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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 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.)
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Figure 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.)

Mentions: We studied 4- to 6-month-old h-IAPP transgenic rats (HIP rat) versus wild-type rats. At this age, HIP rats have increased β-cell apoptosis but do not yet have diabetes, allowing us to avoid the confounding effects of glucose toxicity (research design and methods) (10,14). The percentage of β-cells immunostained for ubiquitin was increased in HIP rats (32 ± 5% vs. 8 ± 5%, P < 0.01) (Fig. 2A and B). The staining was mostly cytoplasmic, although nuclear staining was also evident in some cells (Fig. 2A). To draw a parallel between obese individuals with type 2 diabetes and the rat model, we also examined pancreatic sections from HFD-fed HIP rats (6). Within this group of HFD-fed HIP rats, which have diabetes and are insulin resistant, the percentage of β-cells stained for ubiquitin was increased in comparison to HIP rats fed with a regular diet (50 ± 3% vs. 32 ± 5%, P < 0.05) (Fig. 2B). Ubiquitin immunostaining was predominantly cytoplasmic, although nuclear staining was present in some cells, comparable to islets in type 2 diabetes (Fig. 1A). Impaired proteasome function may cause accumulation of ubiquitinated proteins (18). However, proteasome activity was comparable in islets of HIP, HFD-fed HIP, and wild-type rats (Fig. 2C).


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 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.)
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Related In: Results  -  Collection

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Figure 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.)
Mentions: We studied 4- to 6-month-old h-IAPP transgenic rats (HIP rat) versus wild-type rats. At this age, HIP rats have increased β-cell apoptosis but do not yet have diabetes, allowing us to avoid the confounding effects of glucose toxicity (research design and methods) (10,14). The percentage of β-cells immunostained for ubiquitin was increased in HIP rats (32 ± 5% vs. 8 ± 5%, P < 0.01) (Fig. 2A and B). The staining was mostly cytoplasmic, although nuclear staining was also evident in some cells (Fig. 2A). To draw a parallel between obese individuals with type 2 diabetes and the rat model, we also examined pancreatic sections from HFD-fed HIP rats (6). Within this group of HFD-fed HIP rats, which have diabetes and are insulin resistant, the percentage of β-cells stained for ubiquitin was increased in comparison to HIP rats fed with a regular diet (50 ± 3% vs. 32 ± 5%, P < 0.05) (Fig. 2B). Ubiquitin immunostaining was predominantly cytoplasmic, although nuclear staining was present in some cells, comparable to islets in type 2 diabetes (Fig. 1A). Impaired proteasome function may cause accumulation of ubiquitinated proteins (18). However, proteasome activity was comparable in islets of HIP, HFD-fed HIP, and wild-type rats (Fig. 2C).

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