Translation attenuation through eIF2alpha phosphorylation prevents oxidative stress and maintains the differentiated state in beta cells

Abstract

Accumulation of unfolded protein within the endoplasmic reticulum (ER) attenuates mRNA translation through PERK-mediated phosphorylation of eukaryotic initiation factor 2 on Ser51 of the alpha subunit (eIF2alpha). To elucidate the role of eIF2alpha phosphorylation, we engineered mice for conditional expression of homozygous Ser51Ala mutant eIF2alpha. The absence of eIF2alpha phosphorylation in beta cells caused a severe diabetic phenotype due to heightened and unregulated proinsulin translation; defective intracellular trafficking of ER cargo proteins; increased oxidative damage; reduced expression of stress response and beta-cell-specific genes; and apoptosis. However, glucose intolerance and beta cell death in these mice were attenuated by a diet containing antioxidant. We conclude that phosphorylation of eIF2alpha coordinately attenuates mRNA translation, prevents oxidative stress, and optimizes ER protein folding to support insulin production. The finding that increased proinsulin synthesis causes oxidative damage in beta cells may reflect events in the beta cell failure associated with insulin resistance in type 2 diabetes.

Figure 1. Ubiquitous expression of a floxed wt eIF2α transgene (fTg) in homozygous eIF2αA/A mice prevents lethality, preserves beta cell mass, and is required for glucose-regulated protein synthesis

(A) Diagram depicts the four genotypes of mice used in these experiments. Heterozygous eIF2α S/A mice harbor Ser51Ala (*) mutation in exon 2 of one eIF2α allele. fTg/0 represents the floxed wt eIF2α transgene driven by the CMV- enhancer and chicken β-actin promoter (Enh-Pro). LoxP sequences (black arrowheads) allow excision of eIF2α coding sequence and coordinate expression of EGFP. CreER/0 represents the CreER recombinase transgene driven by the rat insulin II promoter (RIP). The deletion of fTg catalyzed by Tam treatment is represented. (B) Tam-induced Cre recombinase deletion of wt eIF2α fTg is specific. Pancreatic tissue sections obtained from mice 3 wks after Tam administration were triple immunostained for EGFP, insulin, and glucagon and representative single channel fluorescence images are shown individually and merged (lower right image of each group). The scale bar represents 20 µm. (C) Tam-induced Cre recombinase deletion of wt eIF2α fTg is efficient. The wt eIF2α fTg directs high-level conditional expression of wt eIF2α mRNA in beta cells. Tam was administered to all mice and after 3 wks total RNA was prepared from isolated islets. Results from quantitative RT-PCR analyses of transgenic, total, and endogenous eIF2α mRNAs are shown. n= 3 mice per group. (D–F) eIF2α phosphorylation is required to attenuate protein synthesis in beta cells. Islets from tamoxifen-injected A/A;fTg/0 and A/A;fTg/0;CreER/0 mice were isolated and preincubated for 1 hr in Kreb’s buffer containing basal 2.8 mM glucose, followed by incuation for 1 hr in buffer containing 2.8 mM, 16.7 mM glucose, or 16.7 mM glucose and 1µM thapsigargin (Tg). Metabolic labeling with [³⁵S] methionine and cysteine was performed during the last 20 min. of incubation as described in EXPERIMENTAL PROCEDURES. (D and E), Proinsulin synthesis was measured by immunoprecipitation from labeled extracts. (D) Immunoprecipitated samples were subjected to acrylamide gel electrophoresis and autoradiography. (E) Quantitation of labeled proinsulin by phosphorimaging is expressed as the fold change relative to A/A;fTg/0 islets incubated with 2.8 mM glucose. (F) Total protein synthesis was quantified by TCA precipitation of labeled extracts and results were normalized to total protein. n=3 samples per group. Data are Mean ± SEM, (C), (E), and (F).

Figure 2. Beta cell-specific deletion of wt eIF2α fTg causes glucose intolerance due to reduced islet mass

(A and B) At the indicated weeks after Tam injection, blood glucose (A) and body mass (B) measurements were performed in mice fasted for 5–6 hrs. n=5–6 mice per group. Significant differences between A/A;fTg/0 versus A/A;fTg/0;CreER/0 mice are indicated. (C) GTTs were performed at 3–15 wks after Tam administration. n=5–6 mice per group. Significant differences between A/A;fTg/0 versus A/A;fTg/0;CreER/0 mice are indicated. (D) Pancreatic tissue sections obtained from mice at the indicated times after Tam administration were triple immunostained for insulin, EGFP, and glucagon. Representative single channel fluorescence images are shown individually and merged (lower right image of each group). The scale bar represents 20 µm. (E) Serum insulin levels and pancreatic insulin content were measured in mice at 3 and 15 wks after Tam injection. Insulin (Ins) measurements were normalized to glucagon (Glu). n=3–7 mice per group. (F and G) TUNEL staining was performed on pancreatic sections obtained from mice at 3 and 8 wks after Tam injection and the number (#) of positive (+) cells per islet area was quantified. n = 5–7 mice per group. The scale bars represent 50 µm. Data are Mean ± SEM, (A–C), (E), and (G).

Figure 2. Beta cell-specific deletion of wt eIF2α fTg causes glucose intolerance due to reduced islet mass

(A and B) At the indicated weeks after Tam injection, blood glucose (A) and body mass (B) measurements were performed in mice fasted for 5–6 hrs. n=5–6 mice per group. Significant differences between A/A;fTg/0 versus A/A;fTg/0;CreER/0 mice are indicated. (C) GTTs were performed at 3–15 wks after Tam administration. n=5–6 mice per group. Significant differences between A/A;fTg/0 versus A/A;fTg/0;CreER/0 mice are indicated. (D) Pancreatic tissue sections obtained from mice at the indicated times after Tam administration were triple immunostained for insulin, EGFP, and glucagon. Representative single channel fluorescence images are shown individually and merged (lower right image of each group). The scale bar represents 20 µm. (E) Serum insulin levels and pancreatic insulin content were measured in mice at 3 and 15 wks after Tam injection. Insulin (Ins) measurements were normalized to glucagon (Glu). n=3–7 mice per group. (F and G) TUNEL staining was performed on pancreatic sections obtained from mice at 3 and 8 wks after Tam injection and the number (#) of positive (+) cells per islet area was quantified. n = 5–7 mice per group. The scale bars represent 50 µm. Data are Mean ± SEM, (A–C), (E), and (G).

Figure 3. Translational control is required for optimal expression of UPR genes, antioxidant response genes, and beta cell-specific genes

(A–C, E, and F) Total RNA was extracted from islets isolated from mice at 3 wks after Tam administration. Expression levels of mRNA were measured using quantitative RT-PCR. Data are Mean ± SEM, n= 5–6 mice per group. (A) Genes regulated through eIF2α phosphorylation. (B) Genes regulated through IRE1 α signaling and genes of ERAD machinery. (C) Pancreatic beta or alpha cell-specific genes. (D) PDX1, MafA, and insulin immunolabeling of pancreatic sections was conducted on samples isolated from mice at 6 wks after Tam administration. Representative merged images of PDX1 with insulin (upper panels) and MafA with insulin (lower panels) are shown. The scale bars represent 20 µm. (E) Antioxidant response genes. (F) Anti- and pro-apoptotic genes.

Figure 4. Translation attenuation is required for ER and mitochondrial integrity and proper protein trafficking

(A and B) TEM was performed on pancreatic sections from A/A;fTg/0 and A/A;fTg/0;CreER/0 mice at 3 wks after Tam administration and representative images are shown. In the A/A;fTg/0 image, arrows indicate normal ER regions and arrowheads demark normal mitochondria. In A/A;fTg/0;CreER/0 images, arrows indicate regions of severe ER distention, asterisks indicate abnormal structures of low electron density, and arrowheads demark swollen mitochondria. The scale bars represent 2 µm. (B) The percentage of beta cells with dilated ER at 3 wks was quantified. Data are Mean ± SEM, n = 5–7 mice per group. (C–E) Pancreata from mice at 3 and 8 wks after Tam administration were analyzed by immunofluorescence for proinsulin (C), E-Cad (D), and GLUT2 and EGFP (E). The scale bars represent 20 µm.

Figure 5. Translation attenuation is required to prevent oxidative stress in beta cells

(A and B) Immunohistochemistry was performed to detect nitrotyrosine in pancreatic sections from mice at 3 and 8 wks after Tam administration. Representative images and quantitation of the number (#) of nitrotyrosine-positive (+) beta cells per islet area are shown. n = 5–7 mice per group. The scale bars represent 50 µm. (C and D) Protein carbonyls (C) and HODEs (D) were quantified in extracts from islets freshly isolated from mice at 3 wks after Tam injection. n= 7 mice per group. Islets were isolated from wt S/S mice and in vitro incubated in the absence or presence of tunicamycin (Tm, 2 µg/ml) for 5 hrs prior to analysis of oxidation products. Data are Mean ± SEM, (B–D).

Figure 6. Antioxidant treatment preserves insulin production, reduces beta cell death and improves glucose intolerance in A/A;fTg/0;CreER/0 mice

(A) GTTs were performed on mice fed chow supplemented with or without BHA for 3–15 wks after Tam treatment. The area under the curve (A.U.C.) from the GTTs was quantified. n=5–6 mice per group. (B) Blood glucose (left panel), serum insulin (middle panel), and pancreatic insulin (right panel) were measured in A/A;fTg/0 and A/A;fTg/0;CreER/0 mice at 6 weeks after Tam injection with and without BHA feeding. Insulin measurements were normalized to glucagon (Glu). n=7–9 mice per group. (C and D) TUNEL staining was performed on pancreatic sections obtained from mice at 6 wks after Tam injection and the number (#) of positive (+) cells per islet area was quantified. n = 7–9 mice per group. The scale bars represent 50 µm. Data are Mean ± SEM, (A), (B), and (D).

Figure 7. Mechanism for dysfunction and death of eIF2α phosphorylation-deficient beta cells

(A) Fluctuations in glucose regulate eIF2α kinases and phosphatases to control eIF2α phosphorylation and the rate of mRNA translation in the beta cell. A feedback loop attenuates mRNA translation through PERK activation in response to misfolded proteins. Phosphorylation of eIF2α is required for translation of Atf4 mRNA that leads to transcriptional induction. Increased expression of GADD34 causes dephosphorylation of eIF2α to increase mRNA translation. The eIF2α A/A mutation prevents glucose repression of protein synthesis and causes unrestricted high rates of mRNA translation. Elevated protein synthesis leads to the accumulation of misfolded proteins in the ER. The absence of eIF2α phosphorylation reduces expression of UPR genes and antioxidant response genes, thereby exacerbating the protein-folding defect. Accumulation of misfolded proteins in the ER initiates production of ROS. ROS may inactivate beta cell-specific transcription factors, leading to insulin granule depletion. Beta cells ultimately succumb to apoptosis.