Drosophila contain an insulin receptor homologue, encoded by the inr gene located at position 93E4-5 on the third chromosome. The receptor protein is strikingly homologous to the human receptor, exhibiting the same alpha2beta2 subunit structure and containing a ligand- activated tyrosine kinase in its cytoplasmic domain. Chemical mutagenesis was used to induce mutations in the inr gene and six independent mutations that lead to a loss of expression or function of the receptor protein were identified. These mutations are recessive, embryonic, or early larval lethals, but some alleles exhibit heteroallelic complementation to yield adults with a severe developmental delay (10 days), growth-deficiency, female-sterile phenotype. Interestingly, the severity of the mutant phenotype correlates with biochemical measures of loss of function of the receptor tyrosine kinase. The growth deficiency appears to be due to a reduction in cell number, suggesting a role for inr in regulation of cell proliferation during development. The phenotype is reminiscent of those seen in syndromes of insulin-resistance or IGF-I and IGF-I receptor deficiencies in higher organisms, suggesting a conserved function for this growth factor family in the regulation of growth and body size.

These findings provide strong evidence suggesting an important role of ER stress and the UPR in CS-related oxidative injury of RPE cells. Thus, the modulation of the UPR signaling may provide a promising target for the treatment of AMD.

Damage to the retinal pigment epithelium (RPE) is an early event in the pathogenesis of age-related macular degeneration (AMD). X-box binding protein 1 (XBP1) is a key transcription factor that regulates endoplasmic reticulum (ER) homeostasis and cell survival. This study aimed to delineate the role of endogenous XBP1 in the RPE. Our results show that in a rat model of light-induced retinal degeneration, XBP1 activation was suppressed in the RPE/choroid complex, accompanied by decreased anti-oxidant genes and increased oxidative stress. Knockdown of XBP1 by siRNA resulted in reduced expression of SOD1, SOD2, catalase, and glutathione synthase and sensitized RPE cells to oxidative damage. Using Cre/LoxP system, we generated a mouse line that lacks XBP1 only in RPE cells. Compared to wildtype littermates, RPE-XBP1 KO mice expressed less SOD1, SOD2, and catalase in the RPE, and had increased oxidative stress. At age 3 months and older, these mice exhibited apoptosis of RPE cells, decreased number of cone photoreceptors, shortened photoreceptor outer segment, reduced ONL thickness, and deficit in retinal function. Electron microscopy showed abnormal ultrastructure, Bruch's membrane thickening, and disrupted basal membrane infolding in XBP1-deficient RPE. These results indicate that XBP1 is an important gene involved in regulation of the anti-oxidant defense in the RPE, and that impaired activation of XBP1 may contribute to RPE dysfunction and cell death during retinal degeneration and AMD.

Aims/hypothesis Müller glia (MG) are major sources of retinal cytokines, and their activation is closely linked to retinal inflammation and vascular leakage in diabetic retinopathy. Previously, we demonstrated that X-box binding protein 1 (XBP1), a transcription factor activated by endoplasmic reticulum (ER) stress in diabetic retinopathy, is involved in regulation of inflammation in retinal endothelial cells. Now, we have explored the role of XBP1 and ER stress in the regulation of MGderived proinflammatory factors, and their influence on vascular permeability in diabetic retinopathy. Methods MG-specific conditional Xbp1 knockout (Xbp1 Müller−/− ) mice were generated by crossing Xbp1 flox/flox mice with Müller-Cre transgenic mice. Diabetes was modelled by induction with streptozotocin, and retinal vascular permeability was measured with FITC-conjugated dextran 2 months after induction. Primary Müller cells were isolated from Xbp1 Müller−/− and Xbp1 Müller+/+ mice and exposed to hypoxia and high levels of glucose. Levels of ER-stress and inflammatory factors were examined by real-time PCR, western blotting or immunohistochemistry. Results Xbp1 Müller−/− mice exhibited normal retinal development and retinal function and expressed similar levels of ER-stress and inflammatory genes to Xbp1 Müller+/+ littermates. In diabetes-inducing conditions, compared with Xbp1 Müller+/+ mice, Xbp1 Müller−/− mice had higher mRNA levels of retinal Vegf (also known as Vegfa) and Tnf-α (also known as Tnf) and ERstress marker genes Grp78 (also known as Hspa5), Atf4, Chop (also known as Ddit3) and Atf6 and higher protein levels of vascular endothelial growth factor (VEGF), TNF-α, phospho-c-Jun N-terminal kinase (JNK), 78 kDa glucose-regulated protein (GRP78), phospho-eukaryotic translation initiation factor (eIF)2α and activating transcription factor (ATF)6. Retinal vascular permeability was significantly higher in diabetic Xbp1 Müller−/− mice than in diabetic Xbp1 Müller+/+ mice (p < 0.01). Results obtained in vitro with primary Müller cells isolated from Xbp1 Müller−/− mice confirmed higher expression levels of inflammatory and ER-stress markers (but not GRP78) than in cells from Xbp1 Müller+/+ mice. Moreover, XBP1-deficient Müller cells were more susceptible to high-glucose-or hypoxia-induced ER stress and inflammation than cells from Xbp1 Müller+/+ mice. Inhibition of ER stress with chemical chaperones suppressed hypoxia-induced VEGF and TNF-α production in XBP1-deficient Müller cells.Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00125-018-4776-y) contains peer-reviewed but unedited supplementary material, which is available to authorised users.