Human-IAPP disrupts the autophagy/lysosomal pathway in pancreatic β-cells: protective role of p62-positive cytoplasmic inclusions - PubMed (original) (raw)

Human-IAPP disrupts the autophagy/lysosomal pathway in pancreatic β-cells: protective role of p62-positive cytoplasmic inclusions

J F Rivera et al. Cell Death Differ. 2011 Mar.

Abstract

In type II diabetes (T2DM), there is a deficit in β-cells, increased β-cell apoptosis and formation of intracellular membrane-permeant oligomers of islet amyloid polypeptide (IAPP). Human-IAPP (h-IAPP) is an amyloidogenic protein co-expressed with insulin by β-cells. IAPP expression is increased with obesity, the major risk factor for T2DM. In this study we report that increased expression of human-IAPP led to impaired autophagy, due at least in part to the disruption of lysosome-dependent degradation. This action of IAPP to alter lysosomal clearance in vivo depends on its propensity to form toxic oligomers and is independent of the confounding effect of hyperglycemia. We report that the scaffold protein p62 that delivers polyubiquitinated proteins to autophagy may have a protective role against human-IAPP-induced apoptosis, apparently by sequestrating protein targets for degradation. Finally, we found that inhibition of lysosomal degradation increases vulnerability of β-cells to h-IAPP-induced toxicity and, conversely, stimulation of autophagy protects β-cells from h-IAPP-induced apoptosis. Collectively, these data imply an important role for the p62/autophagy/lysosomal degradation system in protection against toxic oligomer-induced apoptosis.

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Figures

Figure 1

High expression of h-IAPP increases LC3-II and p62 protein levels and induces p62-positive cytoplasmic inclusions in HIP rats. (a) Protein levels of LC3, p62 and IAPP were assessed by western blot using islet protein lysates obtained from 4- to 6-month-old wild type (WT, _n_=6) and HIP rats (HIP, _n_=6). GAPDH was used as loading control. The graph represents the quantification of p62 protein levels. (b) Levels of p62 mRNA were evaluated by RT-qPCR in islets isolated from WT (_n_=3) and HIP rats (_n_=3). Data are expressed as mean±S.E.M.; *P<0.05. (c) p62 protein levels were assessed by immunofluorescence (p62, red; insulin, green; nuclei, blue) in pancreatic tissue from 4- to 6-month-old WT and HIP rats. High magnification of p62-positive structures found in HIP rats (i, ii and iii)

Figure 2

High expression of h-IAPP, but not r-IAPP, increases LC3-II and p62 protein levels and induces formation of p62-positive cytoplasmic inclusions in h-IAPP homozygous transgenic mice. (a) Protein levels of LC3 and p62 were assessed by western blot using islet protein lysates obtained from WT (WT, _n_=4), r-IAPP transgenic (r-TG, _n_=3) and h-IAPP transgenic mice (h-TG, _n_=4). GAPDH was used as loading control. The graph represents the quantification of LC3-II protein levels. (b) The graph represents the quantification of p62 protein levels. (c) Levels of p62 mRNA were evaluated by RT-qPCR in islets isolated from WT (_n_=3), r-TG (_n_=3) and h-TG mice (_n_=3). Data are expressed as mean±S.E.M.; *P<0.05. (d) p62 protein levels were assessed by immunofluorescence (p62, red; insulin, green; nuclei, blue) in pancreatic tissue from WT, r-TG and h-TG mice. High magnification of p62-positive structures found in h-TG mice (i, ii and iii)

Figure 3

Interaction of obesity and h-IAPP expression, adaptive and maladaptive changes in autophagy. (a) Protein levels of LC3 and p62 were assessed by western blot using islet protein lysates obtained from lean non-transgenic (LNT, _n_=5), lean transgenic (LT, _n_=4), obese non-transgenic (ONT, _n_=5) and obese transgenic (OT, _n_=4–6) 10- to 12-week-old mice. Insulin and GAPDH were used as control. The graph represents the quantification of LC3-II protein levels. (b) The graph represents the quantification of p62 protein levels. (c) Levels of p62 mRNA were evaluated by RT-qPCR in islets isolated from LNT (_n_=5), LT (_n_=4), ONT (_n_=5) and OT mice (_n_=5). Data are expressed as mean±S.E.M.; *P<0.05; **P<0.01; ***P<0.001. (d) p62 protein levels were assessed by immunofluorescence (p62, red; insulin, green; nuclei, blue) in pancreatic tissue from LNT, LT, ONT and OT 10- to 12-week-old mice. (e) The graph represents the quantification of _β_-cells positive for p62 in each group (expressed in percentage). (f) The graph represents the quantification of _β_-cell area positive for p62 aggregates in each group (expressed in percentage). Data are expressed as mean±S.E.M.; *P<0.05; **P<0.01. The mathematical transformation ‘logarithm (value+1)' was applied before statistical analysis of LC3-II, p62 protein levels and _β_-cells positive for p62. (g) Fluorescence confocal images of p62 at magnification × 63 (p62, red; insulin, green) in pancreatic tissue from LNT, LT, ONT and OT 10- to 12-week-old mice

Figure 3

Figure 4

High expression of h-IAPP in INS 832/13 cells increases LC3-II and affects p62 protein levels. INS 832/13 cells were transduced at 400 MOI with r-IAPP or h-IAPP adenoviruses and cell lysates were collected at different time points as indicated (time 0, non-transduced cells). Protein levels of LC3, p62, cleaved caspase-3 (Cl. Casp-3) and IAPP were assessed by western blot. GAPDH was used as loading control. The graphs represent the quantification of LC3-II and p62 protein levels (_n_=4). Data are expressed as mean±S.E.M.; *P<0.05; **P<0.01; ***P<0.001, significant differences versus non-transduced cells (Ctrl)

Figure 5

Downregulation of p62 by shRNA lentivirus induces apoptosis in INS 832/13 cells and exacerbates h-IAPP-induced _β_-cell apoptosis. (a) INS 832/13 cells were transduced for 72 h with control shRNA or p62 shRNA lentivirus at increasing concentrations. Levels of p62 and cleaved caspase-3 (Cl. Casp-3) were analyzed by western blot. GAPDH was used as loading control. The graphs represent the quantification of p62 protein levels and the cleaved form of caspase-3 (_n_=3). Data are expressed as mean±S.E.M.; *P<0.05; **P<0.01; ***P<0.001, significant differences versus Ctrl shRNA-transduced cells (at the corresponding concentration). (b) INS 832/13 cells were transduced for 48 h with control shRNA or p62 shRNA lentivirus (0.1 _μ_g/ml) and transduced at 400 MOI with r-IAPP (R) or h-IAPP (H) adenoviruses for the last 27 h (C, non-transduced cells). Levels of cleaved caspase-3 and p62 were analyzed by western blot. GAPDH was used as loading control. The graph represents the quantification of the cleaved form of caspase-3 (_n_=4). Data are expressed as mean±S.E.M.; ***P<0.001

Figure 6

Overexpression of p62 in INS 832/13 cells reduces h-IAPP-induced _β_-cell apoptosis. (a) INS 832/13 cells were transfected with GFP plasmid or p62-GFP plasmid (0.1_μ_g) for 24 h and observed under fluorescence microscope (p62, green; nuclei, blue). (b) INS 832/13 cells were transduced at 400 MOI with r-IAPP (R) or h-IAPP (H) adenoviruses for 36 h (C, non-transduced cells). Cells were transfected with GFP plasmid or p62-GFP plasmid (0.1 _μ_g) for the last 22 h. Levels of p62 and cleaved caspase-3 (Cl. Casp-3) were analyzed by western blot. GAPDH was used as loading control. The graph represents the quantification of the cleaved form of caspase-3 (_n_=4). Data are expressed as mean±S.E.M.; *P<0.05

Figure 7

Inhibition of lysosomal degradation exacerbates h-IAPP-induced _β_-cell apoptosis in INS 832/13 cells and rat islets. (a) INS 832/13 cells were transduced at 400 MOI with r-IAPP (R) or h-IAPP (H) adenoviruses for 36 h (C, non-transduced cells) and treated with lysosomal inhibitors (Lyso I) (E-64-d, 10 _μ_g/ml and pepstatin A, 10 _μ_g/ml) for the last 24 h. Levels of cleaved caspase-3 (Cl. Casp-3) were analyzed by western blot. GAPDH was used as loading control. The graph represents the quantification of the cleaved form of caspase-3 (_n_=4). Data are expressed as mean±S.E.M.; *P<0.05. (b) Isolated islets obtained from wild-type (WT: _n_=3) and HIP rats (HIP: _n_=5) were treated with lysosomal inhibitors (E-64-d, 10 _μ_g/ml and pepstatin A, 10 _μ_g/ml) for 24 h. Levels of cleaved caspase-3 and ubiquitin were analyzed by western blot. GAPDH was used as loading control. The graph represents the quantification of the cleaved form of caspase-3. Data are expressed as mean±S.E.M.; **P<0.01

Figure 8

Enhanced autophagy by treatment with rapamycin decreases h-IAPP-induced apoptosis in INS 832/13 cells. INS 832/13 cells were transduced at 400 MOI with r-IAPP (R) or h-IAPP (H) adenoviruses for 48 h (C, non-transduced cells) and treated with rapamycin (Rapa, 10 n) for the last 40 h. Levels of P-mTOR and cleaved caspase-3 (Cl. Casp-3) were analyzed by western blot. GAPDH was used as loading control. The graph represents the quantification of the cleaved form of caspase-3 (_n_=4). Data are expressed as mean±S.E.M.; **P<0.01; ***P<0.001

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Human-IAPP disrupts the autophagy/lysosomal pathway in pancreatic β-cells: protective role of p62-positive cytoplasmic inclusions - PubMed (original) (raw)