UCHL1 deficiency exacerbates human islet amyloid polypeptide toxicity in β-cells: evidence of interplay between the ubiquitin/proteasome system and autophagy - PubMed (original) (raw)

UCHL1 deficiency exacerbates human islet amyloid polypeptide toxicity in β-cells: evidence of interplay between the ubiquitin/proteasome system and autophagy

Safia Costes et al. Autophagy. 2014 Jun.

Abstract

The islet in type 2 diabetes mellitus (T2DM) is characterized by a deficit in β-cells and increased β-cell apoptosis attributable at least in part to intracellular toxic oligomers of IAPP (islet amyloid polypeptide). β-cells of individuals with T2DM are also characterized by accumulation of polyubiquitinated proteins and deficiency in the deubiquitinating enzyme UCHL1 (ubiquitin carboxyl-terminal esterase L1 [ubiquitin thiolesterase]), accounting for a dysfunctional ubiquitin/proteasome system. In the present study, we used mouse genetics to elucidate in vivo whether a partial deficit in UCHL1 enhances the vulnerability of β-cells to human-IAPP (hIAPP) toxicity, and thus accelerates diabetes onset. We further investigated whether a genetically induced deficit in UCHL1 function in β-cells exacerbates hIAPP-induced alteration of the autophagy pathway in vivo. We report that a deficit in UCHL1 accelerated the onset of diabetes in hIAPP transgenic mice, due to a decrease in β-cell mass caused by increased β-cell apoptosis. We report that UCHL1 dysfunction aggravated the hIAPP-induced defect in the autophagy/lysosomal pathway, illustrated by the marked accumulation of autophagosomes and cytoplasmic inclusions positive for SQSTM1/p62 and polyubiquitinated proteins with lysine 63-specific ubiquitin chains. Collectively, this study shows that defective UCHL1 function may be an early contributor to vulnerability of pancreatic β-cells for protein misfolding and proteotoxicity, hallmark defects in islets of T2DM. Also, given that deficiency in UCHL1 exacerbated the defective autophagy/lysosomal degradation characteristic of hIAPP proteotoxicity, we demonstrate a previously unrecognized role of UCHL1 in the function of the autophagy/lysosomal pathway in β-cells.

Keywords: SQSTM1/p62; apoptosis; autophagy; diabetes; islet amyloid polypeptide; ubiquitin carboxyl-terminal esterase L1; β-cell.

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Figures

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Figure 1. UCHL1 activity and expression are decreased in islets isolated from Uchl1nm3419 heterozygous mice (Uchl1+/−). Activity of UCHL1 was assessed by active-site labeling of deubiquitinating enzymes. Islet obtained from 8–10-wk-old WT and Uchl1+/− mice were incubated with HA-Ub-VS and lysates were analyzed by western blotting using anti-HA antibody. Levels of UCHL1 were analyzed by western blotting. Levels of GAPDH were shown as loading control.

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Figure 2. UCHL1 deficiency accelerates diabetes progression in _hIAPP_-Tg mice. Body weight (A) and fasting blood glucose (B) were measured in the 4 groups of mice: wild-type (WT: n = 7); UCHL1 deficient (Uchl1+/−: n = 9), hIAPP transgenic (_hIAPP_-Tg: n = 9) and hIAPP transgenic mice deficient for UCHL1 (_hIAPP_-Tg, Uchl1+/−: n = 17 at 5, 6, and 7 wk; n = 7 at 8 wk). Data are expressed as mean ± SEM; *P < 0.05.

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Figure 3.Uchl1 mRNA and UCHL1 protein levels in mouse islets. (A) UCHL1 protein levels were assessed by western blotting using islet protein lysates obtained from 7–8-wk-old WT, Uchl1+/−, _hIAPP_-Tg, and _hIAPP_-Tg, Uchl1+/− mice (n = 3). GAPDH was used as a control. (B) Levels of Uchl1 mRNA were evaluated by RT-qPCR in islets isolated from 7–8-wk-old WT, Uchl1+/−, _hIAPP_-Tg, and _hIAPP_-Tg, Uchl1+/− mice (n = 2–4). Data are expressed as mean ± SEM. The mouse Ppia (peptidylprolyl isomerase A [cyclophilin A]) gene was used for the ratio.

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Figure 4. Deficiency in UCHL1 decreases β-cell mass but does not alter insulin sensitivity in _hIAPP_-Tg mice. (A) Insulin tolerance tests were performed on 7-wk-old mice of the indicated genotypes: _hIAPP_-Tg (n = 10) and _hIAPP_-Tg, Uchl1+/− mice (n = 14). Results represent the blood glucose concentration as a percentage of the starting glucose value and are expressed as mean ± SEM (B) Representative pancreatic islets immunostained for insulin (brown) and counterstained with hematoxylin (blue) from WT; Uchl1+/−; _hIAPP_-Tg; and _hIAPP_-Tg, Uchl1+/− mice. (C) β-cell mass was evaluated in the 4 groups of 7–8-wk-old mice: WT (n = 4), Uchl1+/− (n = 4), _hIAPP_-Tg (n = 3) and _hIAPP_-Tg, Uchl1+/− mice (n = 4). Data are expressed as mean ± SEM; ***P < 0.001, significant differences vs. _hIAPP_-Tg mice.

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Figure 5. UCHL1 deficiency exacerbates β-cell ER stress and apoptosis in _hIAPP_-Tg mice. (A) β-cell apoptosis (percentage of β-cells positive for TUNEL) was evaluated in 7–8-wk-old WT (n = 3), Uchl1+/− (n = 3), _hIAPP_-Tg (n = 3) and _hIAPP_-Tg, Uchl1+/− mice (n = 3). (B) Protein levels of cleaved CASP3 (Cl. CASP3) were assessed by western blotting using islet protein lysates obtained from 7–8-wk-old WT (n = 2), Uchl1+/− (n = 4), _hIAPP_-Tg (n = 4) and _hIAPP_-Tg, Uchl1+/− mice (n = 5). Insulin and GAPDH were used as controls. (C) β-cell ER stress (percentage of β-cells positive for nuclear DDIT3) was evaluated in 7–8-wk-old WT (n = 3), Uchl1+/− (n = 3), _hIAPP_-Tg (n = 3) and _hIAPP_-Tg, Uchl1+/− mice (n = 3). Data are expressed as mean ± SEM; *P < 0.05, significant differences vs. _hIAPP_-Tg mice.

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Figure 6. UCHL1 deficiency exacerbates defects in lysosomal degradation in _hIAPP_-Tg mouse islets. Protein levels of LC3 (A) and SQSTM1 (B) were assessed by western blotting using islet protein lysates obtained from 7–8-wk-old WT (n = 2), Uchl1+/− (n = 2), _hIAPP_-Tg (n = 2) and _hIAPP_-Tg, Uchl1+/− mice (n = 3). GAPDH was used as a control. The graph represents the quantification of LC3-II protein levels. (C) SQSTM1 protein levels were assessed by immunofluorescence (SQSTM1, red; insulin, green; nuclei, blue) in pancreatic tissue from 7–8-wk-old _hIAPP_-Tg mice and _hIAPP_-Tg, Uchl1+/− mice. (D) The graph represents the quantification of β-cells positive for SQSTM1 in each group (expressed in %). (E) The graph represents the quantification of β-cell area positive for SQSTM1 aggregates in each group (expressed in %). Data are expressed as mean ± SEM; **P < 0.01; ***P < 0.001, significant differences vs. _hIAPP_-Tg mice.

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Figure 7. (A) Fluorescence confocal images of LC3 and SQSTM1 at magnification x 63 (LC3, red; SQSTM1, green; insulin, yellow; nuclei, blue) in pancreatic tissue from 7–8-wk-old _hIAPP_-Tg, Uchl1+/− mice. (B) The detection and localization of ubiquitin lysine 63 chains were assessed by immunofluorescence (ubiquitin K63, red; SQSTM1, green; insulin, white; nuclei, blue) in pancreatic tissue from 7–8-wk-old _hIAPP_-Tg mice and _hIAPP_-Tg, Uchl1+/− mice.

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Figure 8. UCHL1 deficiency increases IAPP/proIAPP protein levels in _hIAPP_-Tg mouse islets. Protein levels of IAPP were assessed by western blotting using islet protein lysates obtained from 7–8-wk-old _hIAPP_-Tg mice (n = 3) and _hIAPP_-Tg, Uchl1+/− mice (n = 5). GAPDH was used as a control. The slowly migrating bands represent unprocessed and partially processed proIAPP (8 and 6 kDa, respectively). The graph represents the quantification of IAPP protein levels. Data are expressed as mean ± SEM; *P < 0.05.

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References

    1. Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes. 2003;52:102–10. doi: 10.2337/diabetes.52.1.102. - DOI - PubMed
    1. Haataja L, Gurlo T, Huang CJ, Butler PC. Islet amyloid in type 2 diabetes, and the toxic oligomer hypothesis. Endocr Rev. 2008;29:303–16. doi: 10.1210/er.2007-0037. - DOI - PMC - PubMed
    1. Gurlo T, Ryazantsev S, Huang CJ, Yeh MW, Reber HA, Hines OJ, O’Brien TD, Glabe CG, Butler PC. Evidence for proteotoxicity in beta cells in type 2 diabetes: toxic islet amyloid polypeptide oligomers form intracellularly in the secretory pathway. Am J Pathol. 2010;176:861–9. doi: 10.2353/ajpath.2010.090532. - DOI - PMC - PubMed
    1. Huang CJ, Haataja L, Gurlo T, Butler AE, Wu X, Soeller WC, Butler PC. Induction of endoplasmic reticulum stress-induced beta-cell apoptosis and accumulation of polyubiquitinated proteins by human islet amyloid polypeptide. Am J Physiol Endocrinol Metab. 2007;293:E1656–62. doi: 10.1152/ajpendo.00318.2007. - DOI - PubMed
    1. Huang CJ, Lin CY, Haataja L, Gurlo T, Butler AE, Rizza RA, Butler PC. High expression rates of human islet amyloid polypeptide induce endoplasmic reticulum stress mediated beta-cell apoptosis, a characteristic of humans with type 2 but not type 1 diabetes. Diabetes. 2007;56:2016–27. doi: 10.2337/db07-0197. - DOI - PubMed

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