Human IAPP-induced pancreatic β cell toxicity and its regulation by autophagy

Abstract

Pancreatic islets in patients with type 2 diabetes mellitus (T2DM) are characterized by loss of β cells and formation of amyloid deposits derived from islet amyloid polypeptide (IAPP). Here we demonstrated that treatment of INS-1 cells with human IAPP (hIAPP) enhances cell death, inhibits cytoproliferation, and increases autophagosome formation. Furthermore, inhibition of autophagy increased the vulnerability of β cells to the cytotoxic effects of hIAPP. Based on these in vitro findings, we examined the pathogenic role of hIAPP and its relation to autophagy in hIAPP-knockin mice. In animals fed a standard diet, hIAPP had no toxic effects on β cell function; however, hIAPP-knockin mice did not exhibit a high-fat-diet-induced compensatory increase in β cell mass, which was due to limited β cell proliferation and enhanced β cell apoptosis. Importantly, expression of hIAPP in mice with a β cell-specific autophagy defect resulted in substantial deterioration of glucose tolerance and dispersed cytoplasmic expression of p62-associated toxic oligomers, which were otherwise sequestrated within p62-positive inclusions. Together, our results indicate that increased insulin resistance in combination with reduced autophagy may enhance the toxic potential of hIAPP and enhance β cell dysfunction and progression of T2DM.

Figure 7. hIAPP inhibits formation of cytoplasmic inclusions immunopositive for p62 in Atg7^fl/fl:Cre islets.

(A–D) Representative section of the pancreas of 22-week-old high-fat diet–fed Atg7^fl/fl (A), hIAPP:Atg7^fl/fl (B), Atg7^fl/fl:Cre (C), and hIAPP:Atg7^fl/fl:Cre (D) mice, immunohistochemically stained for p62. The p62-positive signal accumulated as inclusions (arrows) within islet cells of Atg7^fl/fl:Cre mice, but were dispersed throughout the cytoplasm in hIAPP:Atg7^fl/fl:Cre mice. Scale bar: 200 μm. (E–H) Confocal images of pancreas sections from 22-week-old high-fat diet–fed Atg7^fl/fl (E), hIAPP:Atg7^fl/fl (F), Atg7^fl/fl:Cre (G), and hIAPP:Atg7^fl/fl:Cre (H) mice stained for p62, A11, and cell nuclei (blue). Staining of toxic oligomer was intense in Atg7^fl/fl:Cre and hIAPP:Atg7^fl/fl:Cre islets, but faint in Atg7^fl/fl and hIAPP:Atg7^fl/fl islets. p62 was largely colocalized with A11-positive staining in Atg7^fl/fl:Cre and hIAPP:Atg7^fl/fl:Cre islets. Scale bar: 50 μm.

Figure 6. Islet morphology.

Shown is the morphology of representative islets from Atg7^fl/fl (A and E), hIAPP:Atg7^fl/fl (B and F), hIAPP:Atg7^fl/fl (C and G), and hIAPP:Atg7^fl/fl:Cre (D and H) mice fed high-fat diet from 8 to 20 weeks of age. Arrowheads indicate degenerative “balloon-like” β cells. (A–D) H&E staining. (E–H) Insulin staining. Scale bars: 50 μm.

Figure 5. Effect of hIAPP knockin and autophagy deficiency on β cell mass.

(A) β cell mass of Atg7^fl/fl, Atg7^fl/fl:Cre, hIAPP:Atg7^fl/fl, and hIAPP:Atg7^fl/fl:Cre mice fed standard (STD) or high-fat (HF) diet. (B and C) Number of cleaved caspase-3–positive (B) and Ki-67–positive (C) intraislet cells in high-fat diet–fed mice of each genotype. In A–C, 5 mice per genotype were prepared for morphometric analysis. Data are mean ± SEM. ^§P < 0.05, ^§§P < 0.01, standard vs. high-fat; *P < 0.05, **P < 0.01, Atg7^fl/fl vs. Atg7^fl/fl:Cre; ^†P < 0.05, hIAPP:Atg7^fl/fl vs. hIAPP:Atg7^fl/fl:Cre; ^#P < 0.05, Atg7^fl/fl vs. hIAPP:Atg7^fl/fl; ^¶P < 0.05, Atg7^fl/fl:Cre vs. hIAPP:Atg7^fl/fl:Cre.

Figure 4. Physiological parameters after adjustment for body weight.

Atg7^fl/fl (n = 9), Atg7^fl/fl:Cre (n = 11), hIAPP:Atg7^fl/fl (n = 10), and hIAPP:Atg7^fl/fl:Cre (n = 11) mice were fed high-fat diet from 8 to 20 weeks of age as in Figure 3, E–H. Adjusted values for (A) body weight, (B) nonfasting blood glucose, and (C) glucose tolerance correspond to results in Figure 3, E, G, and H, respectively. Data are mean ± SEM. **P < 0.01, Atg7^fl/fl vs. Atg7^fl/fl:Cre; ^†P < 0.05, ^††P < 0.01, hIAPP:Atg7^fl/fl vs. hIAPP:Atg7^fl/fl:Cre; ^#P < 0.05, Atg7^fl/fl vs. hIAPP:Atg7^fl/fl; ^¶P < 0.05, Atg7^fl/fl:Cre vs. hIAPP:Atg7^fl/fl:Cre.

Figure 3. Physiological parameters.

Mice were fed standard diet (A–D) or high-fat diet (E–H) from 8 to 20 weeks of age. Shown are body weight (A and E), food intake (B and F), nonfasting blood glucose levels (C and G), and glucose tolerance (D and H) of Atg7^fl/fl (n = 31 [standard], 49 [high-fat]), Atg7^fl/fl:Cre (n = 38 [standard], 39 [high-fat]), hIAPP:Atg7^fl/fl (n = 31 [standard], 32 [high-fat]), and hIAPP:Atg7^fl/fl:Cre (n = 19 [standard], 27 [high-fat]) mice. Data are mean ± SEM. *P < 0.05, **P < 0.01, Atg7^fl/fl vs. Atg7^fl/fl:Cre; ^†P < 0.05, ^††P < 0.01, hIAPP:Atg7^fl/fl vs. hIAPP:Atg7^fl/fl:Cre; ^#P < 0.05, ^##P < 0.01, Atg7^fl/fl vs. hIAPP:Atg7^fl/fl; ^¶P < 0.05, ^¶¶P < 0.01, Atg7^fl/fl:Cre vs. hIAPP:Atg7^fl/fl:Cre.

Figure 2. Effects of hIAPP/rIAPP and autophagy deficiency on cell cycle and viability of INS-1 cells.

(A) Association of reduced ATG7 with accumulation of p62 in Atg7-KD INS-1 cells with tetracycline supplementation. GAPDH served as loading control. (B) INS-1 and Atg7-KD INS-1 cells were cultured in the presence of 0–4 μM rIAPP or hIAPP peptide for 48 hours. The number of cells not stained with Trypan Blue was counted; cell number is presented as percentage of control (0 μM IAPP). *P < 0.05, **P < 0.01, INS-1 rIAPP vs. INS-1 hIAPP; ^††P < 0.01, INS-1 rIAPP vs. Atg7-KD rIAPP; ^##P < 0.01, Atg7-KD rIAPP vs. Atg7-KD hIAPP; ^¶P < 0.05, INS-1 hIAPP vs. Atg7-KD hIAPP. (C and D) FACS histograms (C) and stacked bar graphs (D) of INS-1 cells grown under conditions of growth arrest (induced by exposure to 2.8 mMol/l glucose and serum withdrawal for >24 hours) and after release from arrest (by 22.2 mMol/l glucose, 200 nMol/l insulin, hIAPP/rIAPP, and serum withdrawal medium for 24 hours).

Figure 1. Induction of autophagy by hIAPP.

(A) EM examination showed autophagosome formation in INS-1 cells treated for 12 hours with rIAPP or hIAPP peptide. Arrowheads indicate autophagosomes. Scale bar: 2 μm. (B) Number of autophagosomes per cell. **P < 0.01, rIAPP vs. hIAPP. (C) Protein levels of LC3, p62, and GAPDH, assessed by immunoblotting. To visualize accumulation of LC3-II, the lysosomal inhibitors pepstatin A and E64d were added to the culture medium for an additional 6 hours. (D–G) EM examination showed autophagosome formation in β cells of 22-week-old Atg7^fl/fl (D and E) and hIAPP:Atg7^fl/fl (F and G) mice fed high-fat diet from 8 to 20 weeks of age. (E and G) Magnified images of autophagosomes (arrows) in D and F, respectively. Scale bars: 2 μm. (H) Autophagosome counts. ^#P < 0.05, Atg7^fl/fl vs. hIAPP:Atg7^fl/fl.