Autophagy defends pancreatic β cells from human islet amyloid polypeptide-induced toxicity (original) (raw)
The autophagy/lysosome system determines intracellular IAPP content. In a professional secretory cell such as a β cell, intracellular secretory protein content is determined by the balance among protein synthesis, degradation, and secretion. To evaluate whether autophagy contributes to the degradation of IAPP, we investigated the effects of stimulation and inhibition of autophagy on IAPP content in β cells. We previously reported that rapamycin and lysosomal inhibitors are efficient modulators of autophagy in β cells (15). Stimulation of autophagy in INS 832/13 cells with rapamycin reduced β cell IAPP content (54% ± 5.5% decrease versus nontreated cells detected by Western blot, P < 0.001; Figure 1A; and 33% ± 2.5% decrease detected by enzyme immunoassay, P < 0.001; Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI71981DS1). In contrast, treatment of cells with the inhibitors of lysosomal proteases, E-64-d and pepstatin A, increased β cell IAPP content (1.4-fold versus nontreated cells, P < 0.05; Figure 1A and Supplemental Figure 1). We confirmed that these changes in IAPP content were not related to changes in IAPP expression or secretion (Supplemental Figure 2 and Supplemental Figure 3A). Altogether, these data suggest that, in rodent β cells, IAPP content is regulated, at least in part, by autophagy.
Intracellular IAPP levels are modulated by regulators of autophagy. (A) INS 832/13 cells were treated with rapamycin (Rapa, 10 nM) for 40 hours, lysosomal inhibitors (Lyso I) (E-64-d, 10 μg/ml and pepstatin A, 10 μg/ml) for 24 hours, or left untreated (C). Levels of IAPP were assessed by Western blot. GAPDH was used as loading control. The graph represents the quantification of the processed/mature form of IAPP (n = 4). (B) Human islets were treated with rapamycin (10 nM) for 30 hours, lysosomal inhibitors (E-64-d, 10 μg/ml and pepstatin A, 10 μg/ml) for 30 hours, or left untreated. Levels of IAPP were assessed by Western blot. GAPDH was used as loading control. The graph represents the quantification of IAPP protein levels (n = 3). Data are expressed as mean ± SEM; *P < 0.05; ***P < 0.001.
To extend these findings to humans, we repeated the experiments using human islets. Enhancement of autophagy with rapamycin decreased cellular IAPP content (31% ± 12.4% decrease versus nontreated islets, P < 0.05; Figure 1B), while inhibition of lysosome-dependent clearance led to an increase in IAPP cellular content (1.4-fold versus nontreated islets, P < 0.05; Figure 1B). In contrast to IAPP, cellular insulin content was not affected upon treatment with either lysosomal inhibitors or rapamycin (Supplemental Figure 4), suggesting a selective targeting of IAPP to the autophagy/lysosomal pathway.
Besides rapamycin, several FDA-approved compounds with low cytotoxicity promote the degradation of long-lived proteins (16). Amiodarone and trifluoperazine induced autophagic degradation and thus reduced the accumulation of misfolded proteins in human glioblastoma H4 cells (16). Similarly, amiodarone and trifluoperazine stimulated autophagy in pancreatic β cells and rat islets (Supplemental Figure 5, A and B). These compounds decreased cellular IAPP content and reduced h-IAPP–induced apoptosis in islets isolated from h-IAPP transgenic rats (HIP rats, a rodent model for T2D; ref. 4) (Supplemental Figure 5C). We conclude that the autophagy/lysosome system specifically regulates intracellular IAPP content levels in human and rodent β cells.
p62 interacts with IAPP and sequesters IAPP in β cells. The adaptor protein p62 (also known as sequestosome 1 or SQSTM1) is involved in selective autophagic clearance of long-lived and aggregation-prone proteins (8). To test the hypothesis that p62 is required for IAPP degradation by autophagy, we evaluated the interaction between IAPP and p62 in INS 832/13 cells transduced with adenoviruses expressing h-IAPP or the nonamyloidogenic and nontoxic r-IAPP (used as a control for comparable burden of protein expression [Supplemental Figure 6A] as previously published; refs. 17–19). In all 3 conditions (control and r-IAPP– and h-IAPP–transduced cells), IAPP immunoprecipitated with p62 (Figure 2A). However, the proportion of IAPP associated with p62 was markedly increased in β cells with high expression of h-IAPP versus r-IAPP (Figure 2A).
IAPP interacts with p62 in β cells. (A) INS 832/13 cells were transduced at 400 MOI for 36 hours with r-IAPP (R) or h-IAPP (H) adenoviruses. Cell lysates were subjected to immunoprecipitation (IP) with anti-p62 antibody or IgG as control. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted (IB) with anti-IAPP antibody. Levels of GAPDH are shown as internal and loading control. A representative image from 5 independent experiments is shown. C, nontransduced cells (B) Islets were isolated from 9- to 10-week-old WT, r-IAPP (r-TG), and homozygous h-IAPP transgenic (h-TG) mice. Islet lysates were subjected to immunoprecipitation with anti-p62 antibody or IgG as control. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-IAPP antibody. Levels of GAPDH are shown as internal and loading control. A representative image from 3 independent experiments is shown.
To confirm that the IAPP-p62 interaction was relevant in vivo, we performed immunoprecipitation experiments using islets from mice with transgenic β cell expression of h-IAPP (h-TG) and islets from transgenic mice expressing comparable levels of r-IAPP (r-TG) (Supplemental Figure 6B). As we previously reported, toxic oligomers of h-IAPP form intracellularly in β cells of h-TG mice but not r-TG mice, and only h-TG mice develop diabetes (3, 6). Since lysosomal degradation is impaired in β cells of h-TG mice (15), this model characterized by p62 accumulation (Supplemental Figure 6B) provides optimal experimental conditions to investigate whether IAPP interacts with p62 in vivo. We found that, similar to the experiments performed in the INS 832/13 β cell line, IAPP immunoprecipitated with p62 in mouse islets, with a more pronounced interaction in h-TG mice (Figure 2B). The specificity of this interaction between IAPP and p62 was reinforced by the absence of binding among the even more abundantly expressed β cell secretory protein, insulin, and p62 (Supplemental Figure 7).
p62 acts as an autophagic receptor for polyubiquitinated proteins because of its ability to bind both target-associated ubiquitin and LC3 conjugated to the autophagosome membrane (8). We next sought to establish whether IAPP becomes polyubiquitinated in order to be targeted for p62-dependent lysosomal degradation. IAPP was immunoprecipitated from islet lysates from WT and HIP rats and then subjected to Western blotting for the detection of ubiquitin. Polyubiquitinated IAPP was present in WT rat islets and increased in HIP rat islets (Figure 3A). The insoluble protein fraction isolated from HIP rat islets was solubilized as described previously (20), fractionated using high-performance liquid chromatography, and subjected to Western blot analysis. In some fractions (no. 41, no. 43, and no. 44; Figure 3B), we detected high-molecular-weight bands positive for both IAPP and ubiquitin. In conclusion, both experimental approaches confirm the existence of a pool of polyubiquitinated IAPP.
IAPP is ubiquitinated. (A) Islets were isolated from 4- to 6-month-old WT and HIP rats. Islet lysates were subjected to immunoprecipitation with a rabbit anti-IAPP antibody. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with a mouse anti-ubiquitin antibody. Levels of IAPP are shown as control. Arrows indicate polyubiquitinated IAPP (n = 3). (B) Islets were isolated from 4- to 6-month-old HIP rats. Insoluble fraction was obtained by a detergent extraction protocol and dissolved in 6 M guanidine plus 0.5 M DTT for 1 hour at 37°C. Fractions were collected by HPLC and then immunoblotted with a rabbit anti-IAPP antibody. The membrane was then stripped and immunoblotted with a mouse anti-ubiquitin antibody. A representative blot from 2 independent experiments is shown. Boxes indicate bands detected by both anti-IAPP and anti-ubiquitin antibodies.
p62 not only assists autophagic degradation of ubiquitinated proteins, but also, due to its polymeric nature, mediates protein aggregation when lysosomal degradation is limited (8). We thus investigated whether the insoluble cytoplasmic inclusions of p62 present in h-TG β cells (15) also contain insoluble forms of IAPP. To proceed, we separated the detergent-soluble and -insoluble compartments by fractionation of islet lysates. We observed a marked increase in the levels of p62 in both the soluble and insoluble fractions in islets from h-TG mice compared with those from r-TG mice (Figure 4A). In the soluble fraction, IAPP immunoprecipitated with p62 in both r-TG and h-TG mouse islets but with a more pronounced interaction in h-TG mice (Figure 4B). When we examined the insoluble fraction, IAPP immunoprecipitated with p62 exclusively in h-TG mice (Figure 4B). Since insoluble h-IAPP may form amyloid fibrils, we used thioflavin S, a dye that binds to amyloid fibrils but not to soluble monomers (21), to investigate the interaction between p62 and insoluble IAPP fibrils within the cytosolic inclusions. The p62 inclusions were positive for thioflavin S, implying that IAPP sequestered within p62 inclusions in vivo is indeed organized into fibrils within β cells (Figure 4C and Supplemental Figure 8). Since extracellular fibrils of amyloidogenic proteins are relatively inert in contrast to membrane-permeant oligomers (2), p62 may play an important role by favoring fibril formation of potentially toxic h-IAPP and thereby serve as a buffer to protect β cells against h-IAPP toxicity (Supplemental Figures 9 and 10).
Insoluble p62-sequestered IAPP is targeted for lysosomal degradation. (A) Islets were isolated from 9- to 10-week-old r-TG and h-TG mice. Islet lysates were used to separate total cellular protein into soluble and insoluble fractions. Levels of p62 and IAPP were assessed by Western blot. GAPDH was used as control. A representative image from 4 independent experiments is shown. (B) Soluble and insoluble fractions from mouse islets were subjected to immunoprecipitation with anti-p62 antibody. Immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotted with anti-IAPP antibody. (C) Thioflavine S staining in pancreatic sections from 9- to 10-week-old h-TG mice (Thioflavin S, green; p62, red; insulin, yellow; nuclei, blue) (scale bar: 20 μm). A higher magnification of an inclusion is presented on the right (scale bar: 10 μm). The dotted outlines on the insulin panel indicate the position of thioflavin S– and p62-positive inclusions. (D) Fluorescent confocal images of p62-positive inclusion using (p62, yellow; LC3, red; cathepsin D, green) in pancreatic tissue from h-TG mice (original magnification, ×63). Scale bar: 10 μm.
Of interest, confocal images revealed that these inclusions were surrounded by the autophagy machinery involving autophagosomes (LC3 staining) and lysosomes (cathepsin D staining) (Figure 4D). Thus, it is tempting to speculate that the autophagosomes migrate to the much larger p62-bound aggregates and in turn attract the lysosomes. Altogether our data demonstrate that soluble and insoluble h-IAPP are targeted for p62-dependent autophagy.
Intracellular accumulation of toxic oligomers is enhanced in β cells from autophagy-deficient human-IAPP transgenic mice. Since chemical compounds used to manipulate autophagy may have other effects in β cells (22–24), we used a genetic approach to corroborate that autophagy plays a key role in controlling h-IAPP levels in β cells in vivo. We generated mice deficient for ATG7 specifically in β cells with hemizygous expression of h-IAPP. We crossed RIP-Cre Atg7fl/fl mice (referred hereafter as Atg7Δβcell mice) with homozygous h-IAPP transgenic mice to obtain 4 groups of mice: control, h-IAPP+/–, Atg7Δβcell, and h-IAPP+/–:Atg7Δβcell mice. In both Atg7Δβcell and h-IAPP+/–:Atg7Δβcell mice, we confirmed the specific knockout of ATG7 in β cells by Western blot (Figure 5A). Reduction of autophagy in Atg7Δβcell and h-IAPP+/–:Atg7Δβcell mice was confirmed by loss of the ATG7-mediated conversion of microtubule-associated protein 1 light chain-1 (LC3-I) to LC3-II required for formation of autophagosomes (ref. 25 and Figure 5A). Furthermore, deficiency in autophagy/lysosomal degradation was supported by accumulation of p62 in Atg7Δβcell and h-IAPP+/–:Atg7Δβcell mice (ref. 25 and Figure 5A). The higher molecular weight band indicative of tightly aggregated p62 (26) was increased in autophagy-deficient mice with h-IAPP expression compared with that in Atg7Δβcell mice. Since our previous ex vivo data revealed that h-IAPP is targeted for p62-dependent lysosomal degradation, we hypothesized that deficiency in autophagy in h-IAPP–expressing β cells might lead to h-IAPP accumulation and formation of toxic oligomers. The islet content of monomeric IAPP tended to be increased in h-IAPP+/–:Atg7Δβcell mice compared with that in control groups, even though the islet β cell compliment in this model is decreased (Supplemental Figure 11A). To detect toxic oligomers, we used the A11 antibody (3, 21). Although, we observed immunoreactivity for toxic oligomers in both h-IAPP+/– and h-IAPP+/–:_Atg7_Δβcell mice, the percentage of β cells positive for A11 labeling was 5-fold higher in h-IAPP+/–:Atg7Δβcell mice compared with that in h-IAPP+/– mice (Figure 5B and Supplemental Figure 12A). A representative islet of a h-IAPP+/–:Atg7Δβcell mouse with A11 labeling in β cells is shown in Figure 5C. Interestingly, β cells containing p62-positive inclusions had less A11 labeling than cells without inclusions (Figure 5B and Supplemental Figure 12B). These findings further support the hypothesis that p62 inclusions sequester h-IAPP from the intracellular compartments in which toxic oligomers (A11 binding) are prone to form (3). The conditions within the p62 inclusions apparently favor assembly of h-IAPP into relatively inert fibrils that are then presumably subject to lysosomal clearance over time if the autophagy/lysosomal pathway is functional.
h-IAPP toxic oligomers accumulate in β cells of hemizygous h-IAPP transgenic mice deficient in autophagy. (A) Protein levels of ATG7, LC3, and p62 were assessed by Western blot using islet protein lysates obtained from Atg7Δβcell (9 weeks, n = 3) mice and control, h-IAPP+/–, and h-IAPP+/–:Atg7Δβcell (all 9 weeks, n = 4) mice. GAPDH was used as loading control. The asterisk indicates tightly aggregated p62. (B) Quantification of the percentage of β cells positive for cytosolic A11 labeling in h-IAPP+/–:Atg7Δβcell and h-IAPP+/– mice. Included are the percentages of β cells positive (white) or negative (black) for p62 among A11-positive β cells. Data are expressed as mean ± SEM. (C) Confocal images of a representative islet from a h-IAPP+/–:Atg7Δβcell mouse pancreatic section stained with anti-oligomer antibody A11 (oligomers, red; p62, green; IAPP, yellow; nuclei, blue). Scale bar: 24 μm.
Overall, these data show that autophagy deficiency results in increased levels of monomeric h-IAPP and toxic oligomers. These findings are consistent with an important role of the autophagy pathway in clearance of h-IAPP and thus protection of β cells against h-IAPP toxicity.
Deficiency in autophagy leads to diabetes development and loss of β cell mass due to increased β cell apoptosis in human-IAPP transgenic mice. We further tested the postulate that autophagy is important in protecting β cells against h-IAPP oligomer toxicity by evaluating diabetes development in autophagy-deficient h-IAPP transgenic mice. Fasting blood glucose was measured weekly, starting from 6 weeks of age, in all 4 groups of mice described above. Body weight remained comparable among groups (data not shown). As expected, fasting blood glucose concentrations were comparable in control and h-IAPP+/– mice throughout the study period. This confirms that mice hemizygous for β cell transgenic expression of h-IAPP do not develop diabetes, presumably because their β cells are competent to manage the resulting expression rate of amyloidogenic h-IAPP (27). Fasting blood glucose levels in Atg7Δβcell mice were also comparable to those of control mice. In contrast, in h-IAPP+/–:Atg7Δβcell mice, fasting blood glucose increased relative to that of controls at 8 to 9 weeks of age (97.9 ± 7.0 mg/dl versus 70.4 ± 2.7 mg/dl in h-IAPP+/– mice, P < 0.001; Figure 6A). By 12 to 13 weeks of age, these mice had impaired fasting blood glucose (123.7 ± 11.4 mg/dl versus 73.4 ± 3.0 mg/dl in h-IAPP+/– mice, P < 0.001; Figure 6A), and by 14 to 15 weeks of age, h-IAPP+/–:Atg7Δβcell mice had diabetes (178.3 ± 65.6 mg/dl versus 74.6 ± 3.3 mg/dl in h-IAPP+/–, P < 0.001; Figure 6A). Intraperitoneal glucose tolerance tests (IPGTTs) revealed glucose intolerance in 8-week-old h-IAPP+/–:Atg7Δβcell mice before diabetes onset (P < 0.001 versus Atg7Δβcell mice; P < 0.05 versus h-IAPP+/– mice; Figure 6B). In addition, serum insulin/glucose and C-peptide/glucose ratios (Figure 6, C and D) as well as islet and pancreatic insulin contents (Supplemental Figure 11B and Supplemental Figure 13) were decreased in h-IAPP+/–:Atg7Δβcell mice, revealing defective insulin secretion. Consistent with the postulated protective action of autophagy against β cell loss, β cell mass was decreased in h-IAPP+/–:Atg7Δβcell mice compared with that in control groups (P < 0.05; Figure 6E). The mechanism subserving the deficit in β cell mass in h-IAPP+/–:Atg7Δβcell mice was the increased β cell apoptosis (2.5-fold versus h-IAPP+/– mice, P < 0.01; Figure 6F).
Deficiency in autophagy induces diabetes, impaired β cell function, loss of β cell mass, and increased β cell apoptosis in hemizygous h-IAPP transgenic mice. (A) Fasting blood glucose in control, h-IAPP+/–, Atg7Δβcell, and h-IAPP+/–:Atg7Δβcell mice. The number of mice per group of a given age is provided in Supplemental Table 1. ***P < 0.001. (B) IPGTT performed by intraperitoneal injection of 2 g/kg glucose in control and h-IAPP+/–:Atg7Δβcell mice (both 8 weeks, n = 6) and h-IAPP+/– and Atg7Δβcell mice (both 8 weeks, n = 7). The graph represents area under the curve (AUC). #P < 0.05, versus h-IAPP+/– mice; ***P < 0.001, versus Atg7Δβcell mice. (C) Plasma insulin/glucose ratio and (D) C-peptide/glucose ratio in control (14 ± 1 weeks, n = 8), h-IAPP+/– (14 ± 1 weeks, n = 8), Atg7Δβcell (15 weeks, n = 4), and h-IAPP+/–:Atg7Δβcell (12 ± 1 weeks, n = 6) mice. *P < 0.05, versus Atg7Δβcell and h-IAPP+/– mice for C-peptide/glucose ratio; ***P < 0.001. (E) β Cell mass in the 4 groups of mice at given mean age: control (14 ± 1 weeks, n = 4), h-IAPP+/– (14 ± 1 weeks, n = 4), Atg7Δβcell (15 weeks, n = 3), and h-IAPP+/–:Atg7Δβcell mice (12 ± 1 weeks, n = 6). *P < 0.05, versus all groups. (F) β Cell apoptosis (TUNEL) in the 4 groups of mice at given mean age: control (13 ± 1 weeks, n = 3), h-IAPP+/– (13 ± 2 weeks, n = 3), Atg7Δβcell (15 weeks, n = 3), and h-IAPP+/–:Atg7Δβcell (12 ± 1 weeks, n = 3) mice. *P < 0.05, versus Atg7Δβcell mice; **P < 0.01, versus h-IAPP+/– mice. Data are expressed as mean ± SEM.
These in vivo studies corroborate the in vitro data and reveal an important role for autophagy in protection of β cells from toxicity of amyloidogenic h-IAPP, presumably by guarding against the formation and/or accumulation of toxic oligomers.
Oxidative damage and loss of a key antioxidant protective pathway in β cells of autophagy-deficient mice that express oligomeric h-IAPP. Disruption of autophagy promotes oxidative stress (28), and β cells are highly vulnerable to oxidative stress (29). We thus hypothesized that β cells of mice deficient in autophagy that express h-IAPP would be characterized by increased oxidative stress as a contributing mechanism for apoptosis. To test this hypothesis, we evaluated β cell levels of nitrotyrosine, an in situ marker for oxidative stress (30). Interestingly, p62 inclusions were positive for nitrotyrosine in β cells of both Atg7Δβcell and h-IAPP+/–:Atg7Δβcell mice (Supplemental Figure 14); however, cytosolic labeling for nitrotyrosine was more apparent in β cells of h-IAPP+/–:Atg7Δβcell mice (Figure 7A). As shown in Figure 7B, the area positive for nitrotyrosine occupied 4.3% ± 0.3% of the β cell area in h-IAPP+/–:Atg7Δβcell mice in comparison to 2.7% ± 0.3% in Atg7Δβcell mice (P < 0.01), suggesting increased and more widespread oxidative damage.
Deficiency in autophagy increases the oxidative damage in β cells of hemizygous h-IAPP transgenic mice. (A) Nitrotyrosine levels were assessed by immunofluorescence in pancreatic tissue from control, h-IAPP+/–, Atg7Δβcell, and h-IAPP+/–:Atg7Δβcell mice (nitrotyrosine, red; IAPP, white; nuclei, blue). The insets show higher magnification. (B) Quantification of the fractional area of β cell positive for nitrotyrosine (signal above background) in Atg7Δβcell (15 weeks, n = 3) and h-IAPP+/–:Atg7Δβcell mice (12 ± 1 weeks, n = 3) (expressed in percentage). 10–17 islets per section were analyzed. Data are expressed as mean ± SEM; **P < 0.01. Scale bar: 50 μm.
The KEAP1-NRF2 system is one of the main cellular defense mechanisms against oxidative stress (31, 32). In unstressed conditions, the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) is constitutively degraded through the ubiquitin-proteasome system due to the action of its binding partner kelch-like ECH-associated protein 1 (KEAP1), an adaptor of the ubiquitin ligase complex. Autophagy deficiency leads to the formation of p62-positive inclusions that sequester KEAP1, thus preventing NRF2 ubiquitination. As a result, NRF2 becomes stabilized and translocates to the nucleus to induce the transcription of numerous cytoprotective genes (31, 32).
Consistent with findings in autophagy-deficient hepatocytes (31), islets from Atg7Δβcell mice showed an increase in NRF2 protein levels by Western blot (2.2-fold increase versus control mice, P < 0.05; Figure 8A). In contrast, NRF2 levels failed to increase in islets of prediabetic h-IAPP+/–:Atg7Δβcell mice compared with Atg7Δβcell mice (P < 0.05; Figure 8A). This was confirmed by immunofluorescence studies that revealed that h-IAPP+/–:Atg7Δβcell mice had decreased levels of nuclear and cytosolic NRF2 when compared with islets from Atg7Δβcell mice (Figure 8, B and C). Interestingly, a certain amount of NRF2 was localized to p62-positive inclusions in both Atg7Δβcell and h-IAPP+/–:Atg7Δβcell mice. This localization of NRF2 to p62-positive inclusions is not unexpected, since p62 sequesters homodimeric KEAP1 (data not shown), which can simultaneously interact with NRF2 (33). Consistent with the decrease in nuclear and cytosolic levels, NRF2 was also decreased in p62 inclusions of h-IAPP+/–:Atg7Δβcell mice in comparison with those of Atg7Δβcell mice (Figure 8B). The lack of upregulation of the NRF2 transcription factor in prediabetic h-IAPP+/–:Atg7Δβcell mice points to a loss of protection against oxidative stress before diabetes onset.
Antioxidant NRF2 is reduced in hemizygous h-IAPP transgenic mice deficient in autophagy. (A) Protein levels of NRF2 were assessed by Western blot using islet lysates obtained from control (9 weeks, n = 3), h-IAPP+/– (9 weeks, n = 3), Atg7Δβcell (9 weeks, n = 3), and h-IAPP+/–:Atg7Δβcell mice (9 weeks, n = 3). GAPDH was used as loading control. The graph represents the quantification of NRF2 protein levels. Data are expressed as mean ± SEM; *P < 0.05; #P < 0.05, versus control and h-IAPP+/– mice. (B) NRF2 and p62 levels were assessed by immunofluorescence in pancreatic sections from control, h-IAPP+/–, Atg7Δβcell, and h-IAPP+/–:Atg7Δβcell mice (NRF2, red; p62, white; nuclei, blue). Scale bar: 50 μm. (C) Images of islets from Atg7Δβcell and h-IAPP+/–:Atg7Δβcell mice showing cytosolic and nuclear staining of NRF2 (NRF2, red; IAPP, green; nuclei, blue). Arrows indicate nuclei. Scale bar: 12 μm.
To determine whether increased expression of h-IAPP in autophagy-deficient β cells leads to either a general disruption of the antioxidative defense or a specific alteration of the NRF2 system, we evaluated mRNA levels of 2 antioxidant genes: glutathione S-transferase μ (Gstm1), a target of NRF2 (34), and Cu/Zn superoxide dismutase (Sod1), an antioxidant gene, which is not a primary target of NRF2 (35–37). β Cells of Atg7Δβcell mice, which had an increase in NRF2 levels, displayed a corresponding increase in its target antioxidant gene, Gstm1 (P < 0.01 versus control mice; Figure 9). Our data are in accordance with that from a previous publication reporting an increase in GSTμ (Gstm genes) in β cells of Atg7Δβcell mice (9). In contrast, β cells of h-IAPP+/–:Atg7Δβcell mice, which showed a cytosolic targeting of NRF2 and reduced NRF2 protein levels, failed to increase Gstm1 (P < 0.05 versus Atg7Δβcell mice; Figure 9). Interestingly, Sod1 was not similarly regulated (Figure 9). Altogether, these data suggest that increased expression of h-IAPP in autophagy-deficient β cells does not lead to a general disruption of the antioxidative defense but rather specifically attenuates the NRF2-dependent protective mechanisms.
mRNA levels of the antioxidant genes Gstm1 and Sod1 in islets from hemizygous h-IAPP transgenic mice deficient in autophagy. Levels of Gstm1 and Sod1 mRNA were evaluated by RT-qPCR in islets isolated from control (9 weeks, n = 4), h-IAPP+/– (9 weeks, n = 3), Atg7Δβcell (9 weeks, n = 3), and h-IAPP+/–:Atg7Δβcell (9 weeks, n = 3) mice. Cyclophilin was used as housekeeping gene. Data are expressed as mean ± SEM; *P < 0.05; **P < 0.01; #P < 0.05, versus Atg7Δβcell mice.
These data show that autophagy deficiency in the setting of expression of oligomeric IAPP leads to increased levels of oxidative damage in β cells, most likely due to the failed compensatory increase in NRF2. The failed upregulation of this key transcription factor that is expected to protect against oxidative stress likely has deleterious consequences on β cell survival.