Hypoxia reduces ER-to-Golgi protein trafficking and increases cell death by inhibiting the adaptive unfolded protein response in mouse beta cells.

AIMS/HYPOTHESIS: Hypoxia may contribute to beta cell failure in type 2 diabetes and islet transplantation. The adaptive unfolded protein response (UPR) is required for endoplasmic reticulum (ER) homeostasis. Here we investigated whether or not hypoxia regulates the UPR in beta cells and the role the adaptive UPR plays during hypoxic stress. METHODS: Mouse islets and MIN6 cells were exposed to various oxygen (O2) tensions. DNA-damage inducible transcript 3 (DDIT3), hypoxia-inducible transcription factor (HIF)1α and HSPA5 were knocked down using small interfering (si)RNA; Hspa5 was also overexpressed. db/db mice were used. RESULTS: Hypoxia-response genes were upregulated in vivo in the islets of diabetic, but not prediabetic, db/db mice. In isolated mouse islets and MIN6 cells, O2 deprivation (1-5% vs 20%; 4-24 h) markedly reduced the expression of adaptive UPR genes, including Hspa5, Hsp90b1, Fkbp11 and spliced Xbp1. Coatomer protein complex genes (Copa, Cope, Copg [also known as Copg1], Copz1 and Copz2) and ER-to-Golgi protein trafficking were also reduced, whereas apoptotic genes (Ddit3, Atf3 and Trb3 [also known as Trib3]), c-Jun N-terminal kinase (JNK) phosphorylation and cell death were increased. Inhibition of JNK, but not HIF1α, restored adaptive UPR gene expression and ER-to-Golgi protein trafficking while protecting against apoptotic genes and cell death following hypoxia. DDIT3 knockdown delayed the loss of the adaptive UPR and partially protected against hypoxia-induced cell death. The latter response was prevented by HSPA5 knockdown. Finally, Hspa5 overexpression significantly protected against hypoxia-induced cell death. CONCLUSIONS/INTERPRETATION: Hypoxia inhibits the adaptive UPR in beta cells via JNK and DDIT3 activation, but independently of HIF1α. Downregulation of the adaptive UPR contributes to reduced ER-to-Golgi protein trafficking and increased beta cell death during hypoxic stress.

Pancreatic PYY Is Critical in the Control of Insulin Secretion and Glucose Homeostasis in Female Mice.

Insulin secretion is tightly controlled through coordinated actions of a number of systemic and local factors. Peptide YY (PYY) is expressed in α-cells of the islet, but its role in control of islet function such as insulin release is not clear. In this study, we generated a transgenic mouse model (Pyy(tg/+)/Rip-Cre) overexpressing the Pyy gene under the control of the rat insulin 2 gene promoter and assessed the impact of islet-released PYY on ß-cell function, insulin release, and glucose homeostasis in mice. Our results show that up-regulation of PYY in islet ß-cells leads to an increase in serum insulin levels as well as improved glucose tolerance. Interestingly, PYY-overproducing mice show increased lean mass and reduced fat mass with no significant changes in food intake or body weight. Energy expenditure is also increased accompanied by increased respiratory exchange ratio. Mechanistically, the enhanced insulin levels and improved glucose tolerance are primarily due to increased ß-cell mass and secretion. This is associated with alterations in the expression of genes important for ß-cell proliferation and function as well as the maintenance of the ß-cell phenotype. Taken together, these data demonstrate that pancreatic islet-derived PYY plays an important role in controlling glucose homeostasis through the modulation of ß-cell mass and function.

The balance between adaptive and apoptotic unfolded protein responses regulates ß-cell death under ER stress conditions through XBP1, CHOP and JNK.

Endoplasmic reticulum (ER) stress and the subsequent unfolded protein response (UPR) have been implicated in ß-cell death in type 1 and type 2 diabetes. However, the UPR is also a fundamental mechanism required for ß-cell adaptation and survival. The mechanisms regulating the transition from adaptive to apoptotic UPR remain to be clarified. Here, we investigated the relationships between XBP1, CHOP and JNK in the transition from adaptive to apoptotic UPR and ß-cell death in models of type 1 and type 2 diabetes. XBP1 inhibition potentiated cell death induced by pro-inflammatory cytokines or the saturated fatty acid palmitate in MIN6 ß-cells. This response was prevented by CHOP inhibition. IRE1/XBP1 inhibition led to alterations in islets from diabetes-resistant ob/ob mice that resemble those found in diabetes, including increases in cell death and inflammation and antioxidant gene expression. Similarly, IRE1/XBP1 inhibition increased cell death in islets from NOD mice. On the other hand, JNK inhibition: 1) increased adaptive UPR and reduced cell death in islets from diabetic db/db mice, and 2) restored adaptive UPR while protecting against apoptotic UPR gene expression and ß-cell death and dysfunction following cytokine exposure. These findings suggest that the balance between XBP1-mediated adaptive and CHOP-dependent apoptotic UPR is critically important for ß-cell survival during ER stress. JNK activation regulates the transition from adaptive to apoptotic UPR, thus providing a mechanism for ß-cell propensity to cell death rather than ER stress adaptation in type 1 and type 2 diabetes.

NPY signalling in early osteoblasts controls glucose homeostasis.

OBJECTIVE: The skeleton has recently emerged as an additional player in the control of whole-body glucose metabolism; however, the mechanism behind this is not clear. METHODS: Here we employ mice lacking neuropeptide Y, Y1 receptors solely in cells of the early osteoblastic lineage (Y1f3.6Cre), to examine the role of osteoblastic Y1 signalling in glycaemic control. RESULTS: Y1f3.6Cre mice not only have a high bone mass phenotype, but importantly also display altered glucose homeostasis; significantly decreased pancreas weight, islet number and pancreatic insulin content leading to elevated glucose levels and reduced glucose tolerance, but with no effect on insulin induced glucose clearance. The reduced glucose tolerance and elevated bone mass was corrected in Y1f3.6Cre mice by bone marrow transplant from wildtype animals, reinforcing the osteoblastic nature of this pathway. Importantly, when fed a high fat diet, Y1f3.6Cre mice, while equally gaining body weight and fat mass compared to controls, showed significantly improved glucose and insulin tolerance. Conditioned media from Y1f3.6Cre osteoblastic cultures was unable to stimulate insulin expression in MIN6 cells compared to conditioned media from wildtype osteoblast, indicating a direct signalling pathway. Importantly, osteocalcin a secreted osteoblastic factor previously identified as a modulator of insulin secretion was not altered in the Y1f3.6Cre model. CONCLUSION: This study identifies the existence of other osteoblast-derived regulators of pancreas function and insulin secretion and illustrates a mechanism by which NPY signalling in bone tissue is capable of regulating pancreatic function and glucose homeostasis.

Inhibitor of differentiation proteins protect against oxidative stress by regulating the antioxidant-mitochondrial response in mouse beta cells.

AIMS/HYPOTHESIS: Oxidative stress is implicated in beta cell glucotoxicity in type 2 diabetes. Inhibitor of differentiation (ID) proteins are transcriptional regulators induced by hyperglycaemia in islets, but the mechanisms involved and their role in beta cells are not clear. Here we investigated whether or not oxidative stress regulates ID levels in beta cells and the role of ID proteins in beta cells during oxidative stress. METHODS: MIN6 cells were cultured in H2O2 or ribose to induce oxidative stress. ID1, ID3 and small MAF proteins (MAFF, MAFG and MAFK) were inhibited using small interfering RNA. Isolated islets from Id1(-/-), Id3(-/-) and diabetic db/db mice were used. RESULTS: ID1-4 expression was upregulated in vivo in the islets of diabetic db/db mice and stimulated in vitro by ribose and H2O2. Id1/3 inhibition reduced the expression of multiple antioxidant genes and potentiated oxidative stress-induced apoptosis. This finding was associated with increased levels of intracellular reactive oxygen species, altered mitochondrial morphology and reduced expression of Tfam, which encodes a mitochondrial transcription factor, and respiratory chain components. Id1/3 inhibition also reduced the expression of small MAF transcription factors (MafF, MafG and MafK), interacting partners of nuclear factor, erythroid 2-like 2 (NFE2L2), master regulator of the antioxidant response. Inhibition of small MAFs reduced the expression of antioxidant genes and potentiated oxidative stress-induced apoptosis, thus recapitulating the effects of Id1/3 inhibition. CONCLUSIONS/INTERPRETATION: Our study identifies IDs as a novel family of oxidative stress-responsive proteins in beta cells. IDs are crucial regulators of the adaptive antioxidant-mitochondrial response that promotes beta cell survival during oxidative stress through a novel link to the NFE2L2-small MAF pathway.

Influence of chronic hyperglycemia on the loss of the unfolded protein response in transplanted islets.

Chronic hyperglycemia contributes to ß-cell dysfunction in diabetes and with islet transplantation, but the mechanisms remain unclear. Recent studies demonstrate that the unfolded protein response (UPR) is critical for ß-cell function. Here, we assessed the influence of hyperglycemia on UPR gene expression in transplanted islets. Streptozotocin-induced diabetic or control nondiabetic mice were transplanted under the kidney capsule with syngeneic islets either sufficient or not to normalize hyperglycemia. Twenty-one days after transplantation, islet grafts were excised and RT-PCR was used to assess gene expression. In islet grafts from diabetic mice, expression levels of many UPR genes of the IRE1/ATF6 pathways, which are important for adaptation to endoplasmic reticulum stress, were markedly reduced compared with that in islet grafts from control mice. UPR genes of the PERK pathway were also downregulated. The normalization of glycemia restored the changes in mRNA expression, suggesting that chronic hyperglycemia contributes to the downregulation of multiple arms of UPR gene expression. Similar correlations were observed between blood glucose and mRNA levels of transcription factors involved in the maintenance of ß-cell phenotype and genes implicated in ß-cell function, suggesting convergent regulation of UPR gene expression and ß-cell differentiation by hyperglycemia. However, the normalization of glycemia was not accompanied by restoration of antioxidant or pro-inflammatory cytokine mRNA levels, which were increased in islet grafts from diabetic mice. These studies demonstrate that chronic hyperglycemia contributes to the downregulation of multiple arms of UPR gene expression in transplanted mouse islets. Failure of the adaptive UPR may contribute to ß-cell dedifferentiation and dysfunction in diabetes.

Failure of the adaptive unfolded protein response in islets of obese mice is linked with abnormalities in ß-cell gene expression and progression to diabetes.

The normal ß-cell response to obesity-associated insulin resistance is hypersecretion of insulin. Type 2 diabetes develops in subjects with ß-cells that are susceptible to failure. Here, we investigated the time-dependent gene expression changes in islets of diabetes-prone db/db and diabetes-resistant ob/ob mice. The expressions of adaptive unfolded protein response (UPR) genes were progressively induced in islets of ob/ob mice, whereas they declined in diabetic db/db mice. Genes important for ß-cell function and maintenance of the islet phenotype were reduced with time in db/db mice, whereas they were preserved in ob/ob mice. Inflammation and antioxidant genes displayed time-dependent upregulation in db/db islets but were unchanged in ob/ob islets. Treatment of db/db mouse islets with the chemical chaperone 4-phenylbutyric acid partially restored the changes in several ß-cell function genes and transcription factors but did not affect inflammation or antioxidant gene expression. These data suggest that the maintenance (or suppression) of the adaptive UPR is associated with ß-cell compensation (or failure) in obese mice. Inflammation, oxidative stress, and a progressive loss of ß-cell differentiation accompany diabetes progression. The ability to maintain the adaptive UPR in islets may protect against the gene expression changes that underlie diabetes development in obese mice.