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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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Related in: MedlinePlus

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.
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Figure 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.

Mentions: To establish whether UCH-L1 downregulation is caused by h-IAPP, we studied INS 832/13 cells transduced with adenoviruses expressing h-IAPP or the nonamyloidogenic and nontoxic rodent IAPP (r-IAPP), as control for a comparable burden of protein expression (33). Cells transduced with h-IAPP (400 MOI, 48 h) exhibited a 31 ± 10% decrease in UCH-L1 protein expression in comparison to r-IAPP-transduced cells (P < 0.05) (Fig. 4). Therefore, high expression rates of h-IAPP lead to UCH-L1 downregulation in β-cells, and this effect is due to the amyloidogenic properties of h-IAPP rather than just protein overload.


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)

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

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

Figure 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.
Mentions: To establish whether UCH-L1 downregulation is caused by h-IAPP, we studied INS 832/13 cells transduced with adenoviruses expressing h-IAPP or the nonamyloidogenic and nontoxic rodent IAPP (r-IAPP), as control for a comparable burden of protein expression (33). Cells transduced with h-IAPP (400 MOI, 48 h) exhibited a 31 ± 10% decrease in UCH-L1 protein expression in comparison to r-IAPP-transduced cells (P < 0.05) (Fig. 4). Therefore, high expression rates of h-IAPP lead to UCH-L1 downregulation in β-cells, and this effect is due to the amyloidogenic properties of h-IAPP rather than just protein overload.

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