Autophagy defends pancreatic β cells from human islet amyloid polypeptide-induced toxicity

Abstract

Type 2 diabetes (T2D) is characterized by a deficiency in β cell mass, increased β cell apoptosis, and extracellular accumulation of islet amyloid derived from islet amyloid polypeptide (IAPP), which β cells coexpress with insulin. IAPP expression is increased in the context of insulin resistance, the major risk factor for developing T2D. Human IAPP is potentially toxic, especially as membrane-permeant oligomers, which have been observed to accumulate within β cells of patients with T2D and rodents expressing human IAPP. Here, we determined that β cell IAPP content is regulated by autophagy through p62-dependent lysosomal degradation. Induction of high levels of human IAPP in mouse β cells resulted in accumulation of this amyloidogenic protein as relatively inert fibrils within cytosolic p62-positive inclusions, which temporarily averts formation of toxic oligomers. Mice hemizygous for transgenic expression of human IAPP did not develop diabetes; however, loss of β cell-specific autophagy in these animals induced diabetes, which was attributable to accumulation of toxic human IAPP oligomers and loss of β cell mass. In human IAPP-expressing mice that lack β cell autophagy, increased oxidative damage and loss of an antioxidant-protective pathway appeared to contribute to increased β cell apoptosis. These findings indicate that autophagy/lysosomal degradation defends β cells against proteotoxicity induced by oligomerization-prone human IAPP.

Figure 9. 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), Atg7^Δβcell (9 weeks, n = 3), and h-IAPP^+/–:Atg7^Δβcell (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 Atg7^Δβcell mice.

Figure 8. 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), Atg7^Δβcell (9 weeks, n = 3), and h-IAPP^+/–:Atg7^Δβcell 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^+/–, Atg7^Δβcell, and h-IAPP^+/–:Atg7^Δβcell mice (NRF2, red; p62, white; nuclei, blue). Scale bar: 50 μm. (C) Images of islets from Atg7^Δβcell and h-IAPP^+/–:Atg7^Δβcell mice showing cytosolic and nuclear staining of NRF2 (NRF2, red; IAPP, green; nuclei, blue). Arrows indicate nuclei. Scale bar: 12 μm.

Figure 7. 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^+/–, Atg7^Δβcell, and h-IAPP^+/–:Atg7^Δβcell 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 Atg7^Δβcell (15 weeks, n = 3) and h-IAPP^+/–:Atg7^Δβcell 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.

Figure 6. 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^+/–, Atg7^Δβcell, and h-IAPP^+/–:Atg7^Δβcell 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^+/–:Atg7^Δβcell mice (both 8 weeks, n = 6) and h-IAPP^+/– and Atg7^Δβcell 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 Atg7^Δβcell 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), Atg7^Δβcell (15 weeks, n = 4), and h-IAPP^+/–:Atg7^Δβcell (12 ± 1 weeks, n = 6) mice. *P < 0.05, versus Atg7^Δβcell 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), Atg7^Δβcell (15 weeks, n = 3), and h-IAPP^+/–:Atg7^Δβcell 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), Atg7^Δβcell (15 weeks, n = 3), and h-IAPP^+/–:Atg7^Δβcell (12 ± 1 weeks, n = 3) mice. *P < 0.05, versus Atg7^Δβcell mice; **P < 0.01, versus h-IAPP^+/– mice. Data are expressed as mean ± SEM.

Figure 5. 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 Atg7^Δβcell (9 weeks, n = 3) mice and control, h-IAPP^+/–, and h-IAPP^+/–:Atg7^Δβcell (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^+/–:Atg7^Δβcell 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^+/–:Atg7^Δβcell mouse pancreatic section stained with anti-oligomer antibody A11 (oligomers, red; p62, green; IAPP, yellow; nuclei, blue). Scale bar: 24 μm.

Figure 4. 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.

Figure 3. 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.

Figure 2. 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.

Figure 1. 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.