Chop deletion reduces oxidative stress, improves β cell function, and promotes cell survival in multiple mouse models of diabetes (original) (raw)
Chop-null mutation increases obesity but prevents glucose intolerance in HF diet–fed eIF2αS/A mice. Although mice with heterozygous Ser51Ala mutation at the PERK phosphorylation site in eIF2α exhibit reduced attenuation of mRNA translation upon ER stress, they did not exhibit a readily apparent phenotype under standard conditions of diet. Analysis of glucose-stimulated translation in islets isolated from HF diet–fed heterozygous Ser51Ala mice, however, revealed an elevated rate of translation (9). These HF diet–fed mice develop diabetes and represent what we believe to be a novel model of β cell failure that results from ER stress due to elevated proinsulin biosynthesis as a consequence of interaction between genetic (eIF2_α_S/A allele) and environmental (HF diet) factors (9). We asked whether β cell survival and/or function are improved in these heterozygous Ser51Ala mutant mice when the CHOP-mediated death signal is absent. Deletion of the Chop gene modestly increased weight gain in HF diet–fed wild-type eIF2_α_S/S mice, consistent with recent observations (49). In contrast, Chop deletion significantly increased obesity in HF diet–fed eIF2_α_S/A mice (Figure 1A). The enhanced weight gain of eIF2_α_S/AChop–/– animals may be caused by accentuation of the metabolic defect previously reported for the eIF2_α_S/A mice (9), may be a consequence of the deletion of CHOP action as a negative regulator of adipogenesis (50, 51), or may be driven by hyperinsulinemia (see below). Glucose intolerance appeared after 5 weeks of HF diet in eIF2_α_S/AChop+/+ mice compared with HF diet–fed control eIF2_α_S/SChop+/+ mice. In contrast, eIF2_α_S/A mice with the _Chop-_null mutation displayed normal glucose tolerance for up to 32 weeks of HF diet despite their overt obesity (Figure 1B and Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI34587DS1).
_Chop_-null mutation increases β cell mass, improves β cell function, and prevents glucose intolerance in HF diet–fed eIF2_α_S/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 eIF2_α_S/AChop+/+ and eIF2_α_S/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.
Chop-null mutation preserves β cell morphology and function in HF diet–fed eIF2αS/A mice. The improved glucose tolerance observed in the eIF2_α_S/AChop–/– mice was not due to increased insulin sensitivity (Supplemental Figure 2A), but rather was associated with a 6-fold increase in islet mass and pancreas insulin content (Figure 1C and Supplemental Figure 1, B and C). Ultrastructural analysis was performed to monitor the distension of ER cisternae and reduction of secretory granule content characteristic of ER stress and β cell failure. Compared with β cells from HF diet–fed eIF2_α_S/S mice, β cells from HF diet–fed eIF2_α_S/AChop+/+ mice displayed a significantly distended ER and reduced insulin granule number, as previously reported (9). However, strikingly, the number of dense-core insulin granules in eIF2_α_S/AChop–/– mice was not significantly reduced compared with those in eIF2_α_S/SChop+/+ mice or eIF2_α_S/SChop–/– mice, although ER distension was still detectable (Figure 1, D and E).
The improved glucose tolerance and preserved granule content suggested that Chop deletion preserves β cell function by maintaining an adequate pool of secretory granules that were responsive to nutrient stimuli. Consistent with this theory, after 35 weeks of HF diet, the serum insulin levels were increased 2- to 3-fold in eIF2_α_S/AChop–/– mice compared with eIF2_α_S/SChop+/+, eIF2_α_S/SChop–/–, and eIF2_α_S/AChop+/+ mice (Figure 1F). Glucose-stimulated insulin secretion (GSIS) was significantly reduced in islets isolated from eIF2_α_S/AChop+/+ mice compared with wild-type eIF2_α_S/SChop+/+ mice (Figure 1G). In contrast, islets from HF diet–fed eIF2_α_S/AChop–/– mice remained glucose responsive for insulin secretion (Figure 1G). As the GSIS studies were performed with selected islets of similar size and the secretion of insulin was expressed as a percentage of total insulin content, the improved GSIS observed in eIF2_α_S/AChop–/– islets was not due to increased β cell mass, but rather reflected a genuine improvement in β cell function. These findings show that, despite the HF diet and overt obesity, glucose homeostasis was maintained in eIF2_α_S/AChop–/– mice because there was an increase in the number of functional β cells as measured by insulin granule content and GSIS. The results suggest that β cell failure in this eIF2_α_S/A mutant mouse model is mediated through the UPR-inducible gene Chop. This conclusion is also supported by the observation that Chop deletion reduced apoptosis and increased β cell mass in pancreata from homozygous _eIF2_α Ser51Ala embryos (Supplemental Figure 3).
Chop-null mutation prevents loss of β cell mass and diabetes in a HF diet/streptozotocin model of T2D. The previous data indicated that CHOP plays a negative role in β cell function when ER stress signaling is compromised in conjunction with a biosynthetic burden of enhanced proinsulin translation due to insulin resistance. We next evaluated the role of CHOP under conditions that combine the primary determinants of T2D, insulin resistance, and inadequate β cell function and mass in the absence of a genetic predisposition to β cell failure. Wild-type and _Chop_-null mice were fed a 60% HF diet for 5–6 weeks, and β cell mass was reduced by administration of a moderate dose of streptozotocin (STZ). This treatment increases the proinsulin biosynthetic burden upon the remaining β cells and challenges their ability to survive and function. This approach has been successful in evaluating therapeutic strategies that alter insulin resistance or improve β cell function (52–54). In the absence of any additional determinant of obesity, the _Chop_–null mice exhibited a slight increase in weight that was not statistically different from wild-type animals (Figure 2A). However, the fed glucose levels of _Chop_-null mice were significantly lower than those of wild-type mice after 5–6 weeks of HF diet feeding. In addition, the overt hyperglycemia that developed in wild-type mice 4 days after administration of STZ was averted in _Chop_-null mice (Figure 2B).
_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. (G–I) 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.
The improved glycemic control of _Chop_-null HF diet–fed, STZ-treated animals was investigated by analysis of glucose tolerance, insulin tolerance, insulin secretion, insulin content, and islet morphology (Figure 2, C–J). In the absence of HF diet or with 45% HF feeding, glucose tolerance and β cell function were similar between wild-type and _Chop_–/– mice (Figure 1, Supplemental Figure 1, Figure 3, and Supplemental Figure 4). However, under conditions of 60% HF diet, glucose intolerance was more severe in wild-type mice compared with Chop–/– mice (Figure 2C). One week after STZ administration, the wild-type mice were severely hyperglycemic and glucose intolerant, while the Chop–/– mice were only mildly hyperglycemic and glucose intolerant (Figure 2, B and D). Blood glucose levels were 50% lower, and insulin secretion upon fasting and refeeding was significantly elevated in _Chop_-null mice versus wild-type mice (Figure 2, E and F). Analysis of fed insulin levels, pancreatic insulin content, and islet morphology confirmed that the improved glucose homeostasis in the _Chop_-null mice was coincident with elevated basal insulin levels and a larger islet/β cell mass (Figure 2, G–I). There was no significant difference in insulin tolerance (Figure 2J). GSIS was not measured in islets isolated from these mice due to the low β cell mass of the wild-type mice in this model. However, in vitro STZ treatment of islets isolated from wild-type and _Chop-_null mice did not reveal any differences in the acute toxicity of STZ, as GSIS was fully inhibited in both populations of islets (data not shown), suggesting that Chop deletion influences subsequent β cell recovery and/or function.
_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 (B–E) 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.
Chop-null mutation prevents glucose intolerance and improves β cell function in Leprdb/db mice. We proceeded to investigate whether _Chop_-null mutation could prevent β cell loss in the leptin receptor–deficient Leprdb/db mouse, a model of diabetes that encompasses obesity, insulin resistance, and β cell failure. The β cell defects in the Leprdb/db mouse model depend on the C57BL/KsJ strain background. However, we were able to study the Chop contribution to β cell failure by analyzing first-generation littermates from crosses between double heterozygous Chop+/– and Leprdb/+ mice in a mixed C57BL/KsJ and C57BL/6J background. All homozygous Leprdb/db progeny displayed hyperglycemia in the presence of the wild-type Chop allele, indicating there was sufficient C57BL/KsJ genetic contribution to elicit the diabetic phenotype.
Although the obesity of Leprdb/db mice was further increased by _Chop_-null mutation (Figure 3A), development of fasting hyperglycemia and glucose intolerance was dramatically prevented (Figure 3B and Supplemental Figure 4, A and B), similar to the effect of _Chop_-null mutation in the HF diet–fed eIF2_α_S/A mice and HF diet–fed, STZ-treated mice. The improved glucose tolerance was not a consequence of increased sensitivity to insulin (Supplemental Figure 2B). Histological examination indicated that Chop deletion was associated with a 6-fold increase in islet mass in the Leprdb/db mice, and this correlated with serum hyperinsulinemia and glucose-responsive insulin secretion in vitro (Figure 3, C–E, and Supplemental Figure 4C). Ultrastructural analysis demonstrated that islets from Leprdb/db mice contained significantly fewer insulin granules than islets from Leprdb/+ mice (Figure 3, F and G). In contrast, granule depletion was significantly attenuated in Leprdb/db mice that harbor Chop deletion (Figure 3, F and G). In summary, these studies suggest that Chop deletion improves β cell function in the Leprdb/db mouse.
Chop-null mutation causes β cell proliferation and reduces β cell apoptosis in the islets of Leprdb/db mice. To elucidate how Chop deletion may affect β cell mass, we studied β cell replication and apoptosis. TUNEL staining demonstrated that apoptosis was increased about 10-fold in the homozygous Leprdb/db mice compared with heterozygous Leprdb/+ mice, similar to recent observations (55). However, Chop deletion dramatically reduced apoptosis in the Leprdb/db mice to levels observed in control Leprdb/+ mice (Figure 4A). To evaluate β cell proliferation, BrdU-containing water was administered to mice prior to the development of full islet hyperplasia (56). As the rate of proliferation is very low in adult animals, a labeling period of 23 days was implemented to ensure accurate detection of altered proliferation rates. The number of BrdU-positive cells was approximately 2-fold greater in the islets from Leprdb/dbChop–/– mice compared with those from Leprdb/dbChop+/+ mice (Figure 4B). Therefore, a portion of the islet hyperplasia observed in the _Chop_-null mice may be attributed to increased β cell proliferation, possibly a consequence of normal β cell compensation for insulin resistance. Therefore, the islet hyperplasia in Leprdb/dbChop–/– mice was a consequence of reduced apoptosis as well as increased proliferation.
_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.
Chop-null mutation increases expression of adaptive functions and reduces expression of apoptotic functions in Leprdb/db mice. To provide insight into how _Chop_-null mutation might alter the transcriptional profile to preserve β cell function, we analyzed isolated islets for expression of genes encoding functions in the UPR, the oxidative stress response, cell death, and insulin production (Figure 5). The expression of several UPR genes encoding adaptive functions to improve ER folding capacity, such as BiP, Grp94, Fkbp11, and p58IPK, was slightly increased in islets from Leprdb/dbChop+/+ mice compared with control Leprdb/+Chop+/+ mice. The expression levels of these genes were further increased in islets from Leprdb/dbChop–/– mice. In addition, there was increased splicing of Xbp1 mRNA and expression of genes that encode components of the ER-associated protein degradation machinery, such as Edem and Ubc7 (Figure 5A). The expression levels of several targets of CHOP that encode proapoptotic functions, i.e., Gadd34, Dr5, and Trb3, as well as other death pathway–related genes, were reduced in Leprdb/dbChop–/– islets (Figure 5C and Supplemental Figure 5A). The RT-PCR analyses also detected slightly increased expression of genes encoding functions that prevent oxidative stress, including Sod1, Sod2, Gpx1, Pparg, and Ucp2, in Leprdb/dbChop+/+ islets. However, the expression of these genes was significantly elevated in Leprdb/dbChop–/– islets (Figure 5D). In contrast, expression of CHOP-dependent ER oxidoreductase 1 (Ero1a) (42), which generates oxidizing equivalents in the ER (16, 57), was decreased upon Chop deletion. Quantitative analysis of mRNA expression of other genes encoding ER stress proteins, transcription factors, other oxidative stress–related proteins, and β cell–specific genes did not clarify the mechanism by which Leprdb/dbChop–/– β cells adapt but may provide useful information for future studies (Supplemental Figure 5). As β cells express antioxidant functions at low levels (58, 59), these gene expression differences should minimize accumulation of ROS and facilitate adaptation. These findings support the hypothesis that Chop deletion improves β cell function as a consequence of increased expression of UPR adaptive and antioxidative stress response genes and reduced expression of proapoptotic functions.
_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–/–.
Chop-null mutation reduces protein oxidation and lipid peroxidation in response to ER stress. The increased expression of genes encoding antioxidative stress responses suggested that Chop deletion improves the capacity of β cells to accommodate oxidative stress. Therefore, we measured products of protein oxidation (carbonyls) and lipid peroxidation (hydroxyoctadecadienoic acid [HODE]) in isolated islets. Islets from Leprdb/db diabetic mice displayed a 3-fold increase in protein carbonyls and a 2-fold increase in HODEs compared with those from control Leprdb/+ mice (Figure 6A). Therefore, insulin resistance in the Leprdb/db mice was associated with oxidative stress in the islets, an observation consistent with literature that indicates antioxidant molecules can improve glucose homeostasis, restore β cell function, and reduce oxidative stress markers in the islets of Leprdb/db mice (55, 60–63). In contrast, Chop deletion significantly reduced both products of protein oxidation and lipid peroxidation in islets from these obese Leprdb/db mice.
_Chop_-null mutation protects from oxidative stress. (A–C) 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.
The reduction in oxidative damage observed upon Chop deletion may be result from protection of the islets from oxidative damage caused by ROS or it may be an indirect consequence of improved glycemia (8, 64). To discriminate these possibilities, we analyzed islets in the absence of hyperglycemia and insulin resistance contributed by the Leprdb/db mutation. Islets were isolated from wild-type mice and Chop–/– mice and treated with tunicamycin to inhibit _N_-linked glycosylation and induce unfolded protein accumulation in vitro. Tunicamycin treatment increased protein oxidation and lipid peroxidation products 2.5- to 3-fold in wild-type islets (Figure 6B). In contrast, islets from Chop–/– mice displayed significantly reduced levels of carbonyls and HODEs after tunicamycin treatment. In addition, H2O2 treatment increased levels of carbonyls and HODEs to similar extents in islets isolated from Chop+/+ and Chop–/– mice (Figure 6C). Importantly, these results show that the _Chop_-null mutation protects β cells from oxidative damage that occurs in response to ER stress.