Inhibition of deoxyhypusine synthase enhances islet {beta} cell function and survival in the setting of endoplasmic reticulum stress and type 2 diabetes

Abstract

Islet β cell dysfunction resulting from inflammation, ER stress, and oxidative stress is a key determinant in the progression from insulin resistance to type 2 diabetes mellitus. It was recently shown that the enzyme deoxyhypusine synthase (DHS) promotes early cytokine-induced inflammation in the β cell. DHS catalyzes the conversion of lysine to hypusine, an amino acid that is unique to the translational elongation factor eIF5A. Here, we sought to determine whether DHS activity contributes to β cell dysfunction in models of type 2 diabetes in mice and β cell lines. A 2-week treatment of obese diabetic C57BLKS/J-db/db mice with the DHS inhibitor GC7 resulted in improved glucose tolerance, increased insulin release, and enhanced β cell mass. Thapsigargin treatment of β cells in vitro induces a picture of ER stress and apoptosis similar to that seen in db/db mice; in this setting, DHS inhibition led to a block in CHOP (CAAT/enhancer binding protein homologous protein) production despite >30-fold activation of Chop gene transcription. Blockage of CHOP translation resulted in reduction of downstream caspase-3 cleavage and near-complete protection of cells from apoptotic death. DHS inhibition appeared to prevent the cytoplasmic co-localization of eIF5A with the ER, possibly precluding the participation of eIF5A in translational elongation at ER-based ribosomes. We conclude that hypusination by DHS is required for the ongoing production of proteins, particularly CHOP, in response to ER stress in the β cell.

Figures

FIGURE 1.

Glucose homeostasis and islet eIF5A hypusination levels in control and diabetic mice. Obese 12-week-old C57BL6/J-db/db (BL6-db/db) and C57BLKS/J-db/db (BLKS-db/db) mice and lean littermate C57BL6/J-db/+ (BL6-db/+) and C57BLKS/J-db/+ (BLKS-db/+) were studied for glucose homeostasis and islet eIF5A hypusination levels. A, body weights of mice; B, results of fasting blood glucoses; C, results of GTTs (1 mg/kg glucose given intraperitoneally) and corresponding AUC analysis of GTTs for BL6-db/+ and BLKS-db/+ mice; D, results of GTTs (0.25 mg/kg glucose given intraperitoneally) and corresponding AUC analysis of GTTs for BL6-db/db and BLKS-db/db mice; E, PAGE showing results of hypusination assays in vitro using [3H]spermidine and isolated islets from mice and immunoblots from the same protein isolates for total eIF5A protein. Ratios at the bottom of the panel indicate the intensity of the [3H]eIF5AHyp signal to the intensity of the total eIF5A protein signal. Data are presented as mean ± S.E.

FIGURE 2.

Glucose homeostasis in obese diabetic mice receiving GC7, an inhibitor of DHS. Three cohorts of C57BLKS/J-db/db mice (n = 6–8 per group), aged 8–12 weeks, received 4 mg/kg GC7 (or saline control) intraperitoneally for 14 days. A–D, results of fasting blood glucose (A), GTTs (1 mg/kg glucose injection) (B), AUC for the GTTs (C), and insulin tolerance tests (D) in cohort 1. E–H, results of fasting blood glucose (E), fasting plasma insulin (F), GTTs (0.1 mg/kg glucose injection) (G), and AUC for the GTTs (H) in cohort 2. I–L, results of fasting blood glucose (I), fasting plasma insulin (J), GTTs (0.25 mg/kg glucose injection) (K), and AUC for the GTTs (L) in cohort 3. Data are presented as mean ± S.E.

FIGURE 3.

β cell mass and plasma hormone ratios in mice receiving GC7, an inhibitor of DHS. C57BLKS/J-db/db mice (db/db) from cohort 3 (see Fig. 2) and control, age-matched lean C57BLKS/J-db/+ littermates (db/+) were euthanized after an overnight fast, and pancreases and plasma were collected. A, representative pancreas sections from the indicated mice stained for insulin (brown) and counterstained with hematoxylin (blue). Islet β cells are stained brown (original magnification, ×200); B, results of morphometric analysis of β cell mass from the indicated mice. For mass determination, a minimum of three pancreatic sections taken 75 μm apart from three mice per group were quantitated as described under “Materials and Methods.” C, results of plasma proinsulin:insulin ratios from fasted mice in cohort 3.

FIGURE 4.

Tg treatment of INS-1 (832/13) β cells increases hypusination activity and up-regulates UPR genes. INS-1 (832/13) cells were untreated or treated with 1 μm Tg for 6 h or 1 μm Tg for 6 h plus 100 μm GC7 (added 1 h before Tg) (Tg+GC7). A, results of cellular proinsulin:insulin ratio from INS-1 (832/13) cellular extract; B, hypusination assay using [3H]spermidine (top panels) and immunoblot for total DHS and actin levels (bottom two panels); C, results of real-time RT-PCR for UPR genes. Data in A and C represent the results from three independent experiments. DMSO, dimethyl sulfoxide.

FIGURE 5.

DHS inhibition blocks CHOP and caspase-3 production and protects INS-1 (832/13) β cells from apoptotic death. A, results of immunoblot analysis following treatment of INS-1 (832/13) cells with Tg (1 μm) for various times and/or GC7 (100 μm) added 1 h prior to Tg in the culture (−1 h). B, results of immunoblot analysis following treatment of INS-1 (832/13) cells for 6 h with Tg (1 μm), GC7 (100 μm), and/or MG132 (10 μm). C, results of immunoblot analysis following treatment of INS-1 (832/13) cells for 6 h with 1 μm Tg and the indicated concentrations of GC7. D, INS-1 (832/13) cells were transfected with no siRNA, control siRNA (si-Control), or siRNA against eIF5A (si-eIF5A) and then treated with Tg (1 μm) for 6 h, then subjected to the immunoblot analysis shown; E, results of annexin V-FITC/PI flow cytometry analysis of INS-1 (832/13) cell cultures untreated or treated with Tg (1 μm) overnight and with GC7 (10 μm) added 1 h prior to Tg. In E, data represent results from three independent experiments. Casp3, caspase-3.

FIGURE 6.

Intracellular localization of eIF5A and DHS in response to Tg-induced ER stress. A, INS-1 (832/13) β cells were transfected with expression vectors encoding GFP-eIF5A, GFP-eIF5A(K50A), or GFP-DHS, and 24 h later were treated with Tg (1 μm) for 4 h. Images shown are representative live cell images (original magnification, ×400) taken in the green channel (488 nm excitation). Numbers in the lower left corner of each panel represent the cytoplasmic:nuclear ratio of green intensity from at least 10 cells, and an asterisk indicates that the number is statistically different (p < 0.05) from untreated cells. B, results of hypusination assays in vitro using recombinant eIF5A protein, [3H]spermidine, and cytoplasmic (C) or nuclear (N) extracts from INS-1 (832/13) cells. Labeling on the right indicates the positions of [3H]eIF5AHyp and unincorporated [3H]spermidine. C, representative images (original magnification, ×630) of INS-1 (832/13) β cells transfected with RFP-calreticulin and either GFP-eIF5A or GFP-eIF5A(K50A) and then counterstained with DAPI (upper row only). 24 h after transfection, cells were imaged in the green (488 nm excitation), red (580 nm excitation), and blue (DAPI, 365 nm excitation) channels and then merged. Upper and lower rows in C represent different cells viewed from separate transfections.