Search

Increasing evidence supports the role of an augmented immune response in the early development and progression of renal complications caused by diabetes. We recently demonstrated that podocyte-specific expression of stress response protein regulated in development and DNA damage response 1 (REDD1) contributes to activation of the pro-inflammatory transcription factor NF-κB in the kidney of diabetic mice. The studies here were designed to define the specific signaling events whereby REDD1 promotes NF-κB activation in the context of diabetic nephropathy. Streptozotocin (STZ)-induced diabetes promoted activation of glycogen synthase kinase 3β (GSK3β) in the kidney, which was prevented by REDD1 ablation. REDD1 was necessary and sufficient to enhance GSK3β activity in human podocyte cultures exposed to hyperglycemic conditions. GSK3β suppression prevented NF-κB activation and normalized the expression of pro-inflammatory factors in podocytes exposed to hyperglycemic conditions. In the kidneys of diabetic mice and in podocytes exposed to hyperglycemic conditions, REDD1-dependent GSK3β signaling promoted activation of the inhibitor of κB (IκB) kinase (IKK) complex upstream of NF-κB. GSK3β knockdown in podocytes exposed to hyperglycemic conditions reduced macrophage chemotaxis. Similarly, in diabetic mice treated with a GSK3 inhibitor, immune cell infiltration in the kidneys was reduced. Overall, the data support a model wherein hyperglycemia amplifies the activation of GSK3β in a REDD1-dependent manner, leading to canonical NF-κB signaling and an augmented renal immune response in diabetic nephropathy., Competing Interests: Duality of Interest The authors declare no competing interests., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

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 3β (GSK3β) at S9. In human retinal MIO-M1 Müller cell cultures, REDD1 deletion prevented dephosphorylation of GSK3β and increased NF-κB activation in response to hyperglycemic conditions. Expression of a constitutively active GSK3β variant restored NF-κB activation in cells deficient for REDD1. In cells exposed to hyperglycemic conditions, GSK3β 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 GSK3β to promote canonical NF-κB signaling and the development of retinal inflammation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Conflict of interest M. D. D is guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and accuracy of the data analysis. The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Mechanistic target of rapamycin (mTOR) and mTOR complex 1 (mTORC1), linchpins of the nutrient sensing and protein synthesis pathways, are present at relatively high levels in the ganglion cell layer (GCL) and retinal ganglion cells (RGCs) of rodent and human retinas. However, the role of mTORCs in the control of protein synthesis in RGC is unknown. Here, we applied the SUrface SEnsing of Translation (SUnSET) method of nascent protein labeling to localize and quantify protein synthesis in the retinas of adult mice. We also used intravitreal injection of an adeno-associated virus 2 vector encoding Cre recombinase in the eyes of mtor- or rptor-floxed mice to conditionally knockout either both mTORCs or only mTORC1, respectively, in cells within the GCL. A novel vector encoding an inactive Cre mutant (CreΔC) served as control. We found that retinal protein synthesis was highest in the GCL, particularly in RGC. Negation of both complexes or only mTORC1 significantly reduced protein synthesis in RGC. In addition, loss of mTORC1 function caused a significant reduction in the pan-RGC marker, RNA-binding protein with multiple splicing, with little decrease of the total number of cells in the RGC layer, even at 25 weeks after adeno-associated virus-Cre injection. These findings reveal that mTORC1 signaling is necessary for maintaining the high rate of protein synthesis in RGCs of adult rodents, but it may not be essential to maintain RGC viability. These findings may also be relevant to understanding the pathophysiology of RGC disorders, including glaucoma, diabetic retinopathy, and optic neuropathies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Diabetes promotes the posttranslational modification of proteins by O- linked addition of GlcNAc ( O- GlcNAcylation) to Ser/Thr residues of proteins and thereby contributes to diabetic complications. In the retina of diabetic mice, the repressor of mRNA translation, eIF4E-binding protein 1 (4E-BP1), is O- GlcNAcylated, and sequestration of the cap-binding protein eukaryotic translation initiation factor (eIF4E) is enhanced. O- GlcNAcylation has also been detected on several eukaryotic translation initiation factors and ribosomal proteins. However, the functional consequence of this modification is unknown. Here, using ribosome profiling, we evaluated the effect of enhanced O- GlcNAcylation on retinal gene expression. Mice receiving thiamet G (TMG), an inhibitor of the O- GlcNAc hydrolase O- GlcNAcase, exhibited enhanced retinal protein O- GlcNAcylation. The principal effect of TMG on retinal gene expression was observed in ribosome-associated mRNAs ( i.e. mRNAs undergoing translation), as less than 1% of mRNAs exhibited changes in abundance. Remarkably, ∼19% of the transcriptome exhibited TMG-induced changes in ribosome occupancy, with 1912 mRNAs having reduced and 1683 mRNAs having increased translational rates. In the retina, the effect of O- GlcNAcase inhibition on translation of specific mitochondrial proteins, including superoxide dismutase 2 (SOD2), depended on 4E-BP1/2. O- GlcNAcylation enhanced cellular respiration and promoted mitochondrial superoxide levels in WT cells, and 4E-BP1/2 deletion prevented O- GlcNAcylation-induced mitochondrial superoxide in cells in culture and in the retina. The retina of diabetic WT mice exhibited increased reactive oxygen species levels, an effect not observed in diabetic 4E-BP1/2-deficient mice. These findings provide evidence for a mechanism whereby diabetes-induced O- GlcNAcylation promotes oxidative stress in the retina by altering the selection of mRNAs for translation., (© 2019 Dierschke et al.)

Despite recent advances in therapeutics, diabetic retinopathy remains a leading cause of vision impairment. Improvement in the treatment of diabetic retinopathy requires a better understanding of the molecular mechanisms that cause neurovascular complications, particularly in type 2 diabetes. Recent studies demonstrate that rodents fed a high fat diet exhibit retinal dysfunction concomitant with attenuated Akt phosphorylation. The purpose of the present study was to evaluate the impact of a high fat/high sucrose diet on retinal insulin signaling and evaluate the mechanism(s) responsible for the changes. Mice fed a high fat/sucrose diet exhibited attenuated Akt phosphorylation in the retina as compared with mice fed normal chow. Retinas of mice fed a high fat/sucrose diet also exhibited elevated levels of activated JNK as well as enhanced p70S6K1 autoinhibitory domain phosphorylation. In cells, JNK activation enhanced p70S6K1 phosphorylation and mTORC1-dependent activation of the kinase, as evidenced by enhanced phosphorylation of key substrates. Rictor phosphorylation by p70S6K1 was specifically enhanced by the addition of phosphomimetic mutations in the autoinhibitory domain and was more sensitive to inhibition of the kinase as compared with rpS6. Notably, rictor and IRS-1 phosphorylation by p70S6K1 attenuate insulin action through a negative feedback pathway. Indeed, p70S6K1 inhibition prevented the repressive effect of JNK activation on insulin action in retinas. Overall, the results identify the JNK/S6K1 axis as a key molecular mechanism whereby a high fat/sucrose diet impairs insulin action in retina., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Vascular endothelial growth factor (VEGF) is considered a major role player in the pathogenesis of diabetic retinopathy, yet the mechanisms regulating its expression are not fully understood. Our laboratory previously demonstrated that diabetes-induced VEGF expression in the retina was dependent on the repressor of mRNA translation 4E-BP1. Interaction of 4E-BP1 with the cap-binding protein eIF4E regulates protein expression by controlling the selection of mRNAs for translation. The process is regulated by the master kinase mTOR in complex 1 (mTORC1), which phosphorylates 4E-BP1, thus promoting its disassociation from eIF4E. In the present study, we investigated the role of the Akt/mTORC1 repressor REDD1 (regulated in development and DNA damage) in diabetes-induced VEGF expression. REDD1 expression was induced by hyperglycemia in the retina of diabetic rodents and by hyperglycemic conditions in Müller cells concomitant with increased VEGF expression. In Müller cells, hyperglycemic conditions attenuated global rates of protein synthesis and cap-dependent mRNA translation concomitant with up-regulated cap-independent VEGF mRNA translation, as assessed by a bicistronic luciferase reporter assay. Hyperglycemic conditions also attenuated mTORC1 signaling and enhanced 4E-BP1 binding to eIF4E. Furthermore, ectopic expression of REDD1 in Müller cells was sufficient to promote both increased 4E-BP1 binding to eIF4E and VEGF expression. Whereas the retina of wild-type mice exhibited increased expression of VEGF and tumor necrosis factor alpha (TNF-α) 4 weeks after streptozotocin administration, the retina of REDD1 knock-out mice failed to do so. Overall, the results demonstrate that REDD1 contributes to the pathogenesis of diabetes in the retina by mediating the pathogenic effects of hyperglycemia., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) causes stress to which an unfolded protein response is activated to render cell survival or apoptosis (chronic stress). Transcriptional and translational reprogramming is tightly regulated during the unfolded protein response to ensure specific gene expression. The master regulator of this response is the PERK/eIF2α/ATF4 signaling where eIF2α is phosphorylated (eIF2α-P) by the kinase PERK. This signal leads to global translational shutdown, but it also enables translation of the transcription factor ATF4 mRNA. We showed recently that ATF4 induces an anabolic program through the up-regulation of selected amino acid transporters and aminoacyl-tRNA synthetases. Paradoxically, this anabolic program led cells to apoptosis during chronic ER stress in a manner that involved recovery from stress-induced protein synthesis inhibition. By using eIF2α-P-deficient cells as an experimental system, we identified a communicating network of signaling pathways that contribute to the inhibition of protein synthesis during chronic ER stress. This eIF2α-P-independent network includes (i) inhibition of mammalian target of rapamycin kinase protein complex 1 (mTORC1)-targeted protein phosphorylation, (ii) inhibited translation of a selective group of 5'-terminal oligopyrimidine mRNAs (encoding proteins involved in the translation machinery and translationally controlled by mTORC1 signaling), and (iii) inhibited translation of non-5'-terminal oligopyrimidine ribosomal protein mRNAs and ribosomal RNA biogenesis. We propose that the PERK/eIF2α-P/ATF4 signaling acts as a brake in the decline of protein synthesis during chronic ER stress by positively regulating signaling downstream of the mTORC1 activity. These studies advance our knowledge on the complexity of the communicating signaling pathways in controlling protein synthesis rates during chronic stress.

Endoplasmic reticulum (ER) stress-induced responses are associated with the loss of insulin-producing β-cells in type 2 diabetes mellitus. β-Cell survival during ER stress is believed to depend on decreased protein synthesis rates that are mediated via phosphorylation of the translation initiation factor eIF2α. It is reported here that chronic ER stress correlated with increased islet protein synthesis and apoptosis in β-cells in vivo. Paradoxically, chronic ER stress in β-cells induced an anabolic transcription program to overcome translational repression by eIF2α phosphorylation. This program included expression of amino acid transporter and aminoacyl-tRNA synthetase genes downstream of the stress-induced ATF4-mediated transcription program. The anabolic response was associated with increased amino acid flux and charging of tRNAs for branched chain and aromatic amino acids (e.g. leucine and tryptophan), the levels of which are early serum indicators of diabetes. We conclude that regulation of amino acid transport in β-cells during ER stress involves responses leading to increased protein synthesis, which can be protective during acute stress but can lead to apoptosis during chronic stress. These studies suggest that the increased expression of amino acid transporters in islets can serve as early diagnostic biomarkers for the development of diabetes.

In this study, the interaction of mTORC1 with its downstream targets p70S6K1 and 4E-BP1 was evaluated in both mouse liver and mouse embryonic fibroblasts following combined disruption of the genes encoding 4E-BP1 and 4E-BP2. Phosphorylation of p70S6K1 was dramatically elevated in the livers of mice lacking 4E-BP1 and 4E-BP2 following feeding-induced activation of mTORC1. Immunoprecipitation of mTORC1 suggested that elevated phosphorylation was the result of enhanced interaction of p70S6K1 with raptor. These findings were extended to a cell culture system wherein loss of 4E-BP1 and 4E-BP2 resulted in elevated interaction of p70S6K1 with IGF1-induced activation of mTORC1 in conjunction with an enhanced rate of p70S6K1 phosphorylation at Thr-389. Furthermore, cotransfecting HA-p70S6K1 with 4E-BP1, but not 4E-BP1(F114A), reduced recovery of mTORC1 in HA-p70S6K1 immunoprecipitates. Together, these findings support the conclusion that, in the absence of 4E-BP proteins, mTORC1-mediated phosphorylation of p70S6K1 is elevated by a reduction in competition between the two substrates for interaction with raptor.

Modulation of mRNA binding to the 40 S ribosomal subunit during translation initiation controls not only global rates of protein synthesis but also regulates the pattern of protein expression by allowing for selective inclusion, or exclusion, of mRNAs encoding particular proteins from polysomes. The mRNA binding step is modulated by signaling through a protein kinase known as the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 directly phosphorylates the translational repressors eIF4E binding proteins (4E-BP) 1 and 2, releasing them from the mRNA cap binding protein eIF4E, thereby promoting assembly of the eIF4E·eIF4G complex. mTORC1 also phosphorylates the 70-kDa ribosomal protein S6 kinase 1 (p70S6K1), which subsequently phosphorylates eIF4B, and programmed cell death 4 (PDCD4), which sequesters eIF4A from the eIF4E·eIF4G complex, resulting in repressed translation of mRNAs with highly structured 5'-untranslated regions. In the present study, we compared the role of the 4E-BPs in the regulation of global rates of protein synthesis to that of eIF4B and PDCD4. We found that maintenance of eIF4E interaction with eIF4G was not by itself sufficient to sustain global rates of protein synthesis in the absence of mTORC1 signaling to p70S6K1; phosphorylation of both eIF4B and PDCD4 was additionally required. We also found that the interaction of eIF4E with eIF4G was maintained in the liver of fasted rats as well as in serum-deprived mouse embryo fibroblasts lacking both 4E-BP1 and 4E-BP2, suggesting that the interaction of eIF4G with eIF4E is controlled primarily through the 4E-BPs.

4E-BP1 is a protein that, in its hypophosphorylated state, binds the mRNA cap-binding protein eIF4E and represses cap-dependent mRNA translation. By doing so, it plays a major role in the regulation of gene expression by controlling the overall rate of mRNA translation as well as the selection of mRNAs for translation. Phosphorylation of 4E-BP1 causes it to release eIF4E to function in mRNA translation. 4E-BP1 is also subject to covalent addition of N-acetylglucosamine to Ser or Thr residues (O-GlcNAcylation) as well as to truncation. In the truncated form, it is both resistant to phosphorylation and able to bind eIF4E with high affinity. In the present study, Ins2(Akita/+) diabetic mice were used to test the hypothesis that hyperglycemia and elevated flux of glucose through the hexosamine biosynthetic pathway lead to increased O-GlcNAcylation and truncation of 4E-BP1 and consequently decreased eIF4E function in the liver. The amounts of both full-length and truncated 4E-BP1 bound to eIF4E were significantly elevated in the liver of diabetic as compared with non-diabetic mice. In addition, O-GlcNAcylation of both the full-length and truncated proteins was elevated by 2.5- and 5-fold, respectively. Phlorizin treatment of diabetic mice lowered blood glucose concentrations and reduced the expression and O-GlcNAcylation of 4E-BP1. Additionally, when livers were perfused in the absence of insulin, 4E-BP1 phosphorylation in the livers of diabetic mice was normalized to the control value, yet O-GlcNAcylation and the association of 4E-BP1 with eIF4E remained elevated in the liver of diabetic mice. These findings provide insight into the pathogenesis of metabolic abnormalities associated with diabetes.

In this study, we explored the coordinate regulation of mTORC1 by insulin and amino acids. Rat livers were perfused with medium containing various concentrations of insulin and/or amino acids. At fasting (1×) or 2× (2×AA) concentrations of amino acids, insulin maximally stimulated Akt phosphorylation but had no effect on global rates of protein synthesis. In the absence of insulin, 4×AA produced a moderate stimulation of protein synthesis and activation of mTORC1. The combination of 4×AA and insulin produced a maximal stimulation of protein synthesis and activation of mTORC1. These effects were accompanied by decreases in raptor and PRAS40 and an increase in RagC associated with mTOR (mammalian target of rapamycin). The studies were extended to a cell culture model in which mTORC1 activity was repressed by deprivation of leucine and serum, and resupplementation with the amino acid and insulin acted in an additive manner to restore mTORC1 activation. In deprived cells, mTORC1 was activated by expressing either constitutively active (ca) Rheb or a caRagB·caRagC complex, and coexpression of the constructs had an additive effect. Notably, resupplementation with leucine in cells expressing caRheb or with insulin in cells expressing the caRagB·caRagC complex was as effective as resupplementation with both leucine and insulin in non-transfected cells. Moreover, changes in mTORC1 activity correlated directly with altered association of mTOR with RagB/RagC, Rheb, raptor, and PRAS40. Overall, the results suggest that amino acids signal through the Rag complex and insulin through Rheb to achieve coordinate activation of mTORC1.

Cells respond to a variety of stresses, including unfolded proteins in the endoplasmic reticulum (ER), by phosphorylating a subunit of translation initiation factor eIF2, eIF2α. eIF2α phosphorylation inactivates the eIF2B complex. The inactivation of eIF2B not only suppresses the initiation of protein translation but paradoxically up-regulates the translation and expression of transcription factor ATF-4. Both of these processes are important for the cellular response to ER stress, also termed the unfolded protein response. Here we demonstrate that cellular response resulting from eIF2α phosphorylation is attenuated in several cancer cell lines. The deficiency of the unfolded protein response in these cells correlates with the expression of a specific isoform of a regulatory eIF2B subunit, eIF2Bδ variant 1 (V1). Replacement of total eIF2Bδ with V1 renders cells insensitive to eIF2α phosphorylation; specifically, they neither up-regulate ATF-4 and ATF-4 targets nor suppress protein translation. Expression of variant 2 eIF2Bδ in ER stress response-deficient cells restores the stress response. Our data suggest that V1 does not interact with the eIF2 complex, a requisite for eIF2B inhibition by eIF2α phosphorylation. Together, these data delineate a novel physiological mechanism to regulate the ER stress response with a large potential impact on a variety of diseases that result in ER stress.

Control of translation initiation in a tissue of an intact mammalian organism is a highly complex process requiring the continuous integration of multiple positive and negative stimuli. For a tissue such as skeletal muscle, which has the capacity to undergo dramatic changes in size and protein content, translation initiation contributes importantly to the regulation of global rates of protein synthesis and is controlled by numerous stimuli, including those arising from nutrients and hormones in the circulating blood, as well as from contraction-induced signaling within the tissue. Many of the pathways conveying signals generated by these stimuli converge on mTORC1, a serine-threonine protein kinase that has been termed the nutrient and energy sensor of the cell and that plays a prominent role in the regulation of cell growth. Control of translation initiation by mTORC1 is mediated through phosphorylation of downstream targets that modulate the binding of mRNA to the 43 S preinitiation complex. Control of translation initiation is also mediated through modulation of binding of initiator methionyl-tRNA to the 40 S ribosomal subunit. Together, modulation of these two regulatory steps in translation initiation accounts in large part for changes in protein synthesis in skeletal muscle produced by the integration of inputs from hormones, nutrients, and exercise.

mTORC1 is a complex of proteins that includes the mammalian target of rapamycin (mTOR) and several regulatory proteins. It is activated by a variety of hormones (e.g. insulin) and nutrients (e.g. amino acids) that act to stimulate cell growth and proliferation and repressed by hormones (e.g. glucocorticoids) that act to reduce cell growth. Curiously, mTORC1 signaling is reported to be rapidly (e.g. within 1-2 h) activated by inhibitors of protein synthesis that act on either mRNA translation elongation or gene transcription. However, the basis for the mTORC1 activation has not been satisfactorily delineated. In the present study, mTORC1 signaling was found to be activated in response to inhibition of either the initiation or elongation phases of mRNA translation. Changes in mTORC1 signaling were inversely proportional to alterations in the expression of the mTORC1 repressor, REDD1, but not the expression of TRB3 or TSC2. Moreover the cycloheximide-induced increase in mTORC1 signaling was significantly attenuated in cells lacking REDD1, showing that REDD1 plays an integral role in the response. Finally, the half-life of REDD1 was estimated to be 5 min or less. Overall, the results are consistent with a model in which inhibition of protein synthesis leads to a loss of REDD1 protein because of its rapid degradation, and in part reduced REDD1 expression subsequently leads to de-repression of mTORC1 activity.

The mammalian target of rapamycin (mTOR), a critical modulator of cell growth, acts to integrate signals from hormones, nutrients, and growth-promoting stimuli to downstream effector mechanisms involved in the regulation of protein synthesis. Dexamethasone, a synthetic glucocorticoid that represses protein synthesis, acts to inhibit mTOR signaling as assessed by reduced phosphorylation of the downstream targets S6K1 and 4E-BP1. Dexamethasone has also been shown in one study to up-regulate the expression of REDD1 (also referred to RTP801, a novel stress-induced gene linked to repression of mTOR signaling) in lymphoid, but not nonlymphoid, cells. In contrast to the findings of that study, here we demonstrate that REDD1, but not REDD2, mRNA expression is dramatically induced following acute dexamethasone treatment both in rat skeletal muscle in vivo and in L6 myoblasts in culture. In L6 myoblasts, the effect of the drug on mTOR signaling is efficiently blunted in the presence of REDD1 RNA interference oligonucleotides. Moreover, the dexamethasone-induced assembly of the mTOR regulatory complex Tuberin. Hamartin is disrupted in L6 myoblasts following small interfering RNA-mediated repression of REDD1 expression. Finally, overexpression of Rheb, a downstream target of Tuberin function and a positive upstream effector of mTOR, reverses the effect of dexamethasone on phosphorylation of mTOR substrates. Overall, the data support the conclusion that REDD1 functions upstream of Tuberin and Rheb to down-regulate mTOR signaling in response to dexamethasone.

The contribution of mammalian target of rapamycin (mTOR) signaling to the resistance exercise-induced stimulation of skeletal muscle protein synthesis was assessed by administering rapamycin to Sprague-Dawley rats 2 h prior to a bout of resistance exercise. Animals were sacrificed 16 h postexercise, and gastrocnemius protein synthesis, mTOR signaling, and biomarkers of translation initiation were assessed. Exercise stimulated the rate of protein synthesis; however, this effect was prevented by pretreatment with rapamycin. The stimulation of protein synthesis was mediated by an increase in translation initiation, since exercise caused an increase in polysome aggregation that was abrogated by rapamycin administration. Taken together, the data suggest that the effect of rapamycin was not mediated by reduced phosphorylation of eukaryotic initiation factor 4E (eIF4E) binding protein 1 (BP1), because exercise did not cause a significant change in 4E-BP1(Thr-70) phosphorylation, 4E-BP1-eIF4E association, or eIF4F complex assembly concomitant with increased protein synthetic rates. Alternatively, there was a rapamycin-sensitive decrease in relative eIF2Bepsilon(Ser-535) phosphorylation that was explained by a significant increase in the expression of eIF2Bepsilon protein. The proportion of eIF2Bepsilon mRNA in polysomes was increased following exercise, an effect that was prevented by rapamycin treatment, suggesting that the increase in eIF2Bepsilon protein expression was mediated by an mTOR-dependent increase in translation of the mRNA encoding the protein. The increase in eIF2Bepsilon mRNA translation and protein abundance occurred independent of similar changes in other eIF2B subunits. These data suggest a novel link between mTOR signaling and eIF2Bepsilon mRNA translation that could contribute to the stimulation of protein synthesis following acute resistance exercise.

The opposing actions of glucagon and insulin on glucose metabolism within the liver are essential mechanisms for maintaining plasma glucose concentrations within narrow limits. Less well studied are the counterregulatory actions of glucagon on protein metabolism. In the present study, the effect of glucagon on amino acid-induced signaling through the mammalian target of rapamycin (mTOR), an important controller of the mRNA binding step in translation initiation, was examined using the perfused rat liver as an experimental model. The results show that amino acids enhance signaling through mTOR resulting in phosphorylation of eukaryotic initiation factor 4E-binding protein (4E-BP)1, the 70-kDa ribosomal protein (rp)S6 kinase, S6K1, and rpS6. In contrast, glucagon repressed both basal and amino acid-induced signaling through mTOR, as assessed by changes in the phosphorylation of 4E-BP1 and S6K1. The repression was associated with the activation of protein kinase A and enhanced phosphorylation of LKB1 and the AMP-activated protein kinase (AMPK). Surprisingly, the phosphorylation of two S6K1 substrates, rpS6 and eukaryotic initiation factor 4B, was not repressed but instead was increased by glucagon treatment, regardless of the amino acid concentration. The latter finding could be explained by the glucagon-induced phosphorylation of the ERK1 and the 90-kDa rpS6 kinase p90(rsk). Thus, glucagon represses phosphorylation of 4E-BP1 and S6K1 through the activation of a protein kinase A-LKB-AMPK-mTOR signaling pathway, while simultaneously enhancing phosphorylation of other downstream effectors of mTOR through the activation of the extracellular signal-regulated protein kinase 1-p90(rsk) signaling pathway. Amino acids also enhance AMPK phosphorylation, although to a lesser extent than glucagon and amino acids combined.

The purpose of this study was to examine the role of the ribosomal protein S6 protein kinase (p70S6K), a protein synthesis regulator, in promoting retinal neuronal cell survival. Differentiated R28 rat retinal neuronal cells were used as an experimental model. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum, and during the period of experimentation were exposed either to the absence or presence of 10 nm insulin. Insulin treatment induced p70S6K, mTOR, and Akt phosphorylation, effects that were completely prevented by the PI3K inhibitor, LY294002. Insulin-induced phosphorylation of p70S6K and mTOR was prevented by the mTOR inhibitor, rapamycin. Apoptosis, induced by serum deprivation and evaluated by Hoechst staining, was inhibited by insulin treatment in R28 cells, but not in L6 muscle cells. This effect of insulin was also largely prevented by rapamycin. Inhibition of p70S6K activity by exogenous expression of a dominant negative mutant of p70S6K prevented insulin-induced cell survival, whereas, overexpression of wild type p70S6K or expression of a rapamycin resistant form of the kinase enhanced the effect of insulin on survival. Enhanced cell survival under the latter condition was accompanied by increased p70S6K activity and phosphorylation. Rapamycin did not inhibit insulin induced p70S6K phosphorylation and activity in cells transfected with the rapamycin-resistant mutant. Together, these results suggest that p70S6K plays a key role in insulin stimulated retinal neuronal cell survival.

AMP-activated protein kinase (AMPK) is viewed as an energy sensor that acts to modulate glucose uptake and fatty acid oxidation in skeletal muscle. Given that protein synthesis is a high energy-consuming process, it may be transiently depressed during cellular energy stress. Thus, the intent of this investigation was to examine whether AMPK activation modulates the translational control of protein synthesis in skeletal muscle. Injections of 5-aminoimidazole-4-carboxamide 1-beta-d-ribonucleoside (AICAR) were used to activate AMPK in male rats. The activity of alpha1 AMPK remained unchanged in gastrocnemius muscle from AICAR-treated animals compared with controls, whereas alpha2 AMPK activity was significantly increased (51%). AICAR treatment resulted in a reduction in protein synthesis to 45% of the control value. This depression was associated with decreased activation of protein kinases in the mammalian target of rapamycin (mTOR) signal transduction pathway as evidenced by reduced phosphorylation of protein kinase B on Ser(473), mTOR on Ser(2448), ribosomal protein S6 kinase on Thr(389), and eukaryotic initiation factor eIF4E-binding protein on Thr(37). A reduction in eIF4E associated with eIF4G to 10% of the control value was also noted. In contrast, eIF2B activity remained unchanged in response to AICAR treatment and therefore would not appear to contribute to the depression in protein synthesis. This is the first investigation to demonstrate changes in translation initiation and skeletal muscle protein synthesis in response to AMPK activation.

Protein metabolism in eukaryotic organisms is defined by a synthesis-degradation equilibrium that is subject to regulation by hormonal and nutritional signals. In mammalian tissues such as skeletal muscle, glucocorticoid hormones specify a catabolic response that influences both protein synthetic and protein degradative pathways. With regard to the former, glucocorticoids attenuate mRNA translation at two levels: translational efficiency, i.e. translation initiation, and translational capacity, i.e. ribosome biogenesis. Glucocorticoids may impair translational capacity through the ribosomal S6 protein kinase (p70 S6K), a recognized glucocorticoid target and an effector of ribosomal protein synthesis. We demonstrate here that the reduction in growth factor-activated p70 S6K activity by glucocorticoids depends upon a functional glucocorticoid receptor (GR) and that the GR is both necessary and sufficient to render p70 S6K subject to glucocorticoid regulation. Furthermore, the DNA binding and transcriptional activation but not repression properties of the GR are indispensable for p70 S6K regulation. Finally, a mutational analysis of the p70 S6K carboxyl terminus indicates that this region confers glucocorticoid sensitivity, and thus glucocorticoids may facilitate autoinhibition of the enzyme ultimately reducing the efficiency with which T389 is phosphorylated.

Leucine, glutamine, and tyrosine, three amino acids playing key modulatory roles in hepatic proteolysis, were evaluated for activation of signaling pathways involved in regulation of liver protein synthesis. Furthermore, because leucine signals to effectors that lie distal to the mammalian target of rapamycin, these downstream factors were selected for study as candidate mediators of amino acid signaling. Using the perfused rat liver as a model system, we observed a 25% stimulation of protein synthesis in response to balanced hyperaminoacidemia, whereas amino acid imbalance due to elevated concentrations of leucine, glutamine, and tyrosine resulted in a protein synthetic depression of roughly 50% compared with normoaminoacidemic controls. The reduction in protein synthesis accompanying amino acid imbalance became manifest at high physiologic concentrations and was dictated by the guanine nucleotide exchange activity of translation initiation factor eIF2B. Paradoxically, this phenomenon occurred concomitantly with assembly of the mRNA cap recognition complex, eIF4F as well as activation of the 70-kDa ribosomal S6 kinase, p70(S6k). Dual and reciprocal modulation of eIF4F and eIF2B was leucine-specific because isoleucine, a structural analog, was ineffective in these regards. Thus, we conclude that amino acid imbalance, heralded by leucine, initiates a liver-specific translational fail-safe mechanism that deters protein synthesis under unfavorable circumstances despite promotion of the eIF4F complex.

Regulation of translation of mRNAs coding for specific proteins plays an important role in controlling cell growth, differentiation, and transformation. Two proteins have been implicated in the regulation of specific mRNA translation: eukaryotic initiation factor eIF4E and ribosomal protein S6. Increased phosphorylation of eIF4E as well as its overexpression are associated with stimulation of translation of mRNAs with highly structured 5'-untranslated regions. Similarly, phosphorylation of S6 results in preferential translation of mRNAs containing an oligopyrimidine tract at the 5'-end of the message. In the present study, leucine stimulated phosphorylation of the eIF4E-binding protein, 4E-BP1, in L6 myoblasts, resulting in dissociation of eIF4E from the inactive eIF4E.4E-BP1 complex. The increased availability of eIF4E was associated with a 1.6-fold elevation in ornithine decarboxylase relative to global protein synthesis. Leucine also stimulated phosphorylation of the ribosomal protein S6 kinase, p70(S6k), resulting in increased phosphorylation of S6. Hyperphosphorylation of S6 was associated with a 4-fold increase in synthesis of elongation factor eEF1A. Rapamycin, an inhibitor of the protein kinase mTOR, prevented all of the leucine-induced effects. Thus, leucine acting through an mTOR-dependent pathway stimulates the translation of specific mRNAs both by increasing the availability of eIF4E and by stimulating phosphorylation of S6.

The present study was designed to investigate the mechanism through which leucine and histidine regulate translation initiation in L6 myoblasts. The results show that both amino acids stimulate initiation and coordinately regulate the activity of eukaryotic initiation factor eIF2B. The changes in eIF2B activity could be explained in part by modulation of the phosphorylation state of the alpha-subunit of eIF2. The activity changes might also be a result of modulation of the phosphorylation state of the eIF2B epsilon-subunit, because deprivation of either amino acid caused a decrease in eIF2Bepsilon kinase activity. Leucine, but not histidine, additionally caused a redistribution of eIF4E from the inactive eIF4E.4E-BP1 complex to the active eIF4E.eIF4G complex. The redistribution was a result of increased phosphorylation of 4E-BP1. The changes in 4E-BP1 phosphorylation and eIF4E redistribution associated with leucine deprivation were not observed in the presence of insulin. However, the leucine- and histidine-induced alterations in global protein synthesis and eIF2B activity were maintained in the presence of the hormone. Overall, the results suggest that both leucine and histidine regulate global protein synthesis through modulation of eIF2B activity. Furthermore, under the conditions employed herein, alterations in eIF4E availability are not rate-controlling for global protein synthesis but might be necessary for regulation of translation of specific mRNAs.

The guanine nucleotide exchange activity of eIF2B plays a key regulatory role in the translation initiation phase of protein synthesis. The activity is markedly inhibited when the substrate, i. e. eIF2, is phosphorylated on Ser51 of its alpha-subunit. Genetic studies in yeast implicate the alpha-, beta-, and delta-subunits of eIF2B in mediating the inhibition by substrate phosphorylation. However, the mechanism involved in the inhibition has not been defined biochemically. In the present study, we have coexpressed the five subunits of rat eIF2B in Sf9 cells using the baculovirus system and have purified the recombinant holoprotein to >90% homogeneity. We have also expressed and purified a four-subunit eIF2B complex lacking the alpha-subunit. Both the five- and four-subunit forms of eIF2B exhibit similar rates of guanine nucleotide exchange activity using unphosphorylated eIF2 as substrate. The five-subunit form is inhibited by preincubation with phosphorylated eIF2 (eIF2(alphaP)) and exhibits little exchange activity when eIF2(alphaP) is used as substrate. In contrast, eIF2B lacking the alpha-subunit is insensitive to inhibition by eIF2(alphaP) and is able to exchange guanine nucleotide using eIF2(alphaP) as substrate at a faster rate compared with five-subunit eIF2B. Finally, a double point mutation in the delta-subunit of eIF2B has been identified that results in insensitivity to inhibition by eIF2(alphaP) and exhibits little exchange activity when eIF2(alphaP) is used as substrate. The results provide the first direct biochemical evidence that the alpha- and delta-subunits of eIF2B are involved in mediating the effect of substrate phosphorylation.

Modulation of protein/protein interaction is an important mechanism involved in regulation of translation initiation. Specifically, regulation of the interaction of eIF2 with the guanine nucleotide exchange factor, eIF2B, is a key mechanism for controlling translation under a variety of conditions. Phosphorylation of the alpha-subunit of eIF2 converts the protein into a competitive inhibitor of eIF2B by causing an increase in the binding affinity of eIF2B for eIF2. Consequently, it has been assumed that the alpha-subunit of eIF2 is directly involved in binding to eIF2B. In the present study, eIF2 was found to bind only to the delta- and epsilon-subunits of eIF2B, and eIF2B was shown to bind only to the beta-subunit of eIF2 by far-Western blot analysis. The binding site on eIF2beta for either the eIF2B holoprotein, or the isolated delta- or epsilon-subunits of eIF2B was shown to be located within approximately 70 amino acids of the C terminus of the protein. Phosphorylation of the alpha-subunit of eIF2 did not promote binding of eIF2B to the isolated subunit. However, it did cause an increase in the affinity of eIF2B for eIF2. Finally, phosphorylation by protein kinase A of the beta-subunit of eIF2 in the C-terminal portion of the protein increased the guanine nucleotide exchange activity of eIF2B, whereas phosphorylation by casein kinase II or protein kinase C was without effect.

Eukaryotic initiation factor-2B (eIF-2B) is a guanine nucleotide exchange factor (GEF) that plays a key role in the regulation of protein synthesis. In this study, we have used the baculovirus-infected Sf9 insect cell system to express and characterize the five dissimilar subunits of rat eIF-2B. GEF activity was detected in extracts of Sf9 cells expressing the epsilon-subunit alone and was greatly increased when all five subunits were coexpressed. In addition, high GEF activity was observed in extracts containing a four-subunit complex lacking the alpha-subunit. Assembly of an eIF-2B holoprotein was confirmed by coimmunoprecipitation of all five subunits. Gel filtration chromatography revealed that recombinant eIF-2B had the same molecular mass as eIF-2B purified from rat liver and that it did indeed possess GEF activity. Phosphorylation of the substrate eIF-2 inhibited the GEF activity of the five-subunit eIF-2B; this inhibition required the eIF-2B alpha-subunit. The results demonstrate that eIF-2Balpha functions as a regulatory subunit that is not required for GEF activity, but instead mediates the regulation of eIF-2B by substrate phosphorylation. Furthermore, eIF-2Bepsilon is necessary and is perhaps sufficient for GEF activity in vitro.

Reticulocytes contain an eukaryotic initiation factor-2 alpha (eIF-2 alpha) kinase that is negatively regulated by hemin. This protein kinase, which has been termed heme-regulated inhibitor and heme-controlled repressor (HCR), has a strong inhibitory effect on the initiation of protein synthesis and plays an important role in translational control in these cells. Previous evidence has suggested that HCR may be an erythroid-specific enzyme. As reported herein, we have cloned and characterized the cDNA encoding an eIF-2 alpha kinase from rat brain. The predicted amino acid sequence of the kinase from rat brain shows 82% homology to rabbit reticulocyte HCR with the greatest variation concentrated in a central region of approximately 135 amino acids located between protein kinase subdomains IV and VI. We have expressed the rat brain cDNA in Escherichia coli and have demonstrated that the recombinant enzyme is a hemin-sensitive eIF-2 alpha kinase. We have also shown that mRNA recognized by the rat brain cDNA is expressed in a wide range of non-erythroid rat tissues at a lower but significant level compared with reticulocytes. These findings raise the possibility of additional, uncharacterized roles for HCR in non-erythroid cells.

The studies presented herein were designed to investigate the role of insulin in maintaining the steady-state number of ribosomes in primary cultures of rat hepatocytes. The RNA content of hepatocytes maintained in the presence of insulin did not change during the 7 days of culture. In contrast, the RNA content declined significantly following 2 days of insulin deprivation and after 4 days without insulin reached a new steady-state value that was approximately 60% of that observed for hepatocytes maintained in the presence of the hormone. Following addition of insulin, the RNA content of insulin-deprived hepatocytes started to increase within 6 h and was restored to the control value within 48 h. The amounts of 18 and 28 S RNA, and thus the number of ribosomes, changed in concert with the total RNA content. Furthermore, synthesis of total cellular protein responded in parallel to the changes in RNA content, suggesting that the number of ribosomes was the primary determinant of protein synthesis under the conditions of these experiments. About one-half of the increase in RNA content following the addition of insulin to insulin-deprived cells could be accounted for by a reduction in the rate of degradation of ribosomes. The remainder of the change in RNA content must have resulted from an increase in ribosome biogenesis. This possibility was confirmed by measuring [3H]uridine incorporation into ribosomal RNA present in mature ribosomes. The rate of synthesis of ribosomal RNA was reduced in parallel with the fall in RNA content in insulin-deprived hepatocytes and was restored to the control value within 3 h of the addition of insulin. Alterations in the synthesis of ribosomal RNA in response to insulin deprivation and replacement were associated with parallel changes in transcription of rDNA as measured in nuclear run-on assays using a DNA probe corresponding to the external transcribed spacer region of rat rDNA. The data demonstrate that insulin regulates the number of ribosomes in primary cultures of rat hepatocytes by accelerating the rate of transcription of rDNA and by slowing the rate of ribosome degradation.

In previous studies, initiation of protein synthesis was shown to be inhibited in perfused rat livers deprived of single essential amino acids. In the present study, histidinol, a competitive inhibitor of histidinyl-tRNA synthetase, was used to amplify the effects of histidine deprivation on protein synthesis in perfused liver to facilitate investigation of mechanisms involved in the inhibition of peptide chain initiation. Protein synthesis was reduced to 77% of the control rate in livers deprived of histidine and to 13% of the control rate in livers deprived of histidine and exposed to 2.0 mM histidinol. The inhibition of protein synthesis caused by histidine deprivation alone was accompanied by a 2-fold increase in the number of free ribosomal particles, a 29% decrease in Met-tRNA(i) binding to 43 S preinitiation complexes, and a 31% reduction in activity of eukaryotic initiation factor 2B (eIF-2B). By comparison, histidine deprivation combined with histidinol addition resulted in a 3-fold increase in free ribosomal particles, a 66% decrease in Met-tRNAi binding, and a 78% reduction in eIF-2B activity. The proportion of the alpha-subunit of eukaryotic initiation factor two (eIF-2) in the phosphorylated form increased from 8.9 +/- 0.8% in control livers to 52.4 +/- 5.5% in response to histidinol. The increase in the amount of eIF-2 alpha in the phosphorylated form apparently was not due to an increase in kinase activity, because there was no change in eIF-2 alpha kinase activity in extracts of liver perfused with medium containing histidinol compared to controls. Instead, the increased phosphorylation of eIF-2 alpha was associated with an inhibition of eIF-2 alpha phosphatase activity. Thus, in contrast to other systems that have been examined, the mechanism involved in the increase in the phosphorylation state of eIF-2 alpha appears to involve an inhibition of eIF-2 alpha phosphatase activity rather than activation of an eIF-2 alpha kinase.

A recent study reported that protein synthesis was inhibited in rat livers perfused with medium containing vasopressin (Chin, K. -V., Cade, C., Brostrom, M. A., and Brostrom, C. O. (1988) Int. J. Biochem. 20, 1313-1319). The inhibition of protein synthesis caused by vasopressin was associated with a disaggregation of polysomes, suggesting that peptide chain initiation was slowed relative to elongation. In contrast, Redpath and Proud (Redpath, N. T., and Proud, C. G. (1989) Biochem. J. 262, 69-75) recently reported an inhibition of peptide chain elongation by a calcium/calmodulin-dependent mechanism. Therefore, the question remained whether only peptide chain initiation was inhibited or both initiation and elongation were affected by vasopressin. In the present study, vasopressin was found to inhibit protein synthesis in both perfused rat livers and isolated rat hepatocytes. Ribosomal half-transit times in isolated hepatocytes averaged 1.9 +/- 0.1 min with or without vasopressin present in the media, demonstrating that the rate of peptide chain elongation was unaffected by vasopressin. Instead, the inhibition of protein synthesis induced by vasopressin was manifested at the level of peptide chain initiation. Vasopressin treatment resulted in both a 2-fold increase in the number of free ribosomal particles and a greater than 50% decrease in the amount of [35S]methionine bound to 43 S preinitiation complexes. In addition, the activity of eukaryotic initiation factor (eIF) 2B in crude extracts from perfused livers was reduced to 53% of the control value in response to vasopressin. The inhibition of eIF-2B activity was associated with an increase in the proportion of the alpha-subunit of eIF-2 in the phosphorylated form from 9.6% in control livers to 30.7% in livers perfused with medium containing vasopressin. The results demonstrate the novel finding that the inhibition of protein synthesis in vasopressin-treated livers is caused by a reduction in eIF-2B activity due to an increase in phosphorylation of eIF-2 alpha.

Two polypeptide chain initiation factors, eukaryotic initiation factor 2 (eIF-2) and guanine nucleotide exchange factor (GEF), were isolated from rat liver. Two forms of eIF-2 were identified, one contained three subunits (alpha, beta, and gamma), and the other contained only the alpha- and gamma-subunits. The three-subunit form was similar to eIF-2 from rabbit reticulocytes with respect to the sedimentation coefficient, Stokes radius, molecular weight of the alpha- and gamma-subunits, ability to restore protein synthesis in hemin-deficient reticulocyte lysate, and immunological cross-reactivity of the alpha-subunits using antibodies against liver eIF-2. In contrast, the beta-subunits of the liver and reticulocyte factors were distinct; they had different molecular weights, and antibodies against rat liver eIF-2 beta did not recognize the beta-subunit of the reticulocyte factor. Furthermore, the GDP dissociation constant for reticulocyte eIF-2 was more than twice that of the liver factor. GEF from rat liver reversed GDP inhibition of the ternary complex assay and catalyzed the exchange of eIF-2-bound GDP for free GDP or GTP, characteristics ascribed to the corresponding protein from rabbit reticulocytes. However, its subunit composition and molecular weight were different from those reported for reticulocyte GEF. The T1/2 for GDP exchange mediated by GEF was about 5-fold slower with two-subunit than with three-subunit eIF-2. In addition, the KD for GDP was lower for two-subunit than for three-subunit eIF-2 when GEF was present. Taken together, these data demonstrate species-associated variability in the beta-subunit of eIF-2 and suggest a crucial role for the beta-subunit in the functional interaction of eIF-2 and GEF.