β-cell dysfunctional ERAD/ubiquitin/proteasome system in type 2 diabetes mediated by islet amyloid polypeptide-induced UCH-L1 deficiency

Abstract

Objective: The islet in type 2 diabetes is characterized by β-cell apoptosis, β-cell endoplasmic reticulum stress, and islet amyloid deposits derived from islet amyloid polypeptide (IAPP). Toxic oligomers of IAPP form intracellularly in β-cells in humans with type 2 diabetes, suggesting impaired clearance of misfolded proteins. In this study, we investigated whether human-IAPP (h-IAPP) disrupts the endoplasmic reticulum-associated degradation/ubiquitin/proteasome system.

Research design and methods: We used pancreatic tissue from humans with and without type 2 diabetes, isolated islets from h-IAPP transgenic rats, isolated human islets, and INS 832/13 cells transduced with adenoviruses expressing either h-IAPP or a comparable expression of rodent-IAPP. Immunofluorescence and Western blotting were used to detect polyubiquitinated proteins and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) protein levels. Proteasome activity was measured in isolated rat and human islets. UCH-L1 was knocked down by small-interfering RNA in INS 832/13 cells and apoptosis was evaluated.

Results: We report accumulation of polyubiquinated proteins and UCH-L1 deficiency in β-cells of humans with type 2 diabetes. These findings were reproduced by expression of oligomeric h-IAPP but not soluble rat-IAPP. Downregulation of UCH-L1 expression and activity to reproduce that caused by h-IAPP in β-cells induced endoplasmic reticulum stress leading to apoptosis.

Conclusions: Our results indicate that defective protein degradation in β-cells in type 2 diabetes can, at least in part, be attributed to misfolded h-IAPP leading to UCH-L1 deficiency, which in turn further compromises β-cell viability.

FIG. 1.

Accumulation of polyubiquitinated proteins in β-cells of humans with type 2 diabetes abrogated by insulin therapy. A: The detection and localization of ubiquitinated proteins was assessed by immunofluorescence (ubiquitin, red; insulin, green; nuclei, blue) in human pancreatic tissue obtained at autopsy from lean nondiabetic (a), obese nondiabetic (b), obese subjects with type 2 diabetes treated with diet and/or oral medications (nontreated with insulin) (c), and obese subjects with type 2 diabetes treated with insulin (d). B: Percentage of β-cells positive for ubiquitin in each group. LND, lean nondiabetic; OD, obese subjects with type 2 diabetes; OND, obese nondiabetic. Data are expressed as means ± SE. *P < 0.05; **P < 0.01. C: Accumulation of polyubiquitinated proteins was assessed by Western blot in islets isolated from obese nondiabetic and obese subjects with type 2 diabetes. Actin was used as loading control. Ubiquitin and actin images were obtained by grouping different parts of the same gel. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Accumulation of ubiquitinated proteins in β-cells of HIP rats. A: The presence and localization of ubiquitinated proteins were assessed by immunofluorescence (ubiquitin, red; insulin, green; nuclei, blue) in pancreatic tissue obtained from wild-type rats (WT; n = 4), wild-type rats on HFD for 10 weeks (WT+HFD; n = 4), HIP rats (HIP; n = 5), and HIP rats on HFD for 10 weeks (HIP+HFD; n = 5). B: The graph represents the quantification of β-cells positive for ubiquitin in each group (expressed in percentages). Data are expressed as means ± SE. *P < 0.05; **P < 0.01; ###P < 0.001, significant differences vs. WT+HFD. C: Proteasome activity was evaluated in islets isolated from 4- to 6-month-old wild-type rats (WT; n = 9), wild-type rats on HFD for 10 weeks (WT+HFD; n = 4), HIP rats (n = 9), and HIP rats on HFD for 10 weeks (HIP+HFD; n = 5). The activity was normalized to the total protein content. The proteasome inhibitor, lactacystin (Lacta; 25 μmol/l) was used as negative control. Data are expressed as means ± SE. *P < 0.05; ***P < 0.001, significant differences vs. wild type. ns, nonsignificant. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Expression of h-IAPP decreases UCH-L1 levels in HIP rat islets. A: The detection of polyubiquitinated proteins was assessed by Western blot using islet protein lysates obtained from 4- to 6-month-old wild-type (WT; n = 5) and HIP rats (HIP; n = 5). IAPP and actin were used as control. Ubiquitin image was obtained by grouping different parts of the same gel. B: Protein levels of UCH-L1, CHOP, and cleaved caspase-3 (Cl. caspase-3) were assessed by Western blot using islet protein lysates obtained from wild-type (n = 5) and HIP (n = 5) rats. Insulin and actin were used as control. The graph represents the quantification of UCH-L1 protein levels. C: Levels of UCH-L1 mRNA were evaluated by RT-qPCR in islets isolated from wild-type (n = 9) and HIP (n = 9) rats. Data are expressed as means ± SE. *P < 0.05; **P < 0.01.

FIG. 4.

Expression of h-IAPP, but not r-IAPP, decreases UCH-L1 protein levels in INS 832/13 cells. INS 832/13 cells were transduced at 400 MOI for 48 h with r-IAPP (R) or h-IAPP (H) adenoviruses (Ctrl, nontransduced cells). Protein levels of UCH-L1 and cleaved caspase-3 were analyzed by Western blot. GAPDH was used as loading control. The graph represents the quantification of UCH-L1 protein levels (n = 4). Data are expressed as means ± SE. *P < 0.05.

FIG. 5.

Inhibition of UCH-L1 activity leads to ER stress and apoptosis in INS 832/13 cells. A: INS 832/13 cells were treated for 18 h with UCH-L1 inhibitor, LDN-57444, at increasing concentrations. Apoptosis was assessed by measuring caspase-3 activity in lysates. The activity was normalized to the total protein content. Data are expressed as means ± SE (n = 4). ***P < 0.001, significant differences vs. cells treated with vehicle DMSO (Ctrl). B: INS 832/13 cells were treated or not with LDN-57444 (50 μmol/l) for 18 h. Levels of CHOP and cleaved caspase-3 were analyzed by Western blot. Actin was shown as loading control. C: INS 832/13 cells were treated with LDN-57444 (50 μmol/l) for 8 h (b) or not treated (a). The detection of CHOP (arrows) was assessed by immunofluorescence (CHOP, red; nuclei, blue) (n = 3). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

Downregulation of UCH-L1 by siRNA induces ER stress and apoptosis in INS 832/13 cells. A: INS 832/13 cells were transfected for 48 h with scramble or UCH-L1 siRNA (25 nmol/l). Levels of UCH-L1, PARP, and cleaved caspase-3 were analyzed by Western blot. Activity of UCH-L1 was assessed by active-site labeling of deubiquitinating enzymes. The preparations were incubated with HA-Ub-VS and probed with the anti-HA. Levels of GAPDH were shown as loading control. B: The graphs represent the quantification of UCH-L1 expression, UCH-L1 activity, and the cleaved form of PARP. Data are expressed as means ± SE (n = 3–4). *P < 0.05; **P < 0.01. C: INS 832/13 cells were transfected for 48 h with scramble (a) or UCH-L1 siRNA (25 nmol/l) (b). The detection of CHOP (arrows) was assessed by immunofluorescence (CHOP, red; insulin, green; nuclei, blue) (n = 3). (A high-quality digital representation of this figure is available in the online issue.)

FIG. 7.

Expression of h-IAPP in human islets leads to accumulation of polyubiquitinated proteins associated with decrease of UCH-L1. A: Human islets were transduced at 400 MOI for 72 h with r-IAPP (R) or h-IAPP (H) adenoviruses. Accumulation of polyubiquitinated proteins and IAPP expression were assessed by Western blot. Insulin and actin were used as control. Ubiquitin and actin images were obtained by grouping different parts of the same gel. The graph represents the quantification of the Western blot (n = 3). B: Proteasome activity was measured by the 20S proteasome activity assay (n = 4). The proteasome inhibitor, lactacystin (Lacta, 25 μmol/l), was used as negative control. C: Protein level of UCH-L1 was analyzed by Western blot. Actin was used as loading control. The graph represents the quantification of the Western blot (n = 3). Data are expressed as means ± SE. *P < 0.05; **P < 0.01.

FIG. 8.

UCH-L1 deficiency is characteristic to human islets in type 2 diabetes. A: UCH-L1 protein expression was analyzed by Western blot in islets isolated from obese nondiabetic (OND) and obese subjects with type 2 diabetes (OD). Levels of β-tubulin and insulin were used as control. The graph represents the quantification of the Western blot. Data are expressed as means ± SE. *P < 0.05. B: UCH-L1 protein level was assessed by immunofluorescence (UCH-L1, red; insulin, green; nuclei, blue) in surgical pancreatic tissue from obese nondiabetic (OND) and obese subjects with type 2 diabetes (OD). (A high-quality digital representation of this figure is available in the online issue.)