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

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.

Figure 1. UCHL1 activity and expression are decreased in islets isolated from Uchl1^nm3419 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.

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.

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.

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.

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.

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.

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.

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.