Lipotoxic Stress Induces Pancreatic β-Cell Apoptosis through Modulation of Bcl-2 Proteins by the Ubiquitin-Proteasome System

Abstract

Pancreatic β-cell loss induced by saturated free fatty acids (FFAs) is believed to contribute to type 2 diabetes. Previous studies have shown induction of endoplasmic reticulum (ER) stress, increased ubiquitinated proteins, and deregulation of the Bcl-2 family in the pancreas of type 2 diabetic patients. However, the precise mechanism of β-cell death remains unknown. In the present study we demonstrate that the FFA palmitate blocks the ubiquitin-proteasome system (UPS) and causes apoptosis through induction of ER stress and deregulation of Bcl-2 proteins. We found that palmitate and the proteasome inhibitor MG132 induced ER stress in β-cells, resulting in decreased expression of the prosurvival proteins Bcl-2, Mcl-1, and Bcl-XL, and upregulation of the prodeath BH3-only protein PUMA. On the other hand, pharmacological activation of the UPS by sulforaphane ameliorated ER stress, upregulated prosurvival Bcl-2 proteins, and protected β-cells from FFA-induced cell death. Furthermore, transgenic overexpression of Bcl-2 protected islets from FFA-induced cell death in vitro and improved glucose-induced insulin secretion in vivo. Together our results suggest that targeting the UPS and Bcl-2 protein expression may be a valuable strategy to prevent β-cell demise in type 2 diabetes.

Figure 1

Immunohistological images of pancreas slides stained for ubiquitin in lean and obese humans and chow and high fat fed mice after 24 weeks. (a) Pancreases were obtained from human organ donors and then were embedded in paraffin and cut into 5 µm thick sections. Immunohistochemistry was then performed, staining for ubiquitin. (b) Pancreatic sections from mice fed chow or on high fat diet for 24 weeks were stained for ubiquitin. Red arrows indicate islets with higher levels of ubiquitinated protein ((a) and (b)).

Figure 2

Palmitate and MG132 treatment induces a similar increase in levels of ubiquitinated proteins in pancreatic islets and β-cells. ((a)–(c)) Expression of ubiquitinated proteins measured by Western blot in human islets (a), mouse islets (b), or MIN6 cells (c) under control conditions or following 0.5 mM palmitate treatment as indicated. Band intensities were quantified, with values of the specific time points standardised to β-actin loading control levels. Results represent fold induction levels, relative to control. ((d)-(e)) MIN6 cells were treated with 0.5 mM palmitate (d) and 10 μM MG132 (e) for 0 (untreated), 2, 4, and 8 h. Proteasome activity was measured using the Proteasome 20S Activity Assay kit. Results represent fold induction levels relative to the control. (f) MIN6 cells were treated with 10 μM MG132 as indicated and levels of ubiquitinated proteins were measured by Western blot analysis. ((g)-(h)) MIN6 cells were treated with 0.5 mM palmitate (g) and 10 μM MG132 (h) for 24 h and cell death was visualised by HO/PI staining. White arrows indicate cell death. (i) Western blot for cleaved caspase-3 in MIN6 cells after treatment with MG132 as indicated. (j) Human islets isolated from organ donors were treated for 24 h with MG132 and % of β-cells detected by Newport green staining and flow cytometry. Results are the means ± SEM of 3-4 independent experiments, except for Figures 2(a), 2(b), 2(i), and 2(j) (data representative of 2 independent experiments). ^∗ P < 0.05; ^∗∗ P < 0.01.

Figure 3

UPS inactivation induces ER stress in β-cells. ((a)–(f)) MIN6 cells were treated with 0.5 mM palmitate ((a), (c), and (e)) or 10 μM MG132 ((b), (d), and (f)) and ER stress markers (ATF4, Chop, and Bip) measured as indicated. RNA was isolated using NucleoSpin RNA XS kit and cDNA was made with a High Capacity cDNA Reverse Transcriptase Kit. mRNA was analysed via real-time PCR analysis and standardised to β-actin internal control levels. ((g)-(h)) MIN6 cells were treated with 0.5 mM palmitate (g) or 10 μM MG132 (h) as indicated, and levels of Chop protein expression were measured by Western blot. Band intensities were quantified and standardised to β-actin loading control. Results are the means ± SEM of 3–5 independent experiments. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001.

Figure 4

Palmitate and MG132 decrease levels of prosurvival proteins in pancreatic islets and β-cells. ((a)–(c)) MIN6 cells were treated with 0.5 mM palmitate as indicated and expression of prosurvival proteins Bcl-2 (a), Bcl-XL (b), and Mcl-1 (c) was measured by Western blot. Band intensities were quantified and values of the specific time points standardised to β-actin loading controls. Results are the means ± SEM of 3 independent experiments. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001. ((d)-(e)) MIN6 cells were treated with 10 μM MG132 as indicated and expression of prosurvival proteins Bcl-2 (d), Bcl-XL (e), and Mcl-1 (f) was measured by Western blot. Band intensities were quantified and values of the specific time points standardised to β-actin loading controls. Results are the means ± SEM of 3 independent experiments. ^∗ P < 0.05; ^∗∗ P < 0.01. ((g)–(i)) Expression of prosurvival Bcl-2 proteins in mouse islets after palmitate treatment. Cell lysates were subjected to Western blotting with antibodies detecting Bcl-2 (g), Bcl-XL (h), and Mcl-1 (i). Quantification of protein bands is indicated at the bottom as a ratio to β-actin loading control. The results are representative of two independent experiments.

Figure 5

Palmitate and MG132 activate AKT and PUMA in pancreatic β-cells. ((a)-(b)) MIN6 cells were treated with 0.5 mM palmitate (a) or 10 μM MG132 (b) as indicated and expression of p-AKT was measured by Western blot. Band intensities were quantified and values of the specific time points standardised to β-actin loading controls. ((c)-(d)) MIN6 cells were treated with 0.5 mM palmitate (c) or 10 μM MG132 (d) and expression of PUMA was measured by qPCR. (e) Islets were obtained from wild-type C57BL/6 and PUMA −/− mice and treated with 10 μM MG132 for 24 h and subsequent levels of cell death were determined by FACS analysis. Results are the means ± SEM of 3–5 independent experiments. ^∗ P < 0.05, ^∗∗ P < 0.01, and ^∗∗∗ P < 0.001.

Figure 6

SFN protects β-cells from FFA-induced cell death. (a) MIN6 cells were treated with 10 μM MG132 and 2 mM SFN for 24 h and levels of ubiquitinated proteins were measured by Western blot. The result is representative of two independent experiments. (b) MIN6 cells were treated with SFN and palmitate as indicated and Western blot analysis was conducted to detect ubiquitinated proteins. β-actin levels were used as a loading control. The result is representative of two independent experiments. (c) MIN6 cells were treated for 24 h as indicated and viability was detected by HO/PI. Results are the means ± SEM of 4 independent experiments. ^∗∗ P < 0.01. ((d)–(g)) MIN6 cells were treated with 0.5 mM palmitate and/or 2 mM SFN for 24 h and ER stress markers (ATF4, Chop, and Bip) and PUMA measured by qPCR as indicated. Results are the means ± SEM of 3-4 independent experiments. ^∗ P < 0.05. ((h)–(j)) MIN6 cells were treated with 0.5 mM palmitate and/or 2 mM SFN for 24 h and expression of prosurvival Bcl-2 proteins was measured by Western blotting with antibodies detecting Bcl-2 (h), Bcl-XL (i), and Mcl-1 (j). Quantification of protein bands is indicated at the bottom as a ratio to β-actin loading control. The results are representative of two independent experiments.

Figure 7

Overexpression of Bcl-2 improves β-cell function during obesity. (a) Islets were obtained from wild-type C57BL/6 mice and RIP-Bcl-2 transgenic mice. Islets were then treated with 0.5 mM palmitate or 30 mM D-glucose for 5 days and subsequent levels of cell death were determined by FACS analysis. (b) Pancreas sections from wild-type and RIP-Bcl-2 mice fed on high fat for 16 weeks were stained for insulin (green), and quantification of islets from 5 mice per group is provided as percentage of islet area. BAR, 100 μm. (c) Pancreas sections from wild-type and RIP-Bcl-2 mice fed on high fat for 16 weeks were stained for ubiquitin. (d) IV-GTT results are shown in wild-type and RIP-Bcl-2 high fat fed mice. (e) Fasting (8 h) blood glucose levels of wild-type and RIP-Bcl-2 mice fed on high fat for 16 weeks. ^∗ P < 0.05; ^∗∗ P < 0.01.

Figure 8

Proposed model for the role of UPS inactivation in palmitate-induced β-cell apoptosis. Palmitate induces inactivation of the UPS in β-cells resulting in increased ubiquitinated proteins, ER stress, and posttranscriptional inactivation of Bcl-2 and other prosurvival Bcl-2 proteins. In addition, AKT is dephosphorylated leading to upregulated expression of the BH3-only protein PUMA. These signals converge on mitochondrial permeabilization causing β-cell demise.