxidative stress-responsive proteins in beta cells. We also identify an unexpected role of ID expression in the induction of the antioxidant response under oxidative stress conditions. Inhibition of Id1/3 reduces the expression of multiple antioxidant genes and leads to increased ROS production and apoptosis, and altered mitochondrial function and morphology. Our studies also suggest a mechanism for these effects via a previously unrecognised interaction of IDs with the NFE2L2MAF antioxidant pathway. Our results, therefore, suggest that IDs are key regulators of the adaptive antioxidant–mitochondrial response that promote beta cell survival under oxidative stress.
Ribose was obtained from Sigma (St Louis, MI, USA) and 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) and Deep Red Mitotracker were obtained from Invitrogen (Carlsbad, CA, USA). Control non-targeting and ON-TARGETplus SMARTpool small interfering RNA (siRNA) and transfection reagent DharmaFECT3 were sourced from Thermo Fisher Scientific (Lafayette, CO, USA).
C57BL/KsJ db/db and their age-matched lean control db/+ mice were taken from the Garvan Institute (Sydney, NSW, Australia) breeding colonies at the age of 14–16 weeks. Wild-type (C57BL/6/129/Sv), Id1 −/− and Id3 −/− mice were bred in-house using animals provided by Robert Benezra (Memorial Sloan-Kettering Cancer Center, New York, NY, USA) and used at the age of 8–10 weeks. All experiments were approved by the Garvan Institute/St Vincent’s Hospital Animal Experimentation Ethics Committee.
Wild-type, Id1 −/− and Id3 −/− islets were cultured in RPMI medium containing 11.1 mmol/l glucose (Invitrogen), 0.2 mmol/l glutamine, 10% heat-inactivated FBS, 100 units/ml penicillin and 100 mg/ml streptomycin in the presence or absence of 50 mmol/l ribose. To assess mitochondrial morphology, islets were dispersed with trypsin and cells were seeded on poly-L-lysine-treated glass coverslips before treatment.
Cell culture and treatment
MIN6 beta cells (P26-39) were grown in Dulbecco’s modified Eagle’s medium (Invitrogen) containing 25 mmol/l glucose, 10 mmol/l HEPES, 10% FCS, 50 units/ml penicillin and 50 mg/ml streptomycin. Cells were transfected with either control, Id1 and/or Id3 or MafF/G/K siRNA. After 24 h, the transfection medium was changed and cells were cultured for 48 h in the absence or presence of H2O2 or ribose.
Real-time RT-PCR was performed as previously described . Primer sequences are listed in Electronic Supplementary Material (ESM) Table 1. The value obtained for each specific gene product was normalised to the control gene cyclophilin A and expressed as a fold change of the value in control condition.
Glutathione peroxidase (GPX) activity was determined indirectly as the decrease in NADPH absorption at 340 nm as previously described . Cell death was determined with the use of a Cell Death Detection ELISA (Roche Diagnostics, Castle Hill, NSW, Australia) . ATP generation was determined using ATPlite kit (PerkinElmer, Melbourne, VIC, Australia). Oxygen consumption was measured using a Clark-type oxygen electrode system as previously described . For ROS measurement, cells were incubated with 2 μmol/l CM-H2DCFDA or DMSO for 30 min at 37°C. Fluorescence intensity was measured (ex/em at 485/520 nm) using a plate reader (FluoStar OPTIMA, BMG Lab Technologies, Mornington, VIC, Australia).
Immunostaining was performed as previously described . Images were acquired using a Leica SP8 confocal microscope.
Results are given as mean ± SEM for the indicated number of experiments. Statistical significance was assessed by unpaired two-tailed Student’s t test, one-way ANOVA and a post-test of Newman–Keuls or two-way ANOVA and a post-test of Bonferroni.
ID expression is increased in the islets of diabetic mice in parallel with the antioxidant response
Id genes are induced by oxidative stress in beta cells
Furthermore, the ribose-induced upregulation of mRNA levels for Id genes was prevented by treatment with the antioxidant MnTBAP (ESM Fig. 1a–j). Together these results demonstrate that IDs are novel oxidative stress-responsive proteins in MIN6 beta cells.
Inhibition of Id1 and/or Id3 leads to global attenuation of the antioxidant response
Inhibition of Id1 and/or Id3 increases ROS levels, apoptosis and oxidative damage
Inhibition of Id1 and/or Id3 impairs mitochondrial homeostasis
This alteration in mitochondrial function was associated with changes in the expression of electron transport chain (ETC) components. Thus, inhibition of Id1/3 tended to decrease the expression of CI (subunit NDUFB8), CII (subunit SDHB) and CIII (subunit UQCRC2), significantly decreased the expression of CV (subunit ATP5A), and strongly decreased the expression of CIV (subunit MTCO1) (Fig. 6c–h). On the other hand, the expression of Ucp2 was not affected by Id1/3 inhibition (ESM Fig. 3a). MTCO1, a catalytic subunit of cytochrome c oxidase, is regulated by transcription factor A, mitochondrial (TFAM). The mRNA levels of Tfam were upregulated by ribose treatment (Fig. 6i) as well as in the islets of diabetic db/db mice (ESM Fig. 3b). Interestingly, inhibition of Id1/3 reduced the mRNA levels of Tfam (Fig. 6i).
Together, these results show that the inhibition of Id1/3 is associated with altered mitochondrial function and morphology. These alterations were associated with reduced expression of components of the ETC, particularly MTCO1, as well as the upstream transcription factor Tfam.
Inhibition of Id1 and Id3 reduces the expression of MAFs, interacting partners of NFE2L2
Inhibition of MAFs reduces antioxidant gene expression and potentiates apoptosis, recapitulating the effects of Id1/3 inhibition
These results demonstrate for the first time the importance of small MAF proteins (F/K/G) in the regulation of antioxidant gene expression and beta cell survival under conditions of oxidative stress. Our results strongly suggest that Id1/3 regulate antioxidant gene expression via the regulation of Maf expression, thereby providing a novel mechanism of the cellular antioxidant response.
We have unveiled a novel cellular role for IDs in the regulation of redox status, mitochondrial integrity and beta cell survival under oxidative stress conditions. We have shown for the first time that IDs are oxidative stress-responsive genes in beta cells. All four members of the ID family were upregulated in the islets of diabetic db/db mice. This induction was paralleled by a global upregulation of the antioxidant response. Inhibition of Id1/3 in beta cells led to mitochondrial impairment and global attenuation of the antioxidant response. Since oxidative stress is a major downstream mechanism of beta cell glucotoxicity , our results provide important insight into the molecular mechanisms regulating beta cell survival in type 2 diabetes.
IDs and the antioxidant response
NFE2L2 is a key regulator of antioxidant gene expression and cellular homeostasis . Unexpectedly, we found that the inhibition of Id1/3 markedly increased NFE2L2 expression and nuclear localisation. This interesting observation, which is in line with the increased ROS accumulation under these conditions, suggested that Id1/3 may affect an interacting partner rather than NFE2L2 itself. Indeed, we found that inhibition of Id1/3 inhibited the expression of small MAF proteins F, K and G, known interacting partners of NFE2L2 that play an important role in the regulation of antioxidant gene expression [24, 25, 26, 40]. Interestingly, inhibition of MafF/G/K reduced the expression of several antioxidant genes and potentiated ribose-induced apoptosis, thereby mimicking the effects of Id1/3 inhibition.
These striking findings provide a novel regulatory mechanism of the antioxidant response. Our results strongly suggest that Id1/3 regulate antioxidant gene expression through the regulation of small MAF protein expression. Our results highlight the important role of small MAF proteins in beta cells. While recent reports have focused on the role of NFE2L2 in diabetes and beta cell pathophysiology [23, 41], less attention has been given to the role of small MAF proteins. Yet, our results show that increased expression and nuclear accumulation of NFE2L2 alone, in the absence of MAFs, was insufficient to maintain adequate expression of antioxidant genes under oxidative stress.
IDs and mitochondria
Mitochondria have been shown to be a primary target for oxidative damage. Thus, acute exposure of rat islets and INS1 cells to H2O2 has been shown to impair mitochondrial function, reduce Tfam and ETC component expression, especially MTCO1, and increase mitochondrial ROS production and apoptosis . These observations mirror the effects of Id1/3 inhibition. The global attenuation of antioxidant gene expression induced by Id1/3 inhibition may lead to ROS accumulation and mitochondrial alterations that enhance ROS generation, thus further exacerbating oxidative stress/damage and apoptosis. Alternatively, the downregulation of Tfam and ETC components by Id1/3 inhibition may slow electron flow leading to enhanced ROS generation. Indeed, it is well established that beta cells generate high ROS levels when cultured in the presence of a low non-stimulatory glucose concentration in association with increased expression of antioxidant genes and apoptosis [6, 43, 44]. The resulting oxidative stress is further aggravated by the global attenuation of the antioxidant response. This view is supported by the observation that Id1/3 inhibition leads to enhanced ROS formation, increased oxidative damage and potentiation of apoptosis under basal conditions in the absence of any ribose or H2O2 stimulation. Moreover, in our model, chronic stimulation with H2O2 or ribose increases TFAM and ETC component expression. In line with this observation, mRNA levels of Tfam were upregulated in the islets of diabetic mice despite the presence of oxidative stress. Either way, these alterations were associated with altered mitochondrial morphology. The latter has been observed in beta cells of type 2 diabetes both in animal models and in humans, as well as in in vitro models of nutrient oversupply and oxidative stress [15, 19, 42, 45, 46, 47].
Potential exploitation of these new findings to protect beta cells
Although antioxidant supplementation has been shown to be promising in type 2 diabetes animal models, it was overall ineffective in humans. Consequently, the activation of endogenous antioxidant genes could be a more promising strategy to reduce ROS-mediated apoptosis in beta cells. The identification of IDs as key upstream regulators of the antioxidant response, besides their parallel role in mitochondria, may serve for the development of a novel therapeutic strategy to protect beta cells against oxidative stress. IDs are known downstream targets of the bone morphogenetic protein (BMP) signalling pathway. Emerging evidence indicates an important role of this pathway in beta cells [48, 49, 50]. However, one should keep in mind that the activation of IDs may also interfere with beta cell differentiation. We have previously shown that Id1 was induced by elevated lipids in beta cells and plays a role in lipid-induced impairment of insulin secretion and differentiation . Moderate physiological stimulation of this pathway may exert beneficial effects under oxidative stress while avoiding the alteration of the beta cell phenotype. Interestingly, our other studies show that low concentrations of recombinant BMP stimulated the expression of Id1/3 in parallel with a significant protection against ribose-induced apoptosis in MIN6 cells (M. Bensellam, D. R. Laybutt, unpublished results).
In conclusion, we have identified IDs as a novel family of oxidative stress-responsive proteins in beta cells. We have demonstrated that Id1/3 are crucial for the maintenance of an adaptive mitochondrial-antioxidant response that promotes beta cell survival under oxidative stress via a novel link to the NFE2L2–MAF signalling pathway. These novel findings may help the development of therapeutic strategies to protect beta cells against oxidative stress.
This work was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia. DRL is supported by an Australian Research Council (ARC) Future Fellowship. MB was supported by a Post-doctoral Fellowship from the Société Francophone du Diabète (SFD, Paris, France).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
MB and DRL conceived and designed experiments, acquired and analysed data and wrote the manuscript. MKM, JL and JYC designed experiments, acquired and analysed data and critically reviewed the manuscript. All authors approved the final version of the manuscript. DRL is responsible for the integrity of the work as a whole.
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