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, 118 (10), 3378-89

Chop Deletion Reduces Oxidative Stress, Improves Beta Cell Function, and Promotes Cell Survival in Multiple Mouse Models of Diabetes


Chop Deletion Reduces Oxidative Stress, Improves Beta Cell Function, and Promotes Cell Survival in Multiple Mouse Models of Diabetes

Benbo Song et al. J Clin Invest.


The progression from insulin resistance to type 2 diabetes is caused by the failure of pancreatic beta cells to produce sufficient levels of insulin to meet the metabolic demand. Recent studies indicate that nutrient fluctuations and insulin resistance increase proinsulin synthesis in beta cells beyond the capacity for folding of nascent polypeptides within the endoplasmic reticulum (ER) lumen, thereby disrupting ER homeostasis and triggering the unfolded protein response (UPR). Chronic ER stress promotes apoptosis, at least in part through the UPR-induced transcription factor C/EBP homologous protein (CHOP). We assessed the effect of Chop deletion in multiple mouse models of type 2 diabetes and found that Chop-/- mice had improved glycemic control and expanded beta cell mass in all conditions analyzed. In both genetic and diet-induced models of insulin resistance, CHOP deficiency improved beta cell ultrastructure and promoted cell survival. In addition, we found that isolated islets from Chop-/- mice displayed increased expression of UPR and oxidative stress response genes and reduced levels of oxidative damage. These findings suggest that CHOP is a fundamental factor that links protein misfolding in the ER to oxidative stress and apoptosis in beta cells under conditions of increased insulin demand.


Figure 1
Figure 1. Chop-null mutation increases β cell mass, improves β cell function, and prevents glucose intolerance in HF diet–fed eIF2αS/A mice.
Mice of the indicated genotypes were fed a 45% HF diet for 35–41 weeks. (A and B) Body mass and glucose tolerance tests; n = 8–10 mice per condition. Significant differences between eIF2αS/AChop+/+ and eIF2αS/AChop–/– are indicated. (C) Islet morphology shown by H&E and immunofluorescence staining. Scale bars: 400 μm (top), 50 μm (bottom). (D and E) β cell ultrastructure from TEM and insulin granule content quantified by analysis of similar total areas from TEM images from 2 mice per condition. ER, rough ER; M: mitochondria. Scale bar: 1 μm. (F) Analysis of serum insulin levels; n = 8–10 mice per condition. (G) Analysis of GSIS. Islets from 2 animals per condition were analyzed in duplicate. H, high glucose (16.7 mM); L, low glucose (3.3 mM). *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 2
Figure 2. Chop-null mutation prevents hyperglycemia and glucose intolerance by maintaining insulin content and secretion in a HF diet–fed, STZ-treated nongenetic model of T2D.
Chop+/+ and Chop–/– mice were fed a 60% HF diet (HFD) for 5.5 weeks prior to administration of a dosage of 150 mg/kg STZ as described in Methods, and measurements were performed for up to 16 days after STZ with continued HF feeding. (A) Body weight. (B) Fed blood glucose levels. (C and D) Glucose tolerance measurements. Glucose tolerance was tested after HF diet alone for 5.5 weeks (C) and 4 days after STZ treatment and continued HF diet (D). (E and F) Fasting and refed blood glucose and serum insulin levels. Glucose and insulin measurements were taken 13 days after STZ treatment from mice that were fasted overnight and refed for 3.5 hour. (GI) Serum was collected for measurement and mice were sacrificed for determination of pancreatic insulin content and histology 16 days after STZ administration. (G) Fed serum insulin levels, (H) pancreatic insulin content, and (I) islet morphology stained with H&E. Scale bars: 500 μm (top), 100 μm (bottom). (J) Insulin tolerance measurements. Insulin tolerance was tested 15 days after STZ treatment. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3
Figure 3. Chop-null mutation increases obesity and maintains glucose tolerance in Leprdb/db mice through expanded β cell mass and improved cell function.
Analysis was performed on samples collected from mice at 9–10 (BE) or 6 (F and G) months of age. (A) Body mass. Representative mice at 20 wk of age are shown. (B) Glucose tolerance tests; n = 3–5 mice per condition. Significant differences between Leprdb/dbChop+/+ and Leprdb/dbChop–/– are indicated. (C) Islet morphology from H&E and immunofluorescence staining. Scale bars: 400 μm (top), 50 μm (bottom). (D) Serum insulin levels; n = 7–17 mice per condition. (E) GSIS analysis; islets from 2 mice per condition were analyzed in triplicate. (F and G) TEM images of β cells and insulin granule quantitation from similar total areas from 2 mice per condition. Scale bar: 1 μm. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4
Figure 4. Chop-null mutation increases proliferation and reduces apoptosis within the islets of Leprdb/db mice.
(A) Apoptosis. TUNEL staining was performed, and the number of positive cells (arrows) was quantified; n = 2–4 mice per condition. Scale bar: 50 μm. (B) Proliferation. BrdU-positive cells within islet areas were detected by immunohistochemistry, and the darkly stained nuclei were quantified from microscope images. Scale bar: 20 μm. ***P < 0.001.
Figure 5
Figure 5. Chop-null mutation in Leprdb/db mice increases expression of UPR and antioxidative stress response genes and decreases expression of proapoptotic genes.
Real-time RT-PCR analysis of islet mRNA expression. Expression values were normalized to 18S rRNA and are presented as fold-induction compared with wild-type (Leprdb/+Chop+/+) islets. (A) UPR genes and ER-associated protein degradation genes. (B) Control genes. (C) CHOP-regulated genes and other death signaling genes. (D) Antioxidative stress genes. Chop mRNA was not detectable in Leprdb/+Chop–/– or Leprdb/dbChop–/– islets; n = 4–6 mice per condition. *P < 0.05, **P < 0.01, ***P < 0.001 for Leprdb/dbChop+/+ compared with Leprdb/dbChop–/–.
Figure 6
Figure 6. Chop-null mutation protects from oxidative stress.
(AC) Oxidized proteins (carbonyls) and lipids (HODEs). (A) Direct analysis of islets isolated from mice of the indicated genotypes; n = 2–4 mice per condition. (B and C) Oxidation products measured in islets incubated in vitro. Chop+/+ or Chop–/– islets were isolated, cultured overnight, and incubated with control media or media containing tunicamycin (2 μg/ml) for 10 hours (B) or 176 μM H2O2 for 7 hours (C). *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 7
Figure 7. Model depicting interrelationships between protein folding, UPR, CHOP, ROS, and apoptosis in β cells.
The UPR induces genes to improve ER protein folding and reduce oxidative stress and also induces the proapoptotic gene Chop. CHOP enhances ROS formation, possibly through induction of GADD34 or ERO1. Chop-null mutation reduces proapoptotic gene expression to permit increased expression of UPR protective genes and antioxidative stress response genes to minimize ER stress and oxidative stress, thereby improving protein folding to support insulin production (depicted in blue). CHOP may also act, directly or indirectly, to repress transcription of some UPR protective genes or antioxidative stress response genes. Deletion of Chop in combination with insulin resistance increases β cell mass, reduces oxidative stress in islets, and preserves insulin secretion and glucose tolerance.

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