NLRP3 Inflammasome Priming in the Retina of Diabetic Mice Requires REDD1-Dependent Activation of GSK3ß.

Purpose: Inflammasome activation has been implicated in the development of retinal complications caused by diabetes. This study was designed to identify signaling events that promote retinal NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation in response to diabetes. Methods: Diabetes was induced in mice by streptozotocin administration. Retinas were examined after 16 weeks of diabetes. Human MIO-M1 Müller cells were exposed to hyperglycemic culture conditions. Genetic and pharmacological interventions were used to interrogate signaling pathways. Visual function was assessed in mice using a virtual optomotor system. Results: In the retina of diabetic mice and in Müller cell cultures, NLRP3 and interleukin-1ß (IL-1ß) were increased in response to hyperglycemic conditions and the stress response protein Regulated in Development and DNA damage 1 (REDD1) was required for the effect. REDD1 deletion prevented caspase-1 activation in Müller cells exposed to hyperglycemic conditions and reduced IL-1ß release. REDD1 promoted nuclear factor κB signaling in cells exposed to hyperglycemic conditions, which was necessary for an increase in NLRP3. Expression of a constitutively active GSK3ß variant restored NLRP3 expression in REDD1-deficient cells exposed to hyperglycemic conditions. GSK3 activity was necessary for increased NLRP3 expression in the retina of diabetic mice and in cells exposed to hyperglycemic conditions. Müller glia-specific REDD1 deletion prevented increased retinal NLRP3 levels and deficits in contrast sensitivity in diabetic mice. Conclusions: The data support a role for REDD1-dependent activation of GSK3ß in NLRP3 inflammasome transcriptional priming and in the production of IL-1ß by Müller glia in response to diabetes.

REDD1-dependent GSK3ß dephosphorylation promotes NF-κB activation and macrophage infiltration in the retina of diabetic mice.

Increasing evidence supports a role for inflammation in the early development and progression of retinal complications caused by diabetes. We recently demonstrated that the stress response protein regulated in development and DNA damage response 1 (REDD1) promotes diabetes-induced retinal inflammation by sustaining canonical activation of nuclear transcription factor, NF-κB. The studies here were designed to identify signaling events whereby REDD1 promotes NF-κB activation in the retina of diabetic mice. We observed increased REDD1 expression in the retina of mice after 16 weeks of streptozotocin (STZ)-induced diabetes and found that REDD1 was essential for diabetes to suppress inhibitory phosphorylation of glycogen synthase kinase 3ß (GSK3ß) at S9. In human retinal MIO-M1 Müller cell cultures, REDD1 deletion prevented dephosphorylation of GSK3ß and increased NF-κB activation in response to hyperglycemic conditions. Expression of a constitutively active GSK3ß variant restored NF-κB activation in cells deficient for REDD1. In cells exposed to hyperglycemic conditions, GSK3ß knockdown inhibited NF-κB activation and proinflammatory cytokine expression by preventing inhibitor of κB kinase complex autophosphorylation and inhibitor of κB degradation. In both the retina of STZ-diabetic mice and in Müller cells exposed to hyperglycemic conditions, GSK3 inhibition reduced NF-κB activity and prevented an increase in proinflammatory cytokine expression. In contrast with STZ-diabetic mice receiving a vehicle control, macrophage infiltration was not observed in the retina of STZ-diabetic mice treated with GSK3 inhibitor. Collectively, the findings support a model wherein diabetes enhances REDD1-dependent activation of GSK3ß to promote canonical NF-κB signaling and the development of retinal inflammation.

PERK/ATF4-dependent expression of the stress response protein REDD1 promotes proinflammatory cytokine expression in the heart of obese mice.

Endoplasmic reticulum (ER) stress and inflammation are hallmarks of myocardial impairment. Here, we investigated the role of the stress response protein regulated in development and DNA damage 1 (REDD1) as a molecular link between ER stress and inflammation in cardiomyocytes. In mice fed a high-fat high-sucrose (HFHS, 42% kcal fat, 34% sucrose by weight) diet for 12 wk, REDD1 expression in the heart was increased in coordination with markers of ER stress and inflammation. In human AC16 cardiomyocytes exposed to either hyperglycemic conditions or the saturated fatty acid palmitate, REDD1 expression was increased coincident with ER stress and upregulated expression of the proinflammatory cytokines IL-1ß, IL-6, and TNFα. In cardiomyocytes exposed to hyperglycemic/hyperlipidemic conditions, pharmacological inhibition of the ER kinase protein kinase RNA-like endoplasmic reticulum kinase (PERK) or knockdown of the transcription factor ATF4 prevented the increase in REDD1 expression. REDD1 deletion reduced proinflammatory cytokine expression in both cardiomyocytes exposed to hyperglycemic/hyperlipidemic conditions and in the hearts of obese mice. Overall, the findings support a model wherein HFHS diet contributes to the development of inflammation in cardiomyocytes by promoting REDD1 expression via activation of a PERK/ATF4 signaling axis.NEW & NOTEWORTHY Interplay between endoplasmic reticulum stress and inflammation contributes to cardiovascular disease progression. The studies here identify the stress response protein known as REDD1 as a missing molecular link that connects the development of endoplasmic reticulum stress with increased production of proinflammatory cytokines in the hearts of obese mice.

Loss of 4E-BPs prevents the hindlimb immobilization-induced decrease in protein synthesis in skeletal muscle.

The present study was designed to test the hypothesis that upregulating protein synthesis attenuates the loss of muscle mass in a model of disuse atrophy. The studies compared the effect of unilateral hindlimb immobilization in wild-type (WT) mice and double-knockout (DKO) mice lacking the translational regulators 4E-BP1 and 4E-BP2. Immobilization-induced downregulation of protein synthesis occurred in both groups of mice, but protein synthesis was higher in gastrocnemius muscle from the immobilized hindlimb of fasted DKO compared with WT mice. Surprisingly, although protein synthesis was partially elevated in DKO compared with WT mice, atrophy occurred to the same extent in both groups of animals. This may be partially due to impaired leucine-induced stimulation of protein synthesis in DKO compared with WT mice due to downregulated eukaryotic initiation factor eIF4E expression in muscle of DKO compared with WT mice. Expression of the E3 ubiquitin ligases MAFbx and MuRF-1 mRNAs and total protein ubiquitylation was upregulated in the immobilized compared with the nonimmobilized hindlimb of both WT and DKO mice, with little difference in the magnitude of the upregulation between genotypes. Analysis of newly synthesized proteins revealed downregulation of several glycolytic enzymes in the gastrocnemius of DKO mice compared with WT mice, as well as in the immobilized compared with the nonimmobilized hindlimb. Overall, the results suggest that the elevated rate of protein synthesis during hindlimb immobilization in fasted DKO mice is insufficient to prevent disuse-induced muscle atrophy, probably due to induction of compensatory mechanisms including downregulation of eIF4E expression.NEW & NOTEWORTHY Basal rates of protein synthesis are elevated in skeletal muscle in the immobilized leg of mice lacking the translational repressors, 4E-BP1 and 4E-BP2 (knockout mice), compared with wild-type mice. However, disuse-induced muscle atrophy occurs to the same extent in both wild-type and knockout mice suggesting that compensatory mechanisms are induced that overcome the upregulation of muscle protein synthesis. Proteomic analysis revealed that mRNAs encoding several glycolytic enzymes are differentially translated in wild-type and knockout mice.

An integrative approach to assessing effects of a short-term Western diet on gene expression in rat liver.

Consumption of a diet rich in saturated fatty acids and carbohydrates contributes to the accumulation of fat in the liver and development of non-alcoholic steatohepatitis (NASH). Herein we investigated the hypothesis that short-term consumption of a high fat/sucrose Western diet (WD) alters the genomic and translatomic profile of the liver in association with changes in signaling through the protein kinase mTORC1, and that such alterations contribute to development of NAFLD. The results identify a plethora of mRNAs that exhibit altered expression and/or translation in the liver of rats consuming a WD compared to a CD. In particular, consumption of a WD altered the abundance and ribosome association of mRNAs involved in lipid and fatty acid metabolism, as well as those involved in glucose metabolism and insulin signaling. Hepatic mTORC1 signaling was enhanced when rats were fasted overnight and then refed in the morning; however, this effect was blunted in rats fed a WD as compared to a CD. Despite similar plasma insulin concentrations, fatty acid content was elevated in the liver of rats fed a WD as compared to a CD. We found that feeding had a significant positive effect on ribosome occupancy of 49 mRNAs associated with hepatic steatosis (e.g., LIPE, LPL), but this effect was blunted in the liver of rats fed a WD. In many cases, changes in ribosome association were independent of alterations in mRNA abundance, suggesting a critical role for diet-induced changes in mRNA translation in the expression of proteins encoded by those mRNAs. Overall, the findings demonstrate that short-term consumption of a WD impacts hepatic gene expression by altering the abundance of many mRNAs, but also causes wide-spread variation in mRNA translation that potentially contribute to development of hepatic steatosis.

Spleen Tyrosine Kinase Contributes to Müller Glial Expression of Proangiogenic Cytokines in Diabetes.

Purpose: Neuroglial dysfunction occurs early in the progression of diabetic retinopathy. In response to diabetes or hypoxia, Müller glia secrete cytokines and growth factors that contribute to disease progression. This study was designed to examine common signaling pathways activated in Müller glia by both type 1 and pre-/type 2 diabetes. Methods: RiboTag (Pdgfra-cre;HA-Rpl22) mice were used to compare the impact of streptozotocin (STZ) and a high-fat, high-sucrose (HFHS) diet on ribosome association of mRNAs in Müller glia by RNA sequencing analysis. Human MIO-M1 Müller cells were exposed to either hyperglycemic or hypoxic culture conditions. Genetic manipulation and pharmacologic inhibition were used to interrogate signaling pathways. Results: Association of mRNAs encoding triggering receptor expressed on myeloid cells 2 (TREM2), DNAX-activating protein 12 kDa (DAP12), and colony stimulating factor 1 receptor (CSF1R) with ribosomes isolated from Müller glia was upregulated in both STZ diabetic mice and mice fed an HFHS diet. The TREM2/DAP12 receptor-adaptor complex signals in coordination with CSF1R to activate spleen tyrosine kinase (SYK). SYK activation was enhanced in the retina of diabetic mice and in human MIO-M1 Müller cell cultures exposed to hyperglycemic or hypoxic culture conditions. DAP12 knockdown reduced SYK autophosphorylation in Müller cells exposed to hyperglycemic or hypoxic conditions. SYK inhibition or DAP12 knockdown suppressed hypoxia-induced expression of the transcription factor hypoxia-inducible factor 1⍺ (HIF1⍺), as well as expression of vascular endothelial growth factor and angiopoietin-like 4. Conclusions: The findings support TREM2/DAP12 receptor-adaptor complex signaling via SYK to promote HIF1α stabilization and increased angiogenic cytokine production by Müller glia.

Stress response protein REDD1 promotes diabetes-induced retinal inflammation by sustaining canonical NF-κB signaling.

Inflammation contributes to the progression of retinal pathology caused by diabetes. Here, we investigated a role for the stress response protein regulated in development and DNA damage response 1 (REDD1) in the development of retinal inflammation. Increased REDD1 expression was observed in the retina of mice after 16-weeks of streptozotocin (STZ)-induced diabetes, and REDD1 was essential for diabetes-induced pro-inflammatory cytokine expression. In human retinal MIO-M1 Müller cell cultures, REDD1 deletion prevented increased pro-inflammatory cytokine expression in response to hyperglycemic conditions. REDD1 deletion promoted nuclear factor erythroid-2-related factor 2 (Nrf2) hyperactivation; however, Nrf2 was not required for reduced inflammatory cytokine expression in REDD1-deficient cells. Rather, REDD1 enhanced inflammatory cytokine expression by promoting activation of nuclear transcription factor κB (NF-κB). In WT cells exposed to tumor necrosis factor α (TNFα), inflammatory cytokine expression was increased in coordination with activating transcription factor 4 (ATF4)-dependent REDD1 expression and sustained activation of NF-κB. In both Müller cell cultures exposed to TNFα and in the retina of STZ-diabetic mice, REDD1 deletion promoted inhibitor of κB (IκB) expression and reduced NF-κB DNA-binding activity. We found that REDD1 acted upstream of IκB by enhancing both K63-ubiquitination and auto-phosphorylation of IκB kinase complex. In contrast with STZ-diabetic REDD1+/+ mice, IκB kinase complex autophosphorylation and macrophage infiltration were not observed in the retina of STZ-diabetic REDD1-/- mice. The findings provide new insight into how diabetes promotes retinal inflammation and support a model wherein REDD1 sustains activation of canonical NF-κB signaling.

Activation of Disulfide Redox Switch in REDD1 Promotes Oxidative Stress Under Hyperglycemic Conditions.

The stress response protein regulated in development and DNA damage response 1 (REDD1) has been implicated in visual deficits in patients with diabetes. The aim here was to investigate the mechanism responsible for the increase in retinal REDD1 protein content that is observed with diabetes. We found that REDD1 protein expression was increased in the retina of streptozotocin-induced diabetic mice in the absence of a change in REDD1 mRNA abundance or ribosome association. Oral antioxidant supplementation reduced retinal oxidative stress and suppressed REDD1 protein expression in the retina of diabetic mice. In human retinal Müller cell cultures, hyperglycemic conditions increased oxidative stress, enhanced REDD1 expression, and inhibited REDD1 degradation independently of the proteasome. Hyperglycemic conditions promoted a redox-sensitive cross-strand disulfide bond in REDD1 at C150/C157 that was required for reduced REDD1 degradation. Discrete molecular dynamics simulations of REDD1 structure revealed allosteric regulation of a degron upon formation of the disulfide bond that disrupted lysosomal proteolysis of REDD1. REDD1 acetylation at K129 was required for REDD1 recognition by the cytosolic chaperone HSC70 and degradation by chaperone-mediated autophagy. Disruption of REDD1 allostery upon C150/C157 disulfide bond formation prevented the suppressive effect of hyperglycemic conditions on REDD1 degradation and reduced oxidative stress in cells exposed to hyperglycemic conditions. The results reveal redox regulation of REDD1 and demonstrate the role of a REDD1 disulfide switch in development of oxidative stress.

REDD1 Ablation Attenuates the Development of Renal Complications in Diabetic Mice.

Chronic hyperglycemia contributes to development of diabetic kidney disease by promoting glomerular injury. In this study, we evaluated the hypothesis that hyperglycemic conditions promote expression of the stress response protein regulated in development and DNA damage response 1 (REDD1) in the kidney in a manner that contributes to the development of oxidative stress and renal injury. After 16 weeks of streptozotocin-induced diabetes, albuminuria and renal hypertrophy were observed in wild-type (WT) mice coincident with increased renal REDD1 expression. In contrast, diabetic REDD1 knockout (KO) mice did not exhibit impaired renal physiology. Histopathologic examination revealed that glomerular damage including mesangial expansion, matrix deposition, and podocytopenia in the kidneys of diabetic WT mice was reduced or absent in diabetic REDD1 KO mice. In cultured human podocytes, exposure to hyperglycemic conditions enhanced REDD1 expression, increased reactive oxygen species (ROS) levels, and promoted cell death. In both the kidney of diabetic mice and in podocyte cultures exposed to hyperglycemic conditions, REDD1 deletion reduced ROS and prevented podocyte loss. Benefits of REDD1 deletion were recapitulated by pharmacological GSK3ß suppression, supporting a role for REDD1-dependent GSK3ß activation in diabetes-induced oxidative stress and renal defects. The results support a role for REDD1 in diabetes-induced renal complications.

Müller Glial Expression of REDD1 Is Required for Retinal Neurodegeneration and Visual Dysfunction in Diabetic Mice.

Clinical studies support a role for the protein regulated in development and DNA damage response 1 (REDD1) in ischemic retinal complications. To better understand how REDD1 contributes to retinal pathology, we examined human single-cell sequencing data sets and found specificity of REDD1 expression that was consistent with markers of retinal Müller glia. Thus, we investigated the hypothesis that REDD1 expression specifically in Müller glia contributes to diabetes-induced retinal pathology. The retina of Müller glia-specific REDD1 knockout (REDD1-mgKO) mice exhibited dramatic attenuation of REDD1 transcript and protein expression. In the retina of streptozotocin-induced diabetic control mice, REDD1 protein expression was enhanced coincident with an increase in oxidative stress. In the retina of diabetic REDD1-mgKO mice, there was no increase in REDD1 protein expression, and oxidative stress was reduced compared with diabetic control mice. In both Müller glia within the retina of diabetic mice and human Müller cell cultures exposed to hyperglycemic conditions, REDD1 was necessary for increased expression of the gliosis marker glial fibrillary acidic protein. The effect of REDD1 deletion in preventing gliosis was associated with suppression of oxidative stress and required the antioxidant transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2). In contrast to diabetic control mice, diabetic REDD1-mgKO mice did not exhibit retinal thinning, increased markers of neurodegeneration within the retinal ganglion cell layer, or deficits in visual function. Overall, the findings support a key role for Müller glial REDD1 in the failed adaptive response of the retina to diabetes that includes gliosis, neurodegeneration, and impaired vision.

Retinal Protein O-GlcNAcylation and the Ocular Renin-angiotensin System: Signaling Cross-roads in Diabetic Retinopathy.

It is well established that diabetes and its associated hyperglycemia negatively impact retinal function, yet we know little about the role played by augmented flux through the Hexosamine Biosynthetic Pathway (HBP). This offshoot of the glycolytic pathway produces UDP-Nacetyl- glucosamine, which serves as the substrate for post-translational O-linked modification of proteins in a process referred to as O-GlcNAcylation. HBP flux and subsequent protein O-GlcNAcylation serve as nutrient sensors, enabling cells to integrate metabolic information to appropriately modulate fundamental cellular processes including gene expression. Here we summarize the impact of diabetes on retinal physiology, highlighting recent studies that explore the role of O-GlcNAcylation- induced variation in mRNA translation in retinal dysfunction and the pathogenesis of Diabetic Retinopathy (DR). Augmented O-GlcNAcylation results in wide variation in the selection of mRNAs for translation, in part, due to O-GlcNAcylation of the translational repressor 4E-BP1. Recent studies demonstrate that 4E-BP1 plays a critical role in regulating O-GlcNAcylation-induced changes in the translation of the mRNAs encoding Vascular Endothelial Growth Factor (VEGF), a number of important mitochondrial proteins, and CD40, a key costimulatory molecule involved in diabetes-induced retinal inflammation. Remarkably, 4E-BP1/2 ablation delays the onset of diabetes- induced visual dysfunction in mice. Thus, pharmacological interventions to prevent the impact of O-GlcNAcylation on 4E-BP1 may represent promising therapeutics to address the development and progression of DR. In this regard, we discuss the potential interplay between retinal O-GlcNAcylation and the ocular renin-angiotensin system as a potential therapeutic target of future interventions.

Glucagon transiently stimulates mTORC1 by activation of an EPAC/Rap1 signaling axis.

Activation of the protein kinase mechanistic target of rapamycin (mTOR) in both complexes 1 and 2 (mTORC1/2) in the liver is repressed during fasting and rapidly stimulated in response to a meal. The effect of feeding on hepatic mTORC1/2 is attributed to an increase in plasma levels of nutrients, such as amino acids, and insulin. By contrast, fasting is associated with elevated plasma levels of glucagon, which is conventionally viewed as having a counter-regulatory role to insulin. More recently an expanded role for glucagon action in post-prandial metabolism has been demonstrated. Herein we investigated the impact of insulin and glucagon on mTORC1/2 activation. In H4IIE and HepG2 cultures, insulin enhanced phosphorylation of the mTORC1 substrates S6K1 and 4E-BP1. Surprisingly, the effect of glucagon on mTORC1 was biphasic, wherein there was an acute increase in phosphorylation of S6K1 and 4E-BP1 over the first hour of exposure, followed by latent suppression. The transient stimulatory effect of glucagon on mTORC1 was not additive with insulin, suggesting convergent signaling. Glucagon enhanced cAMP levels and mTORC1 stimulation required activation of the glucagon receptor, PI3K/Akt, and exchange protein activated by cAMP (EPAC). EPAC acts as the guanine nucleotide exchange factor for the small GTPase Rap1. Rap1 expression enhanced S6K1 phosphorylation and glucagon addition to culture medium promoted Rap1-GTP loading. Signaling through mTORC1 acts to regulate protein synthesis and we found that glucagon promoted an EPAC-dependent increase in protein synthesis. Overall, the findings support that glucagon elicits acute activation of mTORC1/2 by an EPAC-dependent increase in Rap1-GTP.

The stress response protein REDD1 as a causal factor for oxidative stress in diabetic retinopathy.

Diabetic Retinopathy (DR) is a major cause of visual dysfunction, yet much remains unknown regarding the specific molecular events that contribute to diabetes-induced retinal pathophysiology. Herein, we review the impact of oxidative stress on DR, and explore evidence that supports a key role for the stress response protein regulated in development and DNA damage (REDD1) in the development of diabetes-induced oxidative stress and functional defects in vision. It is well established that REDD1 mediates the cellular response to a number of diverse stressors through repression of the central metabolic regulator known as mechanistic target of rapamycin complex 1 (mTORC1). A growing body of evidence also supports that REDD1 acts independent of mTORC1 to promote oxidative stress by both enhancing the production of reactive oxygen species and suppressing the antioxidant response. Collectively, there is strong preclinical data to support a key role for REDD1 in the development and progression of retinal complications caused by diabetes. Furthermore, early proof-of-concept clinical trials have found a degree of success in combating ischemic retinal disease through intravitreal delivery of an siRNA targeting the REDD1 mRNA. Overall, REDD1-associated signaling represents an intriguing target for novel clinical therapies that go beyond addressing the symptoms of diabetes by targeting the underlying molecular mechanisms that contribute to DR.

Retinol-binding protein 4 mRNA translation in hepatocytes is enhanced by activation of mTORC1.

Increased expression of the peptide hormone retinol-binding protein 4 (RBP4) has been implicated in the development of insulin resistance, type 2 diabetes, and visual dysfunction. Prior investigations of the mechanisms that influence RBP4 synthesis have focused solely on changes in mRNA abundance. Yet, the production of many secreted proteins is controlled at the level of mRNA translation, as it allows for a rapid and reversible change in expression. Herein, we evaluated Rbp4 mRNA translation using sucrose density gradient centrifugation. In the liver of fasted rodents, Rbp4 mRNA translation was low. In response to refeeding, Rbp4 mRNA translation was enhanced and RBP4 levels in serum were increased. In H4IIE cells, refreshing culture medium promoted Rbp4 mRNA translation and expression of the protein. Rbp4 mRNA abundance was not increased by either experimental manipulation. Enhanced Rbp4 mRNA translation was associated with activation of the kinase mechanistic target of rapamycin in complex 1 (mTORC1) and enhanced phosphorylation of the translational repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). In H4IIE cells, expression of a 4E-BP1 variant that is unable to be phosphorylated by mTORC1 or suppression of mTORC1 with rapamycin attenuated activity of a luciferase reporter encoding the Rbp4 mRNA 5'-untranslated region (UTR). Purine substitutions to disrupt a terminal oligopyrimidine (TOP)-like sequence in the Rbp4 5'-UTR prevented the suppressive effect of rapamycin on reporter activity. Rapamycin also prevented upregulation of Rbp4 mRNA translation in the liver and reduced serum levels of RBP4 in response to feeding. Overall, the findings support a model in which nutrient-induced activation of mTORC1 upregulates Rbp4 mRNA translation to promote RBP4 synthesis.NEW & NOTEWORTHY RBP4 plays a critical role in metabolic disease, yet relatively little is known about the mechanisms that regulate its production. Herein, we provide evidence for translational control of RBP4 synthesis. We demonstrate that activation of the nutrient-sensitive kinase mTORC1 promotes hepatic Rbp4 mRNA translation. The findings support the possibility that targeting Rbp4 mRNA translation represents an alternative to current therapeutic interventions that lower serum RBP4 concentration by promoting urinary excretion of the protein.

Diabetes enhances translation of Cd40 mRNA in murine retinal Müller glia via a 4E-BP1/2-dependent mechanism.

Activation of the immune costimulatory molecule cluster of differentiation 40 (CD40) in Müller glia has been implicated in the initiation of diabetes-induced retinal inflammation. Results from previous studies support that CD40 protein expression is elevated in Müller glia of diabetic mice; however, the mechanisms responsible for this increase have not been explored. Here, we evaluated the hypothesis that diabetes augments translation of the Cd40 mRNA. Mice receiving thiamet G (TMG), an inhibitor of the O-GlcNAc hydrolase O-GlcNAcase, exhibited enhanced retinal protein O-GlcNAcylation and increased Cd40 mRNA translation. TMG administration also promoted Cd40 mRNA association with Müller cell-specific ribosomes isolated from the retina of RiboTag mice. Similar effects on O-GlcNAcylation and Cd40 mRNA translation were also observed in the retina of a mouse model of type 1 diabetes. In cultured cells, TMG promoted sequestration of the cap-binding protein eIF4E (eukaryotic translation in initiation factor 4E) by 4E-BP1 (eIF4E-binding protein 1) and enhanced cap-independent Cd40 mRNA translation as assessed by a bicistronic reporter that contained the 5'-UTR of the Cd40 mRNA. Ablation of 4E-BP1/2 prevented the increase in Cd40 mRNA translation in TMG-exposed cells, and expression of a 4E-BP1 variant that constitutively sequesters eIF4E promoted reporter activity. Extending on the cell culture results, we found that in contrast to WT mice, diabetic 4E-BP1/2-deficient mice did not exhibit enhanced retinal Cd40 mRNA translation and failed to up-regulate expression of the inflammatory marker nitric-oxide synthase 2. These findings support a model wherein diabetes-induced O-GlcNAcylation of 4E-BP1 promotes Cd40 mRNA translation in Müller glia.

Glucagon-Dependent Suppression of mTORC1 is Associated with Upregulation of Hepatic FGF21 mRNA Translation.

Fibroblast growth factor 21 (FGF21) is a peptide hormone that acts to enhance insulin sensitivity and reverse many of the metabolic defects associated with consumption of a high-fat diet. Recent studies show that the liver is the primary source of FGF21 in the blood, and that hepatic FGF21 expression is upregulated by glucagon. Interestingly, glucagon acts to upregulate FGF21 production by primary cultures of rat hepatocytes and H4IIE and HepG2 hepatocarcinoma cells independent of changes in FGF21 mRNA abundance, suggesting that FGF21 protein expression is regulated post-transcriptionally. Based on these observations, the goal of the present study was to assess whether or not FGF21 mRNA is translationally regulated. The results show that FGF21 mRNA translation and secretion of the hormone are significantly upregulated in H4IIE cells exposed to 25 nM glucagon, independent of changes in FGF21 mRNA abundance. Furthermore, the glucagon-induced upregulation of FGF21 mRNA translation is associated with suppressed activity of the mechanistic target of rapamycin in complex 1 (mTORC1). Similarly, the results show that rapamycin-induced suppression of mTORC1 leads to upregulation of FGF21 mRNA translation with no change in FGF21 mRNA abundance. In contrast, activation of mTORC1 by refreshing the culture medium leads to downregulation of FGF21 mRNA translation. Notably, re-feeding fasted rats also leads to downregulation of FGF21 mRNA translation concomitantly with activation of mTORC1 in the liver. Overall, the findings support a model in which glucagon acts to upregulate FGF21 production by hepatocytes through suppression of mTORC1 and subsequent upregulation of FGF21 mRNA translation.

The stress response protein REDD1 promotes diabetes-induced oxidative stress in the retina by Keap1-independent Nrf2 degradation.

The transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2) plays a critical role in reducing oxidative stress by promoting the expression of antioxidant genes. Both individuals with diabetes and preclinical diabetes models exhibit evidence of a defect in retinal Nrf2 activation. We recently demonstrated that increased expression of the stress response protein regulated in development and DNA damage 1 (REDD1) is necessary for the development of oxidative stress in the retina of streptozotocin-induced diabetic mice. In the present study, we tested the hypothesis that REDD1 suppresses the retinal antioxidant response to diabetes by repressing Nrf2 function. We found that REDD1 ablation enhances Nrf2 DNA-binding activity in the retina and that the suppressive effect of diabetes on Nrf2 activity is absent in the retina of REDD1-deficient mice compared with WT. In human MIO-M1 Müller cell cultures, REDD1 deletion prevented oxidative stress in response to hyperglycemic conditions, and this protective effect required Nrf2. REDD1 suppressed Nrf2 stability by promoting its proteasomal degradation independently of Nrf2's interaction with Kelch-like ECH-associated protein 1 (Keap1), but REDD1-mediated Nrf2 degradation required glycogen synthase kinase 3 (GSK3) activity and Ser-351/Ser-356 of Nrf2. Diabetes diminished inhibitory phosphorylation of glycogen synthase kinase 3ß (GSK3ß) at Ser-9 in the retina of WT mice but not in REDD1-deficient mice. Pharmacological inhibition of GSK3 enhanced Nrf2 activity and prevented oxidative stress in the retina of diabetic mice. The findings support a model wherein hyperglycemia-induced REDD1 blunts the Nrf2 antioxidant response to diabetes by activating GSK3, which, in turn, phosphorylates Nrf2 to promote its degradation.

Angiotensin-(1-7) Attenuates Protein O-GlcNAcylation in the Retina by EPAC/Rap1-Dependent Inhibition of O-GlcNAc Transferase.

Purpose: O-GlcNAcylation of cellular proteins contributes to the pathophysiology of diabetes and evidence supports a role for augmented O-GlcNAcylation in diabetic retinopathy. The aim of this study was to investigate the impact of the renin-angiotensin system on retinal protein O-GlcNAcylation. Methods: Mice fed a high-fat diet were treated chronically with the angiotensin-converting enzyme inhibitor captopril or captopril plus the angiotensin-(1-7) Mas receptor antagonist A779. Western blotting and quantitative polymerase chain reaction were used to analyze retinal homogenates. Similar analyses were performed on lysates from human MIO-M1 retinal Müller cell cultures exposed to media supplemented with angiotensin-(1-7). Culture conditions were manipulated to influence the hexosamine biosynthetic pathway and/or signaling downstream of the Mas receptor. Results: In the retina of mice fed a high-fat diet, captopril attenuated protein O-GlcNAcylation in a manner dependent on Mas receptor activation. In MIO-M1 cells, angiotensin-(1-7) or adenylate cyclase activation were sufficient to enhance cyclic AMP (cAMP) levels and inhibit O-GlcNAcylation. The repressive effect of cAMP on O-GlcNAcylation was dependent on exchange protein activated by cAMP (EPAC), but not protein kinase A, and was recapitulated by a constitutively active variant of the small GTPase Rap1. We provide evidence that cAMP and angiotensin-(1-7) act to suppress O-GlcNAcylation by inhibition of O-GlcNAc transferase (OGT) activity. In cells exposed to an O-GlcNAcase inhibitor or hyperglycemic culture conditions, mitochondrial superoxide levels were elevated; however, angiotensin-(1-7) signaling prevented the effect. Conclusions: Angiotensin-(1-7) inhibits retinal protein O-GlcNAcylation via an EPAC/Rap1/OGT signaling axis.

ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation.

BACKGROUND: The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase. OBJECTIVE: The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2. METHODS: Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0-16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates. RESULTS: Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted. CONCLUSIONS: The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.

REDD1 Activates a ROS-Generating Feedback Loop in the Retina of Diabetic Mice.

Purpose: The present study was designed to evaluate the role of the stress response protein REDD1 in diabetes-induced oxidative stress and retinal pathology. Methods: Wild-type and REDD1-deficient mice were administered streptozotocin to induce diabetes. Some mice received the antioxidant N-acetyl-l-cysteine (NAC). Visual function was assessed by virtual optometry. Retinas were analyzed by Western blotting. Reactive oxygen species (ROS) were assessed by 2,7-dichlorofluoroscein. Similar analyses were performed on R28 retinal cells in culture exposed to hyperglycemic conditions, NAC, and/or the exogenous ROS source hydrogen peroxide. Results: In the retina of diabetic mice, REDD1 expression and ROS were increased. In cells in culture, hyperglycemic conditions enhanced REDD1 expression, ROS levels, and the mitochondrial membrane potential. However, similar effects were not observed in the retina of diabetic mice or cells lacking REDD1. In the retina of diabetic mice and cells exposed to hyperglycemic conditions, NAC normalized ROS and prevented an increase in REDD1 expression. Diabetic mice receiving NAC also exhibited improved contrast sensitivity as compared to diabetic controls. Hydrogen peroxide addition to culture medium increased REDD1 expression and attenuated Akt/GSK3 phosphorylation in a REDD1-dependent manner. In REDD1-deficient cells exposed to hyperglycemic conditions, expression of a dominant negative Akt or constitutively active GSK3 increased the mitochondrial membrane potential and promoted ROS. Conclusions: The findings provide new insight into the mechanism whereby diabetes-induced hyperglycemia causes oxidative stress and visual dysfunction. Specifically, hyperglycemia-induced REDD1 activates a ROS-generating feedback loop that includes Akt/GSK3. Thus, therapeutic approaches targeting REDD1 expression and ROS may be beneficial for preventing diabetes-induced visual dysfunction.