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

Costes S, Huang CJ, Gurlo T, Daval M, Matveyenko AV, Rizza RA, Butler AE, Butler PC - Diabetes (2010)

Bottom Line: In this study, we investigated whether human-IAPP (h-IAPP) disrupts the endoplasmic reticulum-associated degradation/ubiquitin/proteasome system.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.

View Article: PubMed Central - PubMed

Affiliation: Larry Hillblom Islet Research Center, University of California, Los Angeles, USA. scostes@mednet.ucla.edu

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.

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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.)
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Figure 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.)

Mentions: To test the hypothesis that the ERAD/ubiquitin/proteasome system is dysfunctional in type 2 diabetes, we first examined pancreatic tissue and isolated islets from humans with type 2 diabetes versus BMI-matched control subjects (supplementary Tables 1 and 2). The percentage of β-cells immunostained for ubiquitin was unchanged by obesity in nondiabetic individuals (6.8 ± 2.9% vs. 2.1 ± 1.3%, P = NS, obese nondiabetic vs. lean nondiabetic) (Fig. 1A and B), implying that in health, the β-cell ERAD/ubiquitin/proteasome system adapts to the increased synthetic burden of insulin and IAPP prompted by obesity-mediated insulin resistance (6,7). In contrast, the percentage of β-cells with ubiquitin immunostaining was increased (approximately fourfold) in obese individuals with type 2 diabetes treated with diet and/or sulfonylureas in comparison to obese nondiabetic individuals (25 ± 6% vs. 6.8 ± 2%, P < 0.05) (Fig. 1A and B). Ubiquitin immunostaining in islets was mostly cytoplasmic, although nuclear staining was also found in some cells, consistent with previous reports documenting the presence of components of the ubiquitin-proteasome pathway in both cytoplasmic and nuclear compartments (34) and implying that defective protein degradation may also alter clearance of nuclear proteins.


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

Costes S, Huang CJ, Gurlo T, Daval M, Matveyenko AV, Rizza RA, Butler AE, Butler PC - Diabetes (2010)

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.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3012175&req=5

Figure 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.)
Mentions: To test the hypothesis that the ERAD/ubiquitin/proteasome system is dysfunctional in type 2 diabetes, we first examined pancreatic tissue and isolated islets from humans with type 2 diabetes versus BMI-matched control subjects (supplementary Tables 1 and 2). The percentage of β-cells immunostained for ubiquitin was unchanged by obesity in nondiabetic individuals (6.8 ± 2.9% vs. 2.1 ± 1.3%, P = NS, obese nondiabetic vs. lean nondiabetic) (Fig. 1A and B), implying that in health, the β-cell ERAD/ubiquitin/proteasome system adapts to the increased synthetic burden of insulin and IAPP prompted by obesity-mediated insulin resistance (6,7). In contrast, the percentage of β-cells with ubiquitin immunostaining was increased (approximately fourfold) in obese individuals with type 2 diabetes treated with diet and/or sulfonylureas in comparison to obese nondiabetic individuals (25 ± 6% vs. 6.8 ± 2%, P < 0.05) (Fig. 1A and B). Ubiquitin immunostaining in islets was mostly cytoplasmic, although nuclear staining was also found in some cells, consistent with previous reports documenting the presence of components of the ubiquitin-proteasome pathway in both cytoplasmic and nuclear compartments (34) and implying that defective protein degradation may also alter clearance of nuclear proteins.

Bottom Line: In this study, we investigated whether human-IAPP (h-IAPP) disrupts the endoplasmic reticulum-associated degradation/ubiquitin/proteasome system.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.

View Article: PubMed Central - PubMed

Affiliation: Larry Hillblom Islet Research Center, University of California, Los Angeles, USA. scostes@mednet.ucla.edu

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.

Show MeSH
Related in: MedlinePlus